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Robarts Research Retreat

2016

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TABLE OF CONTENTS

Information

Robarts Research Retreat 2016 Program .................................................................................................................................... 3

Welcome Letter ........................................................................................................................................................................... 4

Keynote Presentation .................................................................................................................................................................. 4

Poster Session 1 Map ................................................................................................................................................................... 5

Poster Session 2 Map ................................................................................................................................................................... 6

Abstracts

Cancer Detection & Local Therapy .............................................................................................................................................. 7

Cancer Metastasis & Progression .............................................................................................................................................. 12

Cardiac Disorders & Infarct ........................................................................................................................................................ 17

Cardiovascular Disorders ........................................................................................................................................................... 20

Cellular Therapies & Immunotherapy........................................................................................................................................ 25

Central Nervous System Injury & Inflammation ........................................................................................................................ 28

Cerebral Vasculature ................................................................................................................................................................. 32

Imaging Safety, Quality & Efficiency .......................................................................................................................................... 36

Movement & Mobility ............................................................................................................................................................... 39

Neurodegeneration, Memory & Attention ................................................................................................................................ 45

Neurological Disorders & Epilepsy ............................................................................................................................................. 51

Pulmonary Health ...................................................................................................................................................................... 55

Author Index .............................................................................................................................................................................. 58

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ROBARTS RESEARCH RETREAT 2016 PROGRAM 8:30 AM Auditorium A Registration and Refreshments

8:40 AM Auditorium A Opening Remarks Dr. Marlys Koschinsky

8:45 AM - 9:45 AM

Auditorium A Oral Presentations - First Session Jacqueline Dron "Elucidating the genetic determinants of extreme high-density lipoprotein phenotypes using next-generation sequencing"

Ashley Makela "Evaluating quantification methods to detect tumour associated macrophages in breast cancer: Cellular MRI using fluorine-19 and iron-based methods"

Louisa Salemi "Mechanism and consequences of HDAC6 regulation by RanBPM"

9:45 AM - 10:45 AM

Auditorium A Oral Presentations - Second Session Trevor Morey "Enhanced Ubiquitination and Proteasomal-degradation of Catalytically-deficient Human Choline Acetyltransferase Mutants"

Kathryn Manning "Brain changes observed with MRI in female athletes after one or more concussion-free seasons"

Dr. Diego Cantor "PERSEUS: enhancing ultrasound imaging with pattern recognition capabilities"

11:00 AM - 12:00 PM

Auditorium A Keynote Presentation Dr. Leonardo G. Cohen "Restoring brain function after injury: strategies in the era of open science"

12:00 PM - 1:00 PM

Robarts 5th Floor Lunch Poster Presentations - First Session Set-Up

1:00 PM - 1:30 PM

Robarts 5th Floor Pitch Presentations - First Session Tom Hrinivich Shirley Liu Kayla Ryan Zachary Hawley

Sara Matovic Warren Winick-Ng John Baxter Jonathan Lau

1:30 PM - 2:15 PM

Robarts 5th Floor Poster Presentations - First Session

2:15 PM - 2:30 PM

Robarts 5th Floor Poster Presentations - First Session Take-Down Poster Presentations - Second Session Set-Up

2:30 PM - 3:00 PM

Robarts 5th Floor Pitch Presentations - Second Session Alex Moszczynski Amy Schranz Trung Le Matthew Lowerison

Jacob Poirer Stephen Sherman Yiwen Xu

3:00 PM - 3:45 PM

Robarts 5th Floor Poster Presentations - Second Session

3:45 PM - 4:15 PM

Robarts 5th Floor Afternoon Break Poster Presentations - Second Session Take-Down

4:15 PM Robarts 5th Floor Closing Remarks Dr. Marlys Koschinsky

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WELCOME LETTER Dear Robarts Research Institute Trainees, Scientists, and Staff, Welcome to the third annual Robarts Research Retreat! Already this year, many big changes have happened to Robarts. We have welcomed our new Scientific and Executive Director, Dr. Marlys Koschinsky, and with her began a new Strategic Plan for the institute. Robarts Clinical Trials have settled into a new location in the heart of downtown London, leaving room here on campus for expansion. Looking forward into the future of Robarts is more exciting now than ever. But sometimes, a glance backwards in inevitable. Two years ago, a group of dedicated Robarts trainees were inspired to create a retreat in which Robarts trainees across disciplines could come together and discuss their work. After months of organization, the 1st Robarts Research Retreat was held. Two years later, the Robarts Research Retreat is still holding strong, with 98 posters, 15 three-minute pitches and 6 top student presentations. In closing, I would like to emphasize that one cannot do research in a vacuum, that the success of our institute is a product of the knowledge, inspiration and dedication brought into Robarts at all levels. This retreat is a product of the hard work of Robarts scientists and staff, but most importantly, its trainees. Thank you all for making this a reality. Sincerely,

John Baxter Robarts Research Retreat 2016 Organizing Committee

KEYNOTE PRESENTATION

Dr. Leonardo G. Cohen, Senior Investigator, National Institute of Neurological Disorders and Stroke (NINDS) Dr. Cohen received his MD from the University of Buenos Aires. He did his neurology residency at Georgetown University and received postdoctoral training in clinical neurophysiology at the Department of Neurology, University of California (Irvine) and in motor control and movement disorders at the Human Motor Control Section, NINDS. In 1998, he became Chief of the Human Cortical Physiology Section, NINDS. He received the prestigious Humboldt Award (1999) from the Republic of Germany and is an elected member of the American Neurological Association. Dr. Cohen's lab is interested in the mechanisms underlying plastic changes in the human central nervous system and in the development of novel therapeutic approaches for recovery of function based on the understanding of these mechanisms.

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POSTER SESSION 1 MAP

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POSTER SESSION 2 MAP

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CANCER DETECTION & LOCAL THERAPY

MRI-CEST AACID DETECT CARIPORIDE INDUCED INTRACELLULAR ACIDIFICATION IN BRAIN TUMOR: IN-VIVO AT 9.4TESLA

Mohammed Albatany1,2

, Alex Li2, Miranda Bellyou

2, Susan Meakin

3, and Robert Bartha

1,2

1Department of Medical Biophysics,

2Centre of Functional and Metabolic Mapping,

3Molecular Medicine Group,

Robarts Research Institute, Western University, London, Ontario Some brain tumours are highly aggressive with few successful treatment options. Chemical exchange saturation transfer (CEST) is an MRI contrast mechanism that is dependent on intracellular pH. Cariporide is a sodium proton exchange inhibitor that is well tolerated by humans in cardiac applications. The goals of this study were to determine whether CEST MRI measurement of tumour pHi could detect acidification after cariporide injection and to measure the magnitude of pHi change. Using a 9.4T MRI scanner two hours after cariporide injection there was a significant decrease in tumour pHi by 0.37±0.03.This use of a pharmacologic agent to induce a measureable physiologic change represents a unique approach to cancer detection that differs from other current molecular imaging techniques.

MEASURING THE SPIN-LATTICE RELAXATION DISPERSION OF RODENT TISSUES USING FAST FIELD-CYCLING MAGNETIC

RESONANCE IMAGING

Araya Y1,2

, Martinez F1,2

, Harris C3, Handler W

3, Chronik B

1-3, Scholl T

1,2

1Robarts Research Institute,


3Department of Physics & Astronomy,

Western University The ability to exploit the native proton spin-lattice relaxation rates (R1) and their associated dispersion over a range of magnetic field shifts using fast field-cycling magnetic resonance imaging (FFC-MRI), is a potential tool to differentiate between normal and atypical tissues. This study quantified the R1-dispersion of rodent tissues using two FFC-MRI relaxomtery methods from 0.23 mT to 3 Tesla. Adult mice tissues were excised for ex vivo R1-dispersion analysis at 37°C. R1-dispersion profiles were acquired using nuclear magnetic resonance dispersion data for magnetic field strengths ranging from 0.23 mT to 1.0 T, using a SpinMaster nuclear magnetic resonance relaxometer. Imaging was performed on a 1.5T GE MRI system outfitted with a fast field-cycling insert magnet to dynamically control B0 prior to imaging. Images were acquired using a field-cycling fast spin-echo inversion recovery pulse sequence, where ΔB0 was modulated for a duration prior to imaging. In vivo whole-body spin-lattice relaxation rates and dispersion maps for mice tissues have been quantified for a magnetic field of shift of ±0.235 T about 1.5 T. These findings emphasize the inherent weak R1 magnetic field dependence of healthy tissues. These are important data as we study atypical or cancerous tissues, which may have a significantly greater field dispersion or may be highlighted by a targeted contrast agent that increases their dispersion.

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MOTION COMPENSATION DURING PROSTATE BIOPSIES USING 2D/3D ULTRASOUND RIGID REGISTRATION

Derek J Gillies1,2

, Ren Zhao3, Lori Gardi

1,2, Aaron Fenster

1,2,3

1Department of Medical Biophysics, Western University, London, Ontario, Canada;

2Robarts Research Institute, Western

University, London, Ontario, Canada; 3Centre for Imaging Technology Commercialization, London, Ontario, Canada;

Of cancers tracked by Canadian registries, prostate cancer has the highest incidence in men and prostate biopsy is the current clinical standard to diagnose this disease. These biopsies use needles to remove small tissue samples in the prostate and are guided by two-dimensional transrectal ultrasound (2D TRUS). We have previously proposed the use of 3D TRUS/MR fusion images during biopsies with the goal of more accurate needle guidance and improved diagnoses by augmenting the 2D TRUS. However, in aligning the 2D and 3D images, a limitation arises due to prostate motion causing misalignment of anatomical and planned targets identified using the 3D TRUS/MR fused images. Here we demonstrate a motion compensation algorithm, leveraging a previously developed implementation, which aligns 2D and 3D ultrasound images to correct for these misalignments near real-time. Image matrix size significantly affects registration accuracy and this was investigated on retrospective prostate images to optimize the registration algorithm. With a mean accuracy of approximately 2.6 ± 1.5 millimeters, this code was accelerated to compute registrations at approximately 110 ± 32 milliseconds per 2D image with a graphics processing unit, verified on 2D and 3D prostate images from 7 biopsy patients. Current and future work involves validating this algorithm on the 3D TRUS system for real-time guidance on prostate phantoms and patients undergoing biopsy procedures.

ULTRASOUND GUIDED HIGH-DOSE-RATE PROSTATE BRACHYTHERAPY: LIVE NEEDLE SEGMENTATION AND 3D IMAGE

RECONSTRUCTION USING THE SAGITTAL TRANSDUCER

Hrinivich WT1,2

, Hoover DA2,4

, Surry K2,4

, Edirisinghe C1, D’Souza D

4, Fenster A

1,2, Wong E

2,3

1Robarts Research Institute; Departments of

2Medical Biophysics and

3Physics and Astronomy, Western University;

4London

Regional Cancer Program

Ultrasound-guided high-dose-rate prostate brachytherapy (HDR-BT) needle segmentation is performed clinically using live-2D sagittal images. Organ segmentation is performed using axial images that are manually registered to the sagittal view, introducing uncertainty in needle positions relative to anatomy. Sagittally-reconstructed 3D (SR3D) ultrasound enables both needle and organ segmentation, but suffers from shadow artifacts. We present a needle segmentation technique augmenting SR3D with live-2D sagittal images using mechanical probe tracking to mitigate image artifacts and compare it to the clinical standard. Seven prostate cancer patients underwent TRUS-guided HDR-BT during which the clinical and proposed segmentation techniques were completed in parallel using dual ultrasound video outputs. Calibrated needle end-length measurements were used to calculate insertion depth errors (IDEs), and the dosimetric impact of IDEs was evaluated by perturbing clinical treatment plan source positions. The proposed and clinical techniques resulted in 84% and 43% of needles with IDEs within ±3 mm, and IDE ranges across all needles of [-7.7 mm, 5.9 mm] and [-9.3 mm, 7.7 mm] respectively. The proposed and clinical IDEs lead to mean±SD changes in the volume of the prostate receiving the prescription dose of -0.6±0.9% and -2.0±5.3% respectively. The proposed technique provides improved HDR-BT needle segmentation accuracy over the clinical technique leading to decreased dosimetric uncertainty by eliminating the axial-to-sagittal registration, and mitigates the effect of shadow artifacts by incorporating mechanically registered live-2D sagittal images.

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MEASURING INTRA- AND EXTRACELLULAR PH IN TUMOURS USING MRI

Heeseung Lim, Mohammed Albatany, Francisco M. Martínez, Robert Bartha, Timothy J. Scholl

Medical Biophysics, Western University, London, ON, Canada.

In tumours, the pH gradient (intracellular pH – extracellular pH) is usually positive and often reversed compared to normal tissue. Given the pharmacokinetics of chemotherapeutic drugs are highly depended on tumour pH, the ability to non-invasively quantify tumour pH is an important aspect of cancer treatment. This study used two magnetic resonance imaging (MRI) techniques to map the intracellular/extracellular pH gradient in a rodent glioma model. The intracellular pH (pHi) was mapped with chemical exchange saturation transfer (CEST) and hyperpolarized 13C bicarbonate magnetic resonance spectroscopic imaging was used to determine regional extracellular pH (pHe).

Longitudinal measurements of the pH gradient for a representative rodent glioma model for days 8, 12 and 15 after surgery are shown in Figure 1. During tumour growth, the extracellular space within the tumour became more acidic, whereas the intracellular compartment became more alkaline leading to an increase in the pH gradient. Overall, the averaged pH gradient in the tumour changed from -0.01 to 0.28 then 0.09. Conversely the pH gradient of contralateral brain tissue changed from -0.34 to -0.24 then -0.47.

The acid-base balance in the brain is tightly controlled by endogenous buffers such as bicarbonate and phosphate. An alkaline tumour pHi increases the activity of several metabolic enzymes that drive cellular proliferation. In contrast, an acidic tumour pHe is established due to increased lactic acid production and the subsequent active transport of protons out of the cell. In this study, the results showed a consistent agreement of increased pH gradient in tumour compared with the contralateral tissue. Also, the pH gradient of tumour increased at later time points. Moreover, the heterogeneity of tumour pH began to increase. Overall, the intracellular/extracellular pH gradients in this rodent glioma model were non-invasively measured to a precision of ~0.1 pH units at three time points. A large difference in pH gradient was observed between tumour and contralateral brain throughout the growth of the tumours. Since most therapeutic agents are weak acids or bases, a priori knowledge of the pH gradient may help guide choice of therapeutic agent. In future work, tumour heterogeneity will be validated using histology.

ENGINEERING GENE VECTORS FOR LONG-TERM TRACKING OF CELL FATE IN ANIMALS WITH BIOLUMINESCENCE IMAGING

Shirley Liu1, Hamilton AM

2, Ronald JA

2-4

1London Central Secondary School,

2Robarts Research Institute, Departments of

3Medical Biophysics and

4Microbiology and

Immunology, Western University

Cellular-genetic imaging can enable the fate of transplanted cells to be visualized in both animals and humans. This requires cells to be tagged using DNA vectors expressing imaging reporter genes prior to implantation into the subject. The ideal DNA vector enables high and persistent reporter expression so that cell number and location can be sensitively tracked over long periods of time. The promoter is a region of DNA on the vector that determines the expression of the encoded gene and is vital for achieving high expression, yet different promoters perform better in different cell types. I hypothesized that DNA cloning could be used to generate a UbiB(h) promoter-driven reporter gene plasmid that would have high and persistent expression over time. The original vector used in this project was the cDNA3.1 (+)/Luc2=tdT plasmid, which expresses a bioluminescent reporter gene (luciferase 2) and a red fluorescent protein (tdTomato) driven by the CMV promoter which has been shown to be silenced over time. I used DNA cloning to insert the UbiB(h) promoter into the pcDNA3.1(+)/Luc2=tdT plasmid and determined successful cloning by restriction enzyme digestion and sequence analysis. I compared the gene expression of the CMV and UbiB(b) promoter plasmids in a murine breast cancer cell line using a luciferase assay and fluorescence microscopy.

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A MECHANICAL 3D ULTRASOUND SYSTEM FOR INTRAOPERATIVE GUIDANCE OF BREAST BRACHYTHERAPY

Michael J1,2

, Batchelar D3, Fenster A

1,2,4

1Imaging Research Laboratories, Robarts Research Institute;

2Biomedical Engineering Graduate Program, Western

University, London ON; 3Department of Medical Physics, British Columbia Cancer Agency, Cancer Center for the Southern

Interior, Kelowna, BC, Canada; 4Departments of Medical Biophysics and Medical Imaging, Western University, London ON

Permanent breast seed implantation (PBSI) is a type of brachytherapy used in treating early stage breast cancer by implanting ‘seeds’ of radioactive Pd-103 using needles inserted under 2D ultrasound guidance. The procedure reduces radiation treatment time to a single visit, which is desirable given the widely hypothesized link between lengthy treatment and underutilization of radiation therapy. A limiting factor to wider adoption of PBSI is operator dependence, largely caused by limitations of 2D ultrasound. Our goal is to develop a 3D ultrasound (3D US) guidance system to reduce operator dependence and increase implantation accuracy. A 3D US system was constructed consisting of a linear, 2D ultrasound transducer that is mechanically articulated and tracked. Images are captured using a laptop and reconstructed into a 3D volume. The scan time is 20s and the 3D image is available immediately. 3D reconstruction was validated, 1) geometrically, using linear measurements of distance between strings known to be 10mm apart in each direction, and 2) volumetrically, using embedded agar phantoms molded after patient contours of the tumor bed and compared to water displacement measurements. Volunteer scans were conducted to demonstrate clinical proof of concept. Geometric validation showed

median measurements within 1.5% of nominal and volumetric validation showed differences of <5% between 3D US and water displacement. Volunteer scans produced clinical quality images with positive user feedback.

SINGLE-SLICE US-MRI REGISTRATION FOR NEUROSURGICAL MRI-GUIDED US

Utsav Pardasani, J.S.H Baxter, A.R. Khan, T. Peters

Imaging Research Laboratories, Robarts Research Institute

The use of tracked ultrasound has been shown to improve patient outcomes in neurosurgery, providing gains comparable to workflows involving an intra-operative MRI according to some studies. Nonetheless, its use is not part of the standard of care for many neurosurgery clinics. Commercially available systems rely on existing optical-tracking systems, but will lose image-registration when line-of- sight is obstructed, or when the brain deforms. The work-flow issues associated with current implementations of tracked ultrasound include set-up time, physical footprint, cabling, and mentally overloading surgical staff. Thus, we posit that a system lighter in workflow may gain more traction in neurosurgical suites.

We will present our work towards developing an MRI-Guided ultrasound system that will be more robust to registration/optical tracking errors as well as brain-shift prior to making the resection cavity. Our envisioned system is based on the use of ultrasound to MR image registration in conjunction with inexpensive off-the- shelf sensors. We will present our progress on evaluating the LC2 similarity metric for the multi-modal 2D-3D registration aspect of this envisioned system with validation on the Brain Images of Tumors for Evaluation (BITE) Database.

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3D ULTRASOUND SYSTEM FOR NEEDLE GUIDANCE DURING HIGH-DOSE-RATE INTERSTITIAL BRACHYTHERAPY OF

GYNAECOLOGICAL CANCERS

J Rodgers1,2

, D Tessier2, D D’Souza

3, E Leung

4, G Hajdok

3, K Surry

3, A Fenster

1,2

1Biomedical Engineering, The University of Western Ontario, London, Ontario, Canada;

2Robarts Imaging Research Lab,

London, Ontario, Canada; 3London Regional Cancer Program, London, Ontario, Canada;

4Odette Cancer Centre, Toronto,

Ontario, Canada

Gynaecological cancers are among the most prevalent in females worldwide with cervical cancer being the fourth most common cancer in women. High-dose-rate (HDR) interstitial brachytherapy is a treatment for these cancers where a radioactive source is inserted via needles into the tumour area through a template. There is no standard imaging modality to guide the needle placement in this procedure and, given the proximity to critical organs, in particular the bladder and rectum, serious complications can arise. We propose improving needle positioning and verification during HDR interstitial gynaecological brachytherapy using a three-dimensional (3D) transrectal ultrasound (TRUS) system. As a proof-of-concept, a 3D US image and a CT scan were acquired following needle insertion and manually, rigidly registered for four patients undergoing HDR interstitial gynaecological brachytherapy at the London Health Sciences Centre. A total of 60 needles were placed; 53 needle paths were visible and 48 needle tips were identified in the 3D US images. The mean errors in needle trajectory were less than 4.00o and the mean errors in needle tip position was less than 4.5 mm for all four patients imaged. Based on the preliminary results, comparison of the 3D US images to post-insertion CT images has shown that 3D TRUS is a feasible technique for correctly visualizing needles, providing the potential for needle guidance during HDR interstitial gynaecological brachytherapy.

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CANCER METASTASIS & PROGRESSION

STRESS-INDUCED NEUROPEPTIDE Y-Y5R AS A MEDIATING FACTOR BREAST CANCER METASTASIS

Kara J1,2

,, Makela A2, Jackson D

1, Foster PJ

2

1

Department of Medical Biophysics, Schulich Medicine & Dentistry, The University of Western Ontario 2 Imaging Research Laboratories, Robarts Research Institute, The University of Western Ontario

Studies have demonstrated a correlation between stress and an increased risk of breast cancer. Neuropeptide Y (NPY) is upregulated in chronic stress and induces proliferation and chemotaxis of 4T1 breast cancer cells via Y5 receptor (Y5R) activation. The objective is to characterize the NPY-Y5R as a regulatory system of breast cancer metastasis. We compared three cancer cell sublines derived from the same murine mammary fat pad tumour, that greatly differ in metastatic potential (67NR, 168FARN, and 4T1) in addition to a 4T1-Y5R knockdown established in our labs. Immunocytochemistry revealed cytoplasmic Y5R expression in 4T1 cells and nuclear Y5R expression in 67NR and 168FARN cells, suggesting Y5R cytoplasmic localization supports an aggressive phenotype. Western blots revealed a significantly greater Y5R expression (p<0.05) in the aggressive 4T1 cell line compared to the 67NR and 168FARN cell lines. An MTS assay revealed NPY stimulation had a significant proliferative effect (p<0.01) on 4T1, 168FARN and 67NR cells at various concentrations. Y5R antagonist addition restored the growth of the cells to basal levels in all three cell lines (p<0.05). Tumour volume, void volumes, and metastatic potential of iron oxidelabelled 4T1 cells were quantified using cellular MRI, setting the grounds to proceed to MRI cell tracking of the 4T1-Y5R knockdown. In this respect, NPY and its Y5 receptor could be therapeutic targets to combat breast cancer metastasis.

TOWARDS THE EVALUATION OF TREATMENT RESPONSE OF GLIOMA IN A RAT MODEL OF CANCER USING HYPERPOLARIZED 13C

MAGNETIC RESONANCE SPECTROSCOPIC IMAGING AND BIOLUMINESCENCE IMAGING.

Trung N.T. Le1, Heeseung Lim

1, Amanda Hamilton

2, Katie Parkins

1, Francisco M. Martínez

2, Timothy J. Scholl

1,2, John A.

Ronald1,2

1Medical Biophysics, Western University, London, ON, Canada.

2Robarts Research Institute, Western University, London,

ON, Canada

Current methods to determine tumour response to treatment rely on anatomical changes and can take weeks to determine treatment effectiveness. Probing the biology of the tumour and the microenvironment will provide information necessary to determine an accurate and timely assessment of therapeutic response of tumour, which is an ability that is extremely important in treating cancer. Our eventual goal is to compare the assessment of therapeutic response of glioblastoma to chemo-radiation using hyperpolarized (HP) [1-13C]pyruvate and [1,4-13C]fumarate magnetic resonance spectroscopic imaging (MRSI), standard multi-parametric 1H magnetic resonance imaging (MRI) and bioluminescence imaging (BLI) in a longitudinal animal study. HP MRSI is an imaging modality capable of measuring metabolism in vivo and non-invasively to determine early treatment response. BLI is a preclinical imaging modality capable of counting live cells in an animal model non-invasively in a longitudinal study. So far, preliminary results suggest that BLI can measure tumour growth over time and can detect changes before standard methods that rely on morphological changes. In addition, HP MRSI can measure metabolism and changes in metabolism which occur sooner than anatomical changes in the tumour. Work is currently being done to assess the ability of HP MRSI and BLI to detect therapeutic response of glioma in a longitudinal animal study.

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PDXOVO: ULTRA-FAST IN VIVO DRUG SENSITIVITY MATRICES FOR RENAL CELL CARCINOMA PATIENTS PRIOR TO

ADMINISTRATION OF TARGETED THERAPY

Lowerison MR1,2

, Leong HS3, Fedyshyn Y

3, Chambers AF

2,4,6, Lacefield JC

1,2,5, and Power NE

3

1Imaging Research Laboratories, Robarts Research Institute, London ON

Depts. of 2Medical Biophysics,

3Surgery,

4Oncology, and

5Electrical & Computer Engineering, Western University

6London Regional Cancer Program, London, Ontario, Canada

Our next generation patient derived xenograft (PDX) model of renal cell carcinoma (RCC) offers the ability to pre-determine de novo drug resistance in fresh patient tumor samples prior to targeted therapy. Implantation of tumor specimens into the chorioallantoic membrane (CAM) of the chicken embryo results in high engraftment efficiencies within two days permitting large scale “tumor avatar” studies due to its angiogenic microenvironment. Functional tumor heterogeneity studies can be performed in context of drug resistance within two weeks, an approach that could guide the selection of drugs and anticipate outcomes for RCC patients. This ultrafast PDX model is mirrored by high-frequency ultrasound imaging that permits quantification of tumor volume and tumor vascularity in a high-throughput manner. Using this “tumor avatar” model paired with a prospective RCC patient cohort, we observe intratumoral functional heterogeneity in the context of Sunitinib treatment as determined by high-frequency ultrasound imaging, highlighting its interventional potential in the clinic.

EXPLORING THE ROLE OF THE CTLH COMPLEX, A NOVEL MAMMALIAN E3 UBIQUITIN LIGASE, IN THE TUMOUR SUPPRESSIVE

FUNCTIONS OF RANBPM.

Maitland M, Salemi L, Wang X, Lajoie G, and Schild-Poulter C

Robarts Research Institute, Department of Biochemistry, Western University

E3 ligases control the tagging of proteins with ubiquitin for degradation by the proteasome, a system that has been successfully therapeutically targeted in some cancers. The C-terminal to LisH (CTLH) complex is a putative E3 ligase whose function and activity are uncharacterized in mammalian cells. A component of the CTLH complex, RanBPM, is a multi-domain, nucleocytoplasmic protein that has known tumour suppressive activities, however, its function is unclear. We postulate that RanBPM tumour suppressive activities stem from the modulation of signaling networks through ubiquitination of target proteins by the CTLH complex. We are at the initial stages of investigating the members of the CTLH complex and characterizing the role of RanBPM in the complex. In Hela and HEK293 cells, transient or stable RanBPM knockdown downregulates CTLH complex members at the protein level, suggesting that RanBPM is critical for complex stability. Using an in vitro auto-ubiquitination assay, we have demonstrated that MAEA, a RING-finger domain-containing CTLH complex member, has E3 ligase activity. We are currently testing the activity of another RING-finger CTLH member, Rmnd5A. We propose to employ a proteomics based approach to determine proteins differentially ubiquitinated in control and RanBPM deficient cells. This should allow us to identify the targets of the CTLH complex, providing a better understanding towards the function of this complex and, ultimately, how RanBPM and its associated E3 ligase complex function to inhibit oncogenic signaling pathways.

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EVALUATING QUANTIFICATION METHODS TO DETECT TUMOUR ASSOCIATED MACROPHAGES IN BREAST CANCER: CELLULAR

MRI USING FLUORINE-19 AND IRON-BASED METHODS

Makela AV1,2

, Gaudet JM1,2

, Foster PJ1,2

Robarts Research Institute1

and Department of Medical Biophysics, Western University

2

Tumour associated macrophages (TAMs) are associated with tumour growth and metastatic spread. Breast tumours can be comprised of up to 50% TAMs and their presence is correlated with a poor outcome. Iron oxide nanoparticles (USPIO) or Fluorine-19 (

19F) cell labeling agents can be used to image TAMs with MRI methods. 4T1 tumours were studied in 4 groups

of mice, separated into imaging method (USPIO & 19

F) and time-point (4 day & 3 week, post injection (pi)). Post iron imaging: 4 day pi tumours display signal loss which encompasses nearly the entire tumour volume. 3 week pi tumours demonstrate signal loss located around the periphery of the tumour and not within the central core.

19F imaging:

19F signal

location appears similar to iron imaging at both time-points, however, it reveals a map of distribution of signal intensity which correlates to density of label. Signal is higher in the central region compared to the periphery at the 4 day pi time-point and is heterogeneous in distribution around the periphery of the tumour at 3 weeks pi. Number of black pixels (iron) may not provide an accurate representation of iron-labeled TAMs due to blooming artifact, as confirmed with histological analysis. Quantification of

19F atoms suggest that a similar number of TAMs are present at both time-points. However a

redistribution of label is seen at later time-points. The use of 19

F-based cell tracking may provide a more accurate representation of TAM infiltration through both the ability to quantify

19F atoms, with a map of distribution, as well as the

lack of blooming artifact as seen with iron-labeled cells.

REGULATION OF C-RAF STABILITY BY THE RANBPM/CTLH COMPLEX

McTavish C, Bérubé-Janzen W, Schild-Poulter C.

Robarts Research Institute, Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University

RanBPM (Ran Binding Protein M) has been implicated in several cellular processes including cellular growth, migration, adhesion, regulation of gene transcription and apoptosis. RanBPM is an evolutionary conserved protein conserved from yeast to mammals. In yeast, RanBPM functions as a scaffold protein of an E3 ubiquitin ligase complex. Currently the precise function of the mammalian orthologous complex, the CTLH (C-terminal to the LisH) complex, remains unknown. Previous studies have shown that RanBPM functions to repress the ERK pathway. RanBPM was found to interact with c-Raf and modulate its expression through a regulation of c-Raf stability. We hypothesize that RanBPM recruits c-Raf to the CTLH complex to promote c-Raf ubiquitination and degradation. Preliminary data suggest that RanBPM interacts directly with c-Raf through its C-terminal domain and we are working towards determining the precise motif responsible for this interaction. Additionally, c-Raf forms a complex with other members of the CLTH complex in HeLa cells. Finally, we show that while proteasomal inhibition through MG132 treatment increases c-Raf levels in normal HeLa cells, it has no significant effect in HeLa RanBPM shRNA cells, suggesting that RanBPM downregulation prevents c-Raf degradation by the ubiquitin-proteasome system. These results shed light on a novel E3 ubiquitin ligase complex that controls c-Raf levels and may play an important role in preventing cancer development.

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INVESTIGATING CANCER CELL DORMANCY WITH CELLULAR MRI

Murrell DH1,2

, Hamilton AM1, Foster PJ

1,2

Robarts Research Institute1, Department of Medical Biophysics

2, Western University

Dormant cancer cells are believed to evade treatment and contribute to tumor recurrence. No biomarkers exist to identify these cells; therefore, critical questions about their nature remain unanswered. Cellular MRI allows surveillance of non-proliferative cancer cells by long-term retention of iron-oxide nanoparticles and provides a unique way to investigate this poorly understood cancer cell population. Here, we use this technology to investigate three novel glioma cell lines with varying dormancy profiles (U373vΙΙΙ, TF U373, EV U373) and the effect of early radiotherapy on proliferative and non-proliferative cancer cell fate (MDA-MB-231-BR-HER2 cell model).

Previous studies of the glioma models suggest U373vIII rapidly grows tumors with no dormant phase, TF U373 has a long latency period before growing tumors, and EV U373 does not grow tumors, remaining dormant. In contrast, cellular MRI co-detected persisting signal voids alongside tumor growth in U373vIII mice, suggesting a subpopulation of dormant cells. Furthermore, a rapid decline in signal voids was observed in both TF and EV U373 mice and neither signal voids nor tumors were detectable at day 70. This reveals TF and EV U373 cells do not grow tumors because cells do not survive and are cleared; it is not because they remain in the brain as dormant cells.

Early radiotherapy (20 Gy/2 on day 1 and 2 post cell injection) prevented brain metastasis growth in the MDA-MB-231-BR-HER2 model; both tumor quantity and tumor volume was less in treated mice than untreated. Notably, the number of signal voids was not significantly different between groups at any time, suggesting non-proliferative cancer cells persist in the brain even though treatment eradicated the macroscopic tumor burden. Further investigation of the dormant cancer cell population is warranted as these may respond to future proliferation signals and contribute to cancer recurrence. Cellular MRI provides an excellent tool for these studies.

A MULTIMODALITY IMAGING MODEL TO MONITOR CANCER METASTASIS

Katie M. Parkins1,2

, AM Hamilton1,2

, AV Makela1,2

, PJ Foster1,2

, JA Ronald1,2



Breast cancer is the second leading cause of cancer related deaths in North America with the majority of deaths due to metastasis. Cellular magnetic resonance imaging (MRI) is an emerging tool that aims to non-invasively visualize and quantify cancer cells in vivo. However, cellular MRI has limited ability to differentiate between dead and viable cells. Thus, complementary bioluminescence imaging (BLI) can provide a more holistic view of cell fate in living subjects by providing a direct readout of cell viability.

Human breast carcinoma cells (JIMT1BR3), expressing a luciferase reporter gene, were incubated with micron-sized iron particles and injected into the left cardiac ventricle of nude mice. On days 0, 8, 21 and 28, serial BLI and MRI was performed. Histological analysis and cryo-imaging were also performed at endpoint to confirm the presence of brain metastases.

The number of signal voids measured on day 0 by MRI showed a correlation with the BLI signal in the brain on day 0 (R2=0.75, p < 0.01). Percent black pixels were also measured from day 0 MRI scans and showed strong correlation with day 0 BLI signal in the brain (R2=0.74, p < 0.01). Total brain tumour burden measured by MRI at endpoint (day 28) showed a strong correlation with BLI signal in the brain (R2=0.80, p < 0.01). BLI complemented our sensitive cellular MRI technologies well, allowing us for the first time to get direct longitudinal measures of cellular viability, as well as screen animals for successful intracardiac injections prior to MRI.

16

MECHANISM AND CONSEQUENCES OF HDAC6 REGULATION BY RANBPM

Salemi L1,2

, Wang X1, Yefet E

2, Hess D

1,3, Schild-Poulter C

1,2


2Department of Biochemistry,

3Department of Physiology and Pharmacology, Western

University

Ran-binding protein M (RanBPM) has been shown to interact with numerous proteins, implicating it in a variety of cellular processes including cell adhesion, migration, microtubule dynamics, and gene transcription. Our studies have suggested a tumour suppressor role for RanBPM, as its downregulation disrupted apoptotic activation, lead to loss of growth factor dependence and increased rates of cell migration. Here we show that RanBPM downregulation promotes tumour formation in subcutaneous xenograft and metastasis mouse models, further suggesting a tumour-suppressive function for RanBPM. Recently, we characterized an interaction between RanBPM and Histone Deacetylase 6 (HDAC6). HDAC6 is a cytoplasmic deacetylase with an important role in cell migration. Its substrates include α-tubulin, cortactin and Hsp90. Increased HDAC6 expression and/or activity has been demonstrated to promote cell migration and tissue invasiveness. HDAC6 has also been shown to be required for oncogenic transformation and tumour formation. We identified that the interaction between RanBPM and HDAC6 mediates the inhibition of HDAC6. RanBPM also interacts with α-tubulin at microtubules and this localization is dependent on HDAC6, whereas HDAC6 localization at microtubules is independent of RanBPM. RanBPM has been identified to be part of a large protein complex, termed the CTLH complex. Components of the complex are conserved from the homologous yeast complex, the Gid complex, which functions as an E3 ubiquitin ligase complex. Here we evaluate HDAC6 regulation by the CTLH complex and asses co-localization of the CTLH complex with microtubules, specifically α-tubulin. Overall, our results suggest that the tumour suppressor functions of RanBPM could stem, at least in part from an inhibition of the oncogenic activities of HDAC6.

REGULATION OF CELL CYCLE CHECKPOINTS AND DNA DAMAGE RESPONSE THROUGH KU70 PHOSPHORYLATION

Walden E1,2

, Fell V1,2

, Schild-Poulter C1,2

Robarts Research Institute1, Department of Biochemistry, Western University2

Ku is the DNA binding component of the classical Non-Homologous End Joining double stranded DNA break (DSB) repair pathway. Recently, a novel function for Ku in DNA damage response (DDR) signaling was identified. Specifically, phosphorylation of the S155 position of Ku70 was identified in signalling in response to DSBs. Ku70 knockout mouse embryonic fibroblasts expressing a phosphomimetic Ku70 S155D are hypersensitive to irradiation, have a growth defect, display altered gene regulation, show constitutive activation of ATM kinase, and are arrested at the G1/S and G2/M cell cycle checkpoints. Co-immunoprecipitation demonstrated that Aurora B interacts with Ku70 S155D and phosphorylated Ku70. Aurora B is a kinase that functions in cell cycle progression through its roles in mitosis and has also been suggested to regulate the G1/S checkpoint. Interestingly, the chemical inhibition of Aurora B in vivo displays the same phenotypes as Ku70 S155D expression. Therefore, we hypothesize that phosphorylated Ku70 inhibits Aurora B following DNA damage, mediating sustained activation of the DDR and cell cycle arrest. Currently we are working to determine how phosphorylated Ku70 inhibits Aurora B following phosphorylation using in vitro kinase assays. Additionally, we are using fluorescent microscopy for DNA damage markers and flow cytometry cell cycle analysis to determine which of the DNA damage response or cell cycle arrest response occurs first in cells expressing Ku70 S155D. This work could lead to the development of therapeutics able to arrest cancer cells through treatment with a Ku70 S155D peptide or manipulation of the endogenous S155 position phosphorylation.

17

CARDIAC DISORDERS & INFARCT

ASCENDING AORTIC DILATION IN PATIENTS WITH BICUSPID AORTIC VALVE DISEASE IS MARKED BY ACCELERATED VASCULAR

SMOOTH MUSCLE CELL AGING

B Balint, H Yin, Z Nong, S Fox, S Rogers, C O'Neil, M.M.W. Chu, J. G. Pickering,

Robarts Research Institute, London, Ontario, Canada

Background: Individuals with a bicuspid aortic valve (BAV) are at increased risk for ascending aorta dilation and rupture. Aortic smooth muscle cell (SMC) loss and extracellular matrix disruption are well-recognized pathologies, but the cellular mechanisms remain elusive. We hypothesized that aortic dilation in BAV patients is marked by accelerated cellular aging.

Methods and Results: Ascending aorta samples were obtained from BAV patients undergoing thoracic aorta replacement (n=40, age 55.5±2.3, aortic diameter 4.8±0.1cm) or patients with a tricuspid aortic valve and nondilated aorta undergoing coronary bypass (n=10, age 64.1±7.6, aortic diameter 3.2±0.2cm). Assessment of fresh aortic tissue for senescence-associated β-galactosidase (SA-ß-Gal) activity revealed that 4.8±1.0% of SMCs in BAV-associated aortas were senescent, whereas SA-ß-Gal activity was rare in non-dilated aortas (0.03±0.02%, p=0.04). Interestingly, γH2A.X immunostaining revealed DNA double-strand breaks in 25.84±2.16% of SMCs in BAV-associated aortas, which was 7.3-fold higher than in controls (p<0.0001). Stress-activated p38 MAPK was activated in 38.9±2.7% of SMCs in BAV-associated aortas, but only in 21.6±3.2% of SMCs in control aortas (P=0.0002). Interestingly, quantitative real-time PCR revealed that expression of all three collagenases (MMP-1, MMP-8 and MMP-13) was increased in BAV SMCs in vitro compared to controls (p<0.05). Remarkably, incubation of BAV SMCs with a clinically relevant inhibitor of p38 MAPK decreased SA-ß-Gal by 51% (p=0.0002), and decreased collagenase expression by 68% (p<0.0001).

Conclusion: These findings identify a previously unrecognized phenomenon of accelerated SMC aging in the BAV-associated aorta. This phenomenon could be a driver of aortic wall degeneration in these patients and a potential therapeutic target.

SHAPE COMPLEXES IN CONTINUOUS MAX-FLOW SEGMENTATION

Baxter JSH1,2

, Yuan J1, Drangova M

1,2,3, Peters TM

1,2,3, Inoue J

1

Robarts Research Institute1, Biomedical Engineering Graduate Program, Department Medical Biophysics

3, Western

University

Optimization-based segmentation approaches deriving from discrete graph-cuts and continuous max-flow have become increasingly nuanced, allowing for topological and geometric constraints on the resulting segmentation while retaining global optimality. However, these two considerations, topological and geometric, have yet to be combined in a unified manner. Previous work required computationally expensive co-ordinate system warping which are ill-defined and ambiguous in the general case. Shape complexes combine geodesic star convexity with extendable continuous max-flow solvers, allowing more complicated shapes such as tubes and walls to be created through the use of multiple labels and super-labels with geodesic star convexity constraints organized in a topological ordering. These problems can be optimized using extendable continuous max-flow solvers. These shape complexes are validated in a set of synthetic images as well as atrial wall segmentation from contrast-enhanced cardiac CT. Synthetic experiments should that shape complexes improve the robustness of continuous max-flow segmentation to the choice of regularization parameter. It's application to contrast-enhanced cardiac CT demonstrates the utility of applying a topological ordering simultaneously with geodesic star convexity constraints. Shape complexes represent a new, extendable tool alongside other continuous max- flow methods that may be suitable for a wide range of medical image segmentation problems.

18

IN VITRO EVALUATION OF A NOVEL CATHETER CONTACT-FORCE CONTROLLER FOR CARDIAC ABLATION THERAPY

Gelman D1, Skanes AC

2, Tavallaei MA

1, Drangova M

1,3

Robarts Research Institute1, Division of Cardiology

2, and Department of Medical Biophysics


Introduction. To treat cardiac arrhythmia, an irregular heart rhythm, ablation catheters are introduced into the heart and manipulated until the distal tip contacts the targeted myocardium. Radiofrequency power is then delivered to form ablation lesions that isolate the heart from electrical pathways responsible for the arrhythmia. Monitoring delivery time and catheter-tip-to-tissue contact force (CF) of the lesion can predict lesion volume, conventionally termed as the force-time integral (FTI). However, varying CF due to inherent cardiac and respiratory motion, as well as catheter instability, make it difficult to clinically assess FTI during treatment. The solution to this problem would be to maintain a prescribed CF level, enabling the interventionalist to deliver a lesion under predetermined parameters as guided by the FTI model. To facilitate CF regulation, a catheter contact force controller (CCFC) has been developed and objective of this work is to evaluate its performance. The CCFC is a hand-held, electromechanical device that monitors changes in CF due to tissue motion and compensates by autonomously adjusting the position of a force-sensing ablation catheter within a steerable sheath in real-time.

Methods. To evaluate the CCFC, a custom-built linear motion phantom was used to impart a series of physiological motion profiles on an experimental catheter. Sixteen motion profiles, corresponding to CF profiles recorded during clinical ablation using a SMARTTOUCH™ catheter, were tested. The CCFC was programmed to deliver prescribed force of 15, 25, and 40 g on the moving target; CCFC-controlled and uncontrolled profiles were compared. FTI values of 500, 1000, and 1500 gs delivered at CF of 25 and 40 g were also prescribed. The CCFC retracted the catheter once the target FTI was reached. Delivery times were recorded and compared with the theoretical duration.

Results. Consistent, narrow CF distributions centered on the prescribed force levels were achieved for all motion profiles. Prescribed CF of 15, 25, and 40 g resulted in mean (± SD of 16 means) of 15.3 ± 0.1, 25.4 ± 0.1, 40.4 ± 0.1g, with root mean squared errors of 3.2 ± 0.6, 3.4 ± 0.7, and 3.9 ± 0.8 g, respectively. During the prescribed FTI experiments, the lesion delivery times recorded were within 1.2 ± 0.6% of the theoretical duration, confirming accurate and effective force control throughout the delivery.

LEFT ATRIAL WALL SEGMENTATION USING HIERARCHICAL MAX-FLOW

Inoue J,1 Baxter J S H,

1 Drangova M

1

1Robarts Research Institute, Western University, London, Ontario, Canada

Introduction. Atrial fibrillation (AF) is the most common cardiac arrhythmia and a major cause of ischemic stroke. Radiofrequency (RF) ablation can be an effective, minimally invasive, image-guided intervention for AF, but often fails, possibly due to insufficient detail in modeling the left atrial (LA) geometry. In particular, the current clinical workflow does not include a LA wall thickness measurement as part of ablation dose planning. Segmenting the wall from preoperative, contrast-enhanced CT based on intensity alone is not possible. A method that incorporates anatomical knowledge is needed to successfully segment the atrial wall. Hypothesis. LA wall segmentation can be achieved by combining an intensity model with anatomical knowledge. Materials and Methods. Hierarchical max-flow (HMF) - a mathematical optimization-based segmentation method - and a user-defined anatomical hierarchy were incorporated into a two-stage segmentation process. The method was validated on ten gated, contrast-enhanced cardiac CT images. Manual segmentations were used as a reference standard. Results. The overlap, by mean Dice similarity coefficient, was 0.79, and the mean root-mean-squared-error of the atrial wall segmentation (epicardial side only) was 0.86 mm. On average, the proposed segmentation pipeline generated segmentations that were 13% larger than manual segmentation. The LA wall was successfully differentiated from aortic wall, and esophagus. Discussion and Conclusions. Using HMF to leverage information provided by the blood in other structures allows the LA wall to be distinguished from nearby muscular structures. This alleviates the need for much of the manual correction required by methods that do not bring this context into the final segmentation. Segmentation accuracy is comparable to the variation between experts and in similar work on ventricles despite the lack of manual correction and minimal interaction.

19

IMAGING SCAR WITH TWO-POINT BIPOLAR DIXON MRI

Junmin Liu,1 Dana Peters,

2 and Maria Drangova

1,3

1Imaging Research Laboratories, Robarts Research Institute, Western University, London, Ontario, Canada

2Department of Diagnostic Radiology, Yale Medical School, New Haven, Connecticut, USA

3Dept. of Medical Biophysics, Schulich School of Medicine & Dentistry, Western University, London,

Ontario,Canada

Purpose: The conventional two-point (2pt) Dixon technique explicitly estimates B0 map by performing phase unwrapping. When signal loss, phase singularity, artifacts, or spatially isolated regions corrupt the measured phase images, this unwrapping-based technique will face difficulty. This work aims to improve the reliability of B0 mapping by performing unwrapping error-correction. Method: To detect the unwrapping-caused phase errors, we determined a magnitude-based fat/water mask and used it as reference to identify any pixels being mismatched by the phase-based mask, which was derived from the B0-corrected phase term of the Hermitian product between echoes. Then, we corrected the afore-determined phase-error on a region-by-region basis. We tested the developed method with nine patients data and the results were compared with a well-established region-growing technique. Results: By adding the step to correct unwrapping-caused error, we improved the robustness of B0 mapping, resulting in better fat-water separation when compared with the conventional 2pt and the phasor-based region-growing techniques. Conclusion: We showed the feasibility of B0 mapping with bipolar 2pt human cardiac data.

MINIMALLY INVASIVE MITRAL VALVE REPAIR USING TRACKED 3D ULTRASOUND NAVIGATION

Adam Rankin(a), John Moore(a), Daniel Bainbridge(b), and Terry M. Peters(a) (a) Robarts Research Institute, Ontario, Canada (b) London Health Sciences Centre, Ontario, Canada Minimally invasive mitral valve repair using tracked 3D ultrasound navigation. Degenerative mitral regurgitation is a consequence of chordae rupture of the mitral valve. The NeoChord DS1000 (NeoChord Inc., Minnetonka, MN) is a device shown to be effective at treating mitral insufficiency by attaching neochordae under off-pump beating heart conditions [1]. The current standard of care for image guidance during neochordae attachment is bi-plane ultrasound. We propose a novel system that uses magnetically tracked 3D TEE ultrasound in an augmented reality interface to provide navigation guidance Duration, path length and optimal path deviation of a feasibility trial are reported. Three novice users performed 6 observations each. Results are presented in figures 1c, 1d and 1e. The mean durations for 3D, AR and US were 11.85s±6.19, 8.95s±3.37, and 26.06s±14.89 respectively. The mean path lengths were 163.73mm±41.13, 142.35mm±18.69, and 427.21mm±221.61. Mean optimal path deviations were 2.77mm±0.38, 2.37mm±0.60, and 6.05mm±2.14. By using magnetically tracked 3D TEE, the system is able to present an intuitive navigation interface to the user, thus providing constant guidance towards the target. Experiments performed with this system showed guidance capabilities equivalent to previously presented augmented reality bi-plane guidance and demonstrated that a less complex workflow is feasible with magnetically tracked 3D TEE imaging. [1] Rucinskas, K., Janusauskas, V., Zakarkaite, D., Aidietiene, S., Samalavicius, R., Speziali, G., and Aidietis, A., “Off-pump transapical implantation of artificial chordae to correct mitral regurgitation: Early results of a single-center experience,” The Journal of Thoracic and Cardiovascular Surgery 147(1), 95 – 99 (2014).

20

CARDIOVASCULAR DISORDERS

REGENERATIVE ANGIOGENESIS IN ISCHEMIC MUSCLE PRODUCES A FLAWED MICROCIRCULATION THAT DOES NOT ADEQUATELY

CONTROL BLOOD FLOW

Arpino JM1,2, Nong Z1, Li F1, Yin H1, Ghonaim N2, Milkovich S2, Ellis CG2,4, Pickering JG1,2,3,4

Robarts Research Institute1, Departments of Medical Biophysics2, Biochemistry3, and Medicine4, Western University

Background: It is well established that angiogenesis can occur following ischemic injury to skeletal muscle, and enhancing this response has been a therapeutic goal. However to appropriately deliver oxygen, a precisely organized and exquisitely responsive microcirculation must form. Whether these network attributes exist in a regenerated microcirculation is known. Methods and Results: To investigate the architecture and functionality of the regenerated microcirculation in skeletal muscle, we dynamically imaged red blood cells in the mouse extensor digitorum longus muscle following femoral artery excision. This revealed complete microcirculatory collapse one day after ischemic insult, together with histological evidence for obliteration of capillaries and widespread skeletal myocyte necrosis. An extensive network of flowing neo-microvessels subsequently regenerated which, after 14 days, structurally resembled that of uninjured muscle. However, the skeletal muscle remained hypoxic. Red blood cell transit analysis revealed slow flow in the regenerated network, a 7-fold increase in capillaries with stalled flow, and the emergence of arteriolarvenular shunts. Furthermore, spatial heterogeneity in red cell transit was highly constrained. To determine if the regenerated microcirculation could regulate flow, the muscle was subjected to local hypoxia using a polydimethylsiloxane membrane. Hypoxia promptly increased red cell velocity and flux in control capillaries but in neo-capillaries the response was blunted and could not be sustained. Three-dimensional confocal imaging revealed that neo-arterioles were aberrantly covered by SMCs, with increased inter-process spacing and haphazard orientation of actin microfilament bundles. Conclusions: Despite robust neovascularization, the microcirculation formed by regenerative angiogenesis in skeletal muscle is profoundly flawed. This network-level dysfunction must be recognized and overcome in order to advance regenerative approaches for ischemic disease.

EFFECT OF IN VITRO BLOOD CLOT HEMATOCRIT AND AGE IN QUANTITATIVE R2* MRI MAPPING

Spencer Christiansen BSc,1,2

J. Liu PhD,2 M. Drangova PhD

1,2


2Imaging Research Laboratories, Robarts Research Institute, Western University

Introduction: Knowledge of thrombus (blood clot) composition, in particular the proportion of red blood cells (RBCs), has been shown to provide useful clinical information in cardiovascular diseases such as heart attack and stroke. Hematocrit (RBC concentration) may be inferred using MRI through the presence of met- and deoxyhemoglobin found in RBCs. Met- and deoxy-hemoglobin are paramagnetic oxygenation states of the hemoglobin molecule and are capable of causing field inhomogeneity effects resulting in local spin dephasing and signal loss, and as thrombi degrade over time the proportion of hemoglobin molecules in these paramagnetic states increases. Materials and methods: A phantom was constructed containing 1.2 cm diameter polystyrene tubes embedded in agar housed in a cylindrical plastic container. Anticoagulated pig’s blood was used to prepare platelet poor plasma (PPP) and seven 2 mL blood samples of varying hematocrit (0 to 60%). One hour following thrombin injection clots were transferred into to individual tubes filled with PPP. The phantom was scanned at 1, 4, 8 hrs and 1, 2, 3, 5, 7 days post-clotting, and kept in a 37°C water bath between scans. The phantom was scanned at 3T with a 32- channel transmit/receive head-coil using a multi-echo GRE sequence. R2* maps, which quantify signal loss, were generated and segmented in three-dimensions in order to determine mean clot R2* values at each time point. Results: Scans of the clot phantom were acquired and showed minimal differences between R2* values at different hematocrit levels at early time points (1-8 hr). However, a trend towards increasing R2* with time was observed in all clots containing RBCs, and the rate of which was proportional to hematocrit. One week post-clotting, mean clot R2* values were dispersed with an average separation of 16.2Hz, where the 60% clot held the highest (129.9Hz) and the 10% clot the lowest (78.6Hz) value. The 0% clot and the control PPP vials had far lower R2* values (average of 1.8 and 2.6Hz, respectively, considering all time points) and did not change over the course of the study. Conclusions: Blood clots of different hematocrit level were generated ex vivo and evaluated over a period of a week. A strong trend was observed towards increasing mean clot R2* values with time, and the rate of this increase was sensitive to hematocrit. This work provides preliminary evidence that clot hematocrit may be inferred using MRI R2* analysis, and further investigation with additional imaging parameters is ongoing.

21

INHIBITION OF ALDEHYDE DEHYDROGENASE ENHANCES THE EX VIVO EXPANSION OF HUMAN HEMATOPOIETIC PROGENITOR

CELLS WITH VASCULAR REGENERATIVE FUNCTIONS IN VIVO.

Tyler T. Cooper1,2

, Stephen E. Sherman1,2

, David A. Hess1,2

1. Department of Physiology and Pharmacology, Western University 2. Molecular Medicine Research Group, Robarts Research Institute

Umbilical cord blood (UCB) cells selected for high aldehyde dehydrogenase (ALDH) activity demonstrate a pro-angiogenic secretome and enhanced the recovery of blood perfusion after intramuscular transplantation into NOD/SCID mice with hind-limb ischemia. Unfortunately, UCB ALDHhi cells are rare (<0.5% of total UCB cells), making ex vivo expansion necessary for the refinement of current cellular therapies using bone marrow-derived cells for vascular regeneration. Paradoxically, purified UCB ALDHhi cells are intrinsically driven towards hematopoietic differentiation during ex vivo expansion via active retinoic acid-signaling; a by-product of ALDH-activity. As a result, the frequency of primitive ALDHhi/CD34+/CD133+ cells is rapidly reduced during prolonged culture (≥ 9 days) coinciding with diminished vascular regenerative functions in vivo. We hypothesize the inhibition of retinoic acid production will enhance the expansion of UCB cells retaining a primitive hematopoietic phenotype and vascular regenerative functions in vivo. UCB ALDHhi cells (n=10) were cultured under basal conditions (X-vivo 15 + SCF, TPO, FLT3L) for 9 days with or without 10μM diethylaminobenzaldehyde (DEAB), a reversible ALDH inhibitor. Compared to basal conditions, UCB ALDHhi cells cultured with DEAB supplementation increased the total number of ALDHhi cells by ≈25-fold and ALDHhi/CD34+/CD133+ cells by ≈5-fold (p<0.01). Furthermore, DEAB-expanded cells seeded in methylcellulose media generated more hematopoietic colonies in vitro (p<0.05). Interestingly, the intramuscular transplantation of DEAB-expanded cells stimulated murine CD31+ proliferation in vivo (p<0.05) leading to a significant recovery of blood perfusion in the ischemic hind-limb of NOD/SCID mice (p<0.01). Overall, we have demonstrated that reversible ALDH-inhibition (DEAB supplementation) during 9-day culture enhanced the number of primitive hematopoietic progenitor cells retaining vascular regenerative functions in vivo.

VASCULAR CONTRAST AGENT FOR PRECLINICAL DUAL ENERGY COMPUTED TOMOGRAPHY IN VIVO

Cruje C1,2

, Nguon O3, Gillies E

3, Drangova M

1,2

Robarts Research Institute1, Departments of Medical Biophysics

2 and Chemistry


Vascular contrast agents are key to distinguishing the blood pool from surrounding tissues in dual energy (DE) computed tomography (CT). Preclinical studies require contrast agents that are optimized for DE micro-CT of small animals. It has been demonstrated ex vivo that the optimized contrast agent has a k-edge between 50-65 keV (i.e. lanthanide-based). It must also be biocompatible and stay in the blood pool in sufficient quantities while imaging for in vivo use (i.e. polymer-coated nanoparticles exceeding 10 nm in size). None of the available contrast agents meet all requirements. We propose that by making a lanthanide-based, long-circulating and biocompatible contrast agent, DE micro-CT can be developed into a valuable in vivo vessel imaging technique. Using this contrast agent, highresolution, quantitative and three-dimensional images of vasculatures can be obtained in live small animals. Lanthanide nanoparticles were made by subjecting gadolinium salt to thermal and chemical treatments. Synthesized nanoparticles were encapsulated in a polymer. The nanoparticles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and single energy micro-CT. Results show that polymer-coated lanthanide nanoparticles that meet the size requirement were successfully made. Work is underway to improve contrast enhancement by nanoparticles to proceed with developing an in vivo DE micro-CT protocol.

22

ELUCIDATING THE GENETIC DETERMINANTS OF EXTREME HIGH-DENSITY LIPOPROTEIN PHENOTYPES USING NEXT-GENERATION SEQUENCING

Dron JS1, 2

, Wang J1, McIntyre A

1, Robinson J

1, Ban M

1, Cao H

1, Rhainds D

3, Lettre G

3, Dubé M-P

3, Tardif J-C

3, & Hegele RA

1, 2

Robarts Research Institute1, Department of Biochemistry

2, Western University; Montréal Heart Institute

3, Université de

Montréal & Beaulieu-Saucier Pharmacogenomics Centre

HDL cholesterol (HDL-C) levels associate with cardiovascular disease risk, and as a complex trait, are influenced by genetic and environmental factors. Extreme HDL-C concentrations are largely genetically determined; monogenic disorders have been well-characterized, including candidate genes driving each extreme phenotype. Within genes causing syndromes of both HDL-C extremes, numerous disease-causing variants have been identified and functionally validated. In a unique cohort of patients with extreme HDL-C profiles, we applied our targeted next-generation sequencing panel LipidSeqTM, which is designed for the re-sequencing of genes associated with dyslipidemia and other metabolic disorders. We found that 20.6% and 11.8% of low (N=136) and high (N=119) HDL-C patients, respectively, carry large-effect mutations in pertinent genes explaining their phenotypes. To further characterize the genetic variation contributing to HDL-C levels, we investigated the polygenic contribution from multiple small-effect variants using a polygenic trait score (PTS). We developed two scores to assess an individual’s burden of variants: one each for lowering and raising HDL-C. Both PTSs are able to differentiate between both extreme HDL-C patient groups and the reference population. When considering the division between large-effect mutation carriers and non-carriers, the non-carriers for each extreme had significantly greater mean PTSs compared to the reference population (P<0.0001). This may suggest a polygenic contribution towards modulating HDL-C levels, and could provide a genetic basis for individuals without large-effect mutations explaining their phenotypes.

NICOTINAMIDE RIBOSIDE ATTENUATES ANGIOTENSIN II-INDUCED NAD+ DECLINE AND DNA DAMAGE

Sina Ghoreishi1,2

, Zengxuan Nong1, Hao Yin

1, Robert Gros

1,3 and J. Geoffrey Pickering

1,2,4,5,6



3Department of Physiology and Pharmacology,

4Department of Biochemistry,

5Department of Medicine, Western University,

6London Heath Sciences Centre, London, ON,

Canada NAD+ (nicotinamide adenine dinucleotide), classically known as coenzyme critical in energy metabolism, also acts a substrate for proteins implicated in stress resistance (sirtuins) and DNA damage repair (Poly (ADP-ribose) polymerases). With aging and during oxidative stress, NAD+ levels decline, impacting the activity of these proteins and thus cellular health. Dietary supplementation with the NAD+ precursor, nicotinamide riboside (NR), has recently been shown to increase NAD+ levels in mammalian cells. Therefore the objective of this project was to determine NR’s effects on vascular stress, in vivo. I hypothesized that nicotinamide riboside administration would increase NAD+ levels in mice, imparting resistance to angiotensin II-mediated (Ang II) vascular stress. To test this, middle aged (10 months) C57Bl/6 mice received NR (400mg/kg/day) or vehicle (chow) treatment for a total of seven weeks. At three weeks vascular stress was induced by continuous angiotensin II (1.44 mg/kg/day) infusion for four weeks. Compared to saline infusion, Ang II infused mice displayed a significant reduction in liver NAD+ content, which was attenuated with NR treatment. NR had no effect over Ang II-induced hypertension, body weight loss, aortic medial thickening, cardiac hypertrophy or fibrosis. However, NR treatment attenuated Ang II-induced DNA damage in endothelial and vascular smooth muscles cells (VSMCs) of the thoracic aorta. Furthermore, a significant negative correlation was observed between liver NAD+ content and VSMC DNA damage. Together these results suggest that NR attenuates DNA damage during oxidative stress by buffering NAD+ decline.

23

THE PARACRINE FUNCTIONS OF ALDHHI MSC IN VASCULAR REGENERATION

Stephen E. Sherman1,2

, Miljan Kuljanin3 and Dr. David A. Hess

1,2

Department of Physiology and Pharmacology1, Robarts Research Institute

2, Department of Biochemistry

3.

High aldehyde dehydrogenase (ALDH) activity is a conserved stem cell function which protects long-lived progenitor cells against oxidative stress. Multipotent stromal cells (MSC) are perivascular-resident mesenchymal stem cells that differentially express ALDH during culture expansion. Therefore, purification based on ALDH-activity may be a useful functional assay to identify regenerative MSC subsets that may provide structural (Pericyte) and paracrine support to direct neovessel formation. After culture expansion of bone marrow-derived MSC, we purified MSC progeny based on high versus low ALDH activity by fluorescence activated cell sorting, and subjected these MSC subsets to microarray and supernatant cytokine protein analyses to obtain insight on secreted effectors differentially expressed. Although the ALDHlo and ALDHhi MSC subsets demonstrated very similar mRNA expression patterns after purification (n=3), the ALDHhi MSC subset showed exclusive secretion of VEGFB, PDGFA, and Angiogenin, factors with documented proangiogenic functions (n=4, p<0.05). The ALDHhi MSC subset also demonstrated a potent proliferative effect on human microvascular endothelial cells (HMVEC) exposed to conditioned endothelial basal media generated by purified MSC subsets (n=3, p<0.05). Furthermore, conditioned media generated from the ALDHhi MSC subset augmented tube-forming capacity of HMVEC in growth factor reduced GeltrexTM after 24 hours under serum-free, growth factor-free conditions (n=3, p<0.05). After subcutaneous transplantation into immunodeficient mice, the ALDHhi MSC subset also demonstrated a heightened capacity to promote angiogenesis as compared to the ALDHlo MSC subset (n=3, p<0.05). Collectively, ALDHhi MSC represents an MSC subset with enhanced pro-angiogenic characteristics in vitro. Transplantation of the ALDHhi and ALDHlo MSC subsets in femoral artery-ligated immunodeficient mice is warranted to address the capacity to recover blood perfusion to the ischemic limb.

NAD+ REGENERATION IN SMOOTH MUSCLE CELLS IS REQUIRED TO MAINTAIN VASCULAR INTEGRITY.

Watson A1,3

, Nong Z1, O’Neil C

1, Pickering JG

1,2,3,4.

Robarts Research Institute1, Departments of Medicine (Cardiology)

2, Biochemistry

3, Medical Biophysics


Vascular smooth muscle cells (SMCs) are vital for repairing damaged arteries but their reparative capacity declines with age and stress. NAD+-consuming enzymes (e.g. sirtuins and PARPs) are critical mediators of cell longevity and, accordingly, molecular systems that generate NAD+ may be critical to vascular health. Nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for NAD+ regeneration, protects SMCs from oxidizing stress and extends SMC lifespan. We have previously shown that decreased Nampt levels in mouse and in human aortopathy patients are associated with a loss aortic integrity. We generated mouse aortic SMCs (mAoSMCs) with a Nampt deletion and assessed for the expression of genes associated with vascular extra-cellular matrix. Nampt-KO mAoSMCs have a 4-fold increase in MMP13 expression and a 10-fold increase in MMP9 expression and there is a small, but significant decrease in the expression of Col1a1. Additionally, the expression of glycoproteins versican, tenascin C, decorin and perlercan were increased from 2-4 fold. These cells also were impaired in their ability to assemble labeled collagen I in vitro. To confirm these findings in vivo, we generated mice with SMC-specific Nampt deletion, using Nampt

flox/flox mice that express Cre under the control of the SMC myosin heavy chain

promoter. We determined that SMC-Nampt KO aortas stained with picrosiruis red and assessed for assembled collagen demonstrate a significant decrease in the % area of collagen in the aorta, along with the degree to which it is organized. Following infusion of angiotensin II, collagen deposition and organization is increased in WT aortas, but this response is blunted in SMC-Nampt KO aortas. Additionally, there is an increase of glycoprotein deposition in SMC-Nampt KO aortas. We conclude that loss of Nampt in vascular SMCs predisposes to a change in the balance of collagen to glycoprotein moieties expressed by the cells, and could contribute to a loss of aortic integrity. Nampt-driven NAD+ generation thus constitutes a novel means of maintaining vascular stability during stress.

24

AUTOMATED 3D MORPHOMETRY OF THE VASCULATURE ON WHOLE-SLIDE DIGITAL HISTOLOGY

Yiwen Xu1, J. Geoffrey Pickering

1,2, Zengxuan Nong

2, Aaron Ward

1,3

Dept. of Medical Biophysics1, Robarts Research Institute

2, Dept. of Oncology


Introduction: Properties of the microvasculature which contribute to tissue perfusion can be assessed using immunohistochemistry on 2D histology sections. The smooth muscle components of the vessel walls are responsible for the control of the vessel lumen calibre; to allow for changes in tissue perfusion. Vessel wall abnormalities can cause vascular disease and organ failure, which is generally assessed in 2D on fields of view resulting in potential under-sampling. Vasculature is inherently 3D and the ability to measure and visualize the vessel wall components in 3D will aid in detecting focal pathologies. Our objectives were (1) to develop a method for 3D measurement and visualization of microvasculature on whole slide histology, (2) to compare the normal and regenerated post-ischemia mouse hind-limb microvasculature, and (3) to compare the 2D and 3D vessel morphology measures. Methods: Vessels were stained for smooth muscle (SM) using 3,3'-Diaminobenzidine for both normal (n = 6) and post-ischemic vasculature (n = 5). The SM component of the vasculature was segmented (Xu, SPIE Medical Imaging 2015), and then connected in 3D through our automated landmark-based whole-slide registration (Xu, Plos One 2015). A surface was fit to the reconstructed vessel contours using the marching cubes algorithm. Measures of vessel wall area, thickness and perimeter were performed in 2D on the tissue sections and in 3D from the vessel surfaces. Results/Discussion: A larger value of area, perimeter, and vessel wall thickness was found in the normal as compared to the post-ischemic vasculature, for both the 2D and 3D measurements (p < 0.01). The post-ischemic tissues are expected to contain regenerated vasculature, and thus contain vessels with different morphological features. We have developed tools for 3D histology vasculature reconstruction, quantification, and visualization, enabling detection and exploration of focal pathologies in individual microvessels.

TELOMERE-BASED ASSESSMENT OF BIOLOGICAL AGE IN PATIENTS WITH ADVANCED VASCULAR DISEASE

Hao Yin1, Oula Akawi

1, Stephanie A. Fox

3, Fuyan Li

1, Caroline O'Neil

1, Jorge Wong

2, John-Michael Arpino

1,5, Brittany Balint

1,5,

Alanna Watson1,4

, Linrui Ray Guo3, MacKenzie A. Quantz

3, A. Dave Nagpal

3, Bob Kiaii

3, Michael W.A. Chu

3, J. Geoffrey

Pickering1,2,4,5

Robarts Research Institute

1, Dept.'s of Cardiology

2, Surgery

3, Biochemistry

4, Medical Biophysics


Introduction: The extent to which telomeres shorten in leukocytes could be a marker of cardiovascular risk and biological age because it reflects the accumulation of replication stresses imposed on leukocyte progenitors. However, because of wide, genetic variability in leukocyte telomere length (TL), a single leukocyte TL measurement does not reliably indicate telomere shortening. Hypothesis: We propose that the difference in length of telomeres in “non-replicating” muscle-rich tissue and that of circulating leukocytes provides a personalized index of telomere shortening in patients with advanced vascular disease. Methods: TL in leukocytes, skeletal muscle, and right atrial cardiac muscle were measured from 134 patients undergoing coronary or thoracic aortic surgery, using quantitative polymerase chain reaction. Relationships between leukocyte TL or the muscle-leukocyte TL difference (ΔTL) and early post-operative outcomes were determined. Results: Telomeres in cardiac muscle and skeletal muscle were significantly longer than those in leukocytes (p<0.001) but with synchrony among these measures in an individual (p<0.001). Leukocyte TL and skeletal muscle TL inversely correlated with chronological age (p<0.001) however cardiac muscle TL did not (p=0.283). There was no relationship between leukocyte TL and either the post-operative length of stay in the intensive care unit (ICU) or major complications. However, one kilobase increase in cardiac muscle-leukocyte ΔTL was associated with 17% higher risk of longer ICU stay (hazard ratio 1.17, p=0.029) and 2.54 fold difference in odds of post-operative complications (odds ratio 2.54, p=0.008). Conclusions: Right atrium-leukocyte ΔTL provides an index of telomere shortening and may inform outcomes in patients with advanced vascular disease. This two-component telomere measurement may reflect the biological age of individuals with chronic vascular disease.

25

CELLULAR THERAPIES & IMMUNOTHERAPY

SULPHAMETHOXAZOLE-HYDROXYLAMINE REDUCES LEVELS OF PEROXIREDOXIN-1 IN JURKAT T CELLS EXPRESSING THE HIV-1

TAT PROTEIN

Adeyanju K 1,2

, Bend JR3, Rieder MJ

1,4,5 and Dekaban GA

1,2

Robarts Research Institute1, Dept's of Microbiology & Immunology

2, Pathology

3, Physiology & Pharmacology

4 and

Pediatrics5, Western University

Adverse drug reactions (ADRs) are a significant problem for HIV patients, with up to 80% of HIV-infected patients experiencing an ADR at some point during the course of the infection. These ADRs can be in response to a variety of medications including antiretroviral agents as well as anticonvulsants and antimicrobials. An example of the latter is Sulphamethoxazole (SMX), which is used as prophylaxis and treatment for pneumocystis pneumonia, a common ailment in HIV/AIDS patients. SMX is also responsible for a high incidence of ADRs in this population. While the pathophysiology behind ADRs in general is unclear, previous studies have implicated the SMX-metabolite sulphamethoxazole-hydroxylamine (SMX-HA) in the development of ADRs to SMX. Furthermore, we have previously demonstrated that thiols modulate the effect of these reactive sulfonamide metabolites by reducing them to the less reactive parent compound. We also have evidence that suggests the HIV-1 Tat protein contributes to ADRs in the HIV population. In this study Jurkat T cell lines expressing Tat were used to elucidate the effect of Tat on the cellular protein thiols, both in the presence and absence of SMX-HA. Western blot analysis showed that the Tat-expressing cells had reduced expression of the thiol protein peroxiredoxin 1 in the absence of SMX-HA compared to the parent cell line. Exposure of the Tat-expressing cells to 200μM SMX-HA, led to further significant reduction in the expression of the peroxiredoxin 1 protein. This suggests the metabolite SMX-HA, in combination with Tat, could alter the cellular homeostasis and thus influence the development of an ADR.

RETINOIC ACID PATHWAY INHIBITION TO GENERATE HEMATOPOIETIC PROGENITOR CELLS FOR INDUCTION OF ISLET

REGENERATION

Elgamal R, Cooper T, Hess D Robarts Research Institute, Department of Physiology & Pharmacology, Western University

Patients with diabetes, a metabolic disorder characterized by loss or dysfunction of ß-cells in the pancreas, are highly susceptible to many complications as a result of inadequate glycemic control. Cellular therapies that induce endogenous islet regeneration are emerging as a novel way to treat diabetes. We have shown that a primitive subset of hematopoietic progenitor cells (HPC) isolated by their aldehyde dehydrogenase activity (ALDH) can stimulate islet cell proliferation and islet revascularization in NOD/SCID mice with ß-cell ablation. This study focuses on using an inverse retinoic acid receptor agonist, BMS 493, during expansion of umbilical cord blood (UCB)-derived ALDHhi cells to prevent differentiation of these primitive cells and preserve islet regenerative function. HPC were isolated from UCB by lineage depletion and fluorescent activated cell sorting (FACS) for cells with high levels of aldehyde dehydrogenase (ALDHhi). UCB ALDHhi cells were expanded in vitro for up to 6 days in serum-free conditions and in the presence of different concentrations of BMS 493. Flow cytometry was then used to measure ALDH retention and frequency of primitive cell surface markers (CD34, CD133). We hypothesize that ALDHhi HPC expanded ex vivo with BMS 493 will retain islet regenerative capacity and will reduce hyperglycemia in STZ-treated NOD/SCID mice via the induction of endogenous islet proliferation and revascularization after transplantation. Expansion of ALDHhi HPC to clinically applicable numbers without loss of islet regenerative functions may provide a clinically feasible approach to tip the balance in favour of islet regeneration versus destruction for the treatment of diabetes mellitus.

26

19FLUORINE (19F) CELLULAR MAGNETIC RESONANCE IMAGING TO MONITOR IN VIVO THERAPEUTIC CELL MIGRATION AND

DISTRIBUTION

Fink C1,2

, Gaudet J1,3

, Foster P1,3

, Dekaban G1,2


1, Departments of Microbiology and Immunology

2 and Medical Biophysics


A major hurdle in the advancement of cell-based cancer vaccines is the inability to track where these therapeutic cells migrate to post injection. Furthermore, with respect to antigen presenting cell-based (APC) cancer vaccines, the quantity of tumor antigen-specific APC reaching a secondary lymphoid organ post-injection is directly proportional to the magnitude of the ensuing anti-cancer immune response. We propose that

19F cellular MRI and a

19F cell labeling agent, CS-1000, permit

quantitative non-invasive tracking of human APC migration in vivo. Therefore, this novel imaging technique can be used as a surrogate marker to assess and improve the effectiveness of APC-based cancer vaccines while also assessing their anatomical fate and longevity. Our laboratory has been able to efficiently label a high percentage (>95%) of human peripheral blood mononuclear cells (PBMC) from prostate cancer patients with CS-1000 and quantify this labeling using NMR spectroscopy. Human PBMC migration post footpad injection to both the popliteal and inguinal lymph nodes has been detected and quantified in a mouse model using

19F cellular MRI. MRI studies using PBMC samples from prostate cancer

patients has allowed for the detection of 19

F-labeled cells at both the injection site and draining inguinal lymph node. Currently, optimization of this novel imaging technique is being performed while awaiting clinical trial approval to image autologous CS-1000-labeled PBMC in humans. By doing so, we would be the first in Canada to conduct

19F cellular MRI in

humans.

DEVELOPMENT OF CLINICAL 19F-MRI CELL TRACKING FOR CANCER IMMUNOTHERAPY

Gaudet J1,2

, Fink C1,3

, Fox M1, Dekaban G

1,3, Foster P

1,2

Robarts Research Institute1, Departments of Medical Biophysics

2 and Microbiology and Immunology


Immunotherapy has the potential to revolutionize modern medicine and clinical trials are already underway. Treatment involves recruiting the immune system to target the tumor using antigen-presenting cells such as dendritic cells (DC). Well over a 1000 cancer patients have been treated with DC vaccines; these trials have shown that DC-based immunotherapy is technically feasible and non-toxic. Nevertheless, results from human clinical trials have been diverse and largely disappointing. One key reason for this failure is the lack of tools to evaluate treatment success following administration. To move forward with cancer immunotherapy we require novel clinical imaging tools for qualitative and quantitative monitoring of transplanted cells in order to monitor their anatomical location, cell numbers, and functional lifespan. Fluorine-19 (

19F)-MRI is an emerging tool for tracking cells in vivo. This technique involves pre-labeling cells with an imaging

probe for non-invasive tracking with MRI. The 19

F-based imaging agent used is FDA approved, non-reactive, and does not emit ionizing radiation. Furthermore, the MRI signal produced by

19F-labeled cells is specific and can be used to quantify the

number of cells located within the region of interest in the body. Our previous work, at high magnetic field, has shown that this technique can be successfully applied in pre-clinical models. This work has shown the label has no impact on cellular phenotype or function. Development is currently focused on translation of this imaging technique to the clinic. Preliminary data has shown that

19F-MRI is feasible at a clinical field strength (3T) and that the MRI signal is linearly proportional to

19F

concentration, a requirement for accurate cell quantification. We anticipate development of a MRI-based cell tracking technique for clinical use will provide a crucial tool for improving outcomes in clinical trials of cancer immunotherapy.

27

FORCE FEEDBACK FOR ULTRASOUND-GUIDED ROBOT IN PRECLINICAL MICRO-INJECTION IMMUNOTHERAPY

Kowal S 1,2

, Dr. James Lacefield1,2,3


1, Departments of Medical Biophysics

2 and Electrical & Computer Engineering

3, Western

University Image-guided mechatronic devices have enabled important improvements in surgical procedures, particularly by supporting minimally-invasive interventions and delicate microsurgery procedures. Dendritic cell cancer vaccines are a point of interest for researchers in cancer immunotherapy. Difficulties are encountered when administering drugs to the lymph nodes of small animals due to their size constraint and target locations. During dendritic cell injection therapy it may take weeks to find out drugs did not reach their target and hundreds of dollars were squandered. Measurements of the force exerted by the needle on a small animal stage can be used to correct for tissue deformation to increase the success rate. During phantom experiments, strain gauge sensors placed underneath a mouse stage in four places will feedback vertical forces applied on the stage to our robot. This calibrated setup will monitor peaks where the needle deforms and punctures thin layers of latex sheets to mimic tissue deformation. Once a peak is detected, our robot will stop the needle. Preliminary tests suggest our force sensor model may detect force peaks during penetration of latex sheets (0.15mm) embedded within gelatin when using a 30 gauge needle. Monitoring the change in force signal showed positive correlation with needle penetration angle. Pilot study suggests force feedback is functional. Injection of a fiducial marker will be used for comparison of our robot to manual injections in further studies. Force feedback will enable the robot to adjust to tissue deformation during in vivo injections. We hypothesize that this will result in significantly greater anti-tumour immune response when used to inject dendritic cell vaccine intranodally (versus manual injections). This system will provide safer and less traumatic procedures for our animal model with less operator dependence that will accelerate preclinical development of cancer vaccines.

INVESTIGATING THE USE OF [19F]-FLUORINE-LABELING TO TRACK THERAPEUTIC DC MIGRATION IN VIVO USING CELLULAR

MRI

Smith M1,2

, Fink C1,2

, Gaudet J1,3

, Foster P1,3

, Dekaban G1,2

. Robarts Research Institute

1, Departments of Microbiology and Immunology



Dendritic cell (DC)-based vaccines are an emerging immunotherapy for the treatment of cancer. However, while their proof-of-principle has been demonstrated, further refinement is needed to improve their efficacy. Critical to the function of these vaccines is the efficient migration of DC to secondary lymphoid organs post-injection. As such, to evaluate both current and potential DC-based vaccine preparations, the need to quantify DC migration in vivo using a non-invasive imaging modality has been identified. Using the combination of

19Fluorine cellular magnetic resonance imaging (MRI) and a

19Fluorine-based perfluorocarbon nanoemulsion called Cell Sense (CS), DC migration from the site of injection can be

imaged and quantified in vivo in a real-time, non-invasive manner. However, it has not yet been established whether CS labeling affects DC function. We hypothesize that CS labeling will not affect the functionality of therapeutic DC, and that migration as assessed by cellular MRI will be predictive of both the magnitude of ensuing tumor-specific CD8 T cell responses and tumor outcome. To assess our hypothesis, this project investigates DC functionality between CS labeled and unlabeled populations with respect to DC migration efficiency, immune activating capability, and tumor outcome in an in vivo mouse model. Furthermore, this project investigates whether migration as assessed by cellular MRI correlates to both the ensuing anti-tumor immune response elucidated by vaccination and subsequent tumor outcome.

28

CENTRAL NERVOUS SYSTEM INJURY & INFLAMMATION

RETROSPECTIVE ASSESSMENT OF R2* FOR PREDICTION OF RADIATION NECROSIS IN A RODENT MODEL

Belliveau, JG 1,2

, Jensen, M 2, Stuart, J

4, Bauman GS

2,3, Menon RS G

1,2

Center for Functional and Metabolic Mapping, Robarts Research Institute1, Departments of Medical Biophysics

2 and

Oncology3, Western University, STTARR, Princess Margaret Hospital, University of Toronto

4

Purpose: To establish a quantitative MRI method that would be capable of predicting radiation necrosis without using a contrast agent. Methods: Healthy male Fischer 344 rats were irradiated using an animal irradiator capable of delivering 2.3 Gy/min with a kVp of 225 V. A dose of 40 Gy was given to half the brain in a single session. Rats were scanned using a 9.4T animal MRI before irradiation and every two weeks following radiation until either necrosis developed or they were sacrificed for health reasons. A multi-echo gradient-echo sequence was acquired at every time point and the apparent transverse relaxation rate R₂* was calculated based on the measured signal decay. At the last time point, an ROI mask with an R₂* value of greater than 45 s⁻1 was applied in the area of the external and internal capsule where radiation necrosis was confirmed. A retrospective analysis was performed to determine whether R₂* values would be able to predict where radiation necrosis would occur. Results: Radiation necrosis was morphologically visible between weeks 22-24 following treatment. Gadolinium MRI and histology confirmed radiation necrosis in the area of MRI enhancement. Our data suggests that there is a trend towards significance in the lesion as early as 10 weeks prior to morphological changes on MRI with significance occurring 6 weeks prior (p≤0.05, p≤0.01 at week 24). Measurements of the R₂* in the hippocampus did not show any significant difference; however, there are areas of visible R₂* change within sub-hippocampal regions. Conclusion: R₂* is a promising method that could be able to predict an underlying disease process that occurs prior to radiation necrosis. The constant increase in R₂* values suggests a possible neuroinflammatory mechanism rather than an acute vascular event where R₂* would tend to decrease in the area.

INVESTIGATING EVOLVING BLOOD LEUKOCYTE POPULATIONS LONGITUDINALLY AFTER CONCUSSION IN ADULT FEMALE

ATHLETES

K. A. BLACKNEY1,3

, L. FISCHER5, T. DOHERTY

4, R. MENON

2, R. BARTHA

2, D. FRASER

6, A. BROWN

1, G. DEKABAN

1,3;

1Mol. Med.,

2Med. Imaging, Robarts Res. Inst., London, ON;

3Microbiology and Immunol.,

4Physical Med. and Rehabil., Univ.

of Western Ontario, London, ON; 5Fowler-Kennedy Sports Med. Clin., London, ON;

6Children’s Hlth. Res. Inst., London, ON

Sports-related mild traumatic brain injuries, also known as concussions, are generating increasing concern due to potential long-term neurological consequences. However, there is currently no universally recognized diagnostic approach for concussion, and many of the current methods used for diagnosis are sub-optimal. This leaves patients with insufficient treatment and at enhanced risk of more severe damage from subsequent head trauma. We hypothesize that a signature temporal response of biomarkers of inflammation in systemic circulation will provide an objective diagnosis of concussion. We predict that this panel of biomarkers could also be used to track patient recovery. The Western University women’s rugby team underwent blood draws at pre-season and post-season as a baseline evaluation, and players determined to have sustained a concussion underwent repeat blood draws at 24-72 hours, 1 week, 1 month, and 3 months post-concussion. Blood samples were analyzed by flow cytometry to profile immune cell populations. Together, this analysis was done alongside accepted concussion assessments, Sport Concussion Assessment Tool 3 (SCAT) and Immediate Post-Concussion Assessment and Cognitive Test (ImPACT), as well as a complete blood count for both baseline and concussed groups. Analysis of the SCAT data revealed a significant increase in self-reported symptoms and symptom severity after concussion, and from the ImPACT, we observed a significant decrease in visual memory 1 month after concussion compared to player baseline. Both of these tests appeared to be plagued by a practice effect with several tests showing significantly improved results in the baseline population from beginning to end of season. Haematology and flow cytometry profiles demonstrated significant alterations in both broad and cell lineage specific immune cell profiles after concussion compared to players pre-season baseline. As a pilot study in concussion biomarker discovery we have been able to identify several potential markers for use in clinical concussion assessment. Further validation of the blood biomarkers identified thus far will be necessary to solidify this as an adequate concussion identification technique.

29

SPINAL DUROTOMY REPAIR SIMULATOR FOR DELIBERATE MICROSURGICAL PRACTICE: TOWARDS INTEGRATION INTO A

RESIDENCY TRAINING MODULE

Lau JC1,2

, Denning L2, Lownie SP

2, Peters TM

1, Chen EC

1

Robarts Research Institute1 and Department of Clinical Neurological Sciences

2

Deliberate practice is one aspect of gaining competency in surgical skills. We present a functional and patient-specific lumbar phantom for the training of spinal durotomy and dura closure under microscopic view, consisting of a lumbar model, pressurized dural surrogate, together immersed in a tissue-mimicking layer simulating fat, muscle and skin. A 3D-printed plastic lumbar spine model was created from a patient computed tomography scan. L2 was manually laminectomized, and paraspinal tissues were simulated using Polyvinyl Chloride (PVC) Plastisol. Harvested bovine pericardium was sewn into tubular form as a dural substitute. The pericardial tubes were tied at either end and attached to intravenous tubing to create a closed loop water system providing a realistic pressurized and closed-loop water system to provide functional cerebrospinal fluid leakage during durotomy. Early experience suggests that this physical simulator would be valuable as a training module in the Drake-Hunterian Neurovascular Laboratory (London, Ontario, Canada). This functional phantom is inexpensive to construct, provides a realistic tactile and visual environment for deliberate practice of spinal durotomy repair, and can be easily extended to simulate other patient-specific spinal interventions.

THE ROLE OF INDUCIBLE NITRIC OXIDE SYNTHASE IN REGULATING STORE OPERATED CALCIUM ENTRY IN MICROGLIA

Maksoud M1,2

, Lu W-Y1,2

, Xiang Y-Y1

Robarts Research Institute1, Department of Neuroscience


Background – Microglia are brain-derived central nervous system macrophages that regulate inflammation and eliminate cellular debris within the brain. During cerebral inflammation, microglia polarize to a proinflammatory phenotype and produce nitric oxide (NO) through the upregulation of the protein inducible nitric oxide synthase (iNOS). Over-activation of this proinflammatory phenotype can lead to neuroinflammation and neurodegeneration. Microglia activity relies heavily on calcium entry through store operated calcium channels (SOCC). While it is known that NO production effects microglia activity, the underlying mechanism is poorly understood. This study sets forth to examine how NO may influence store operated calcium entry and overall microglia activity. Methods – Calcium imaging and whole cell patch clamp electrophysiology on the microglia BV2 cell line was used to examine the effect of NO on store operated calcium conductance. Thapsigargin was used to induce store operated calcium conductance in cells, while the NO-donor drugs; GSNO or SNAP were used to examine the NO effect on store operated calcium conductance. The nonspecific SOCC inhibitor SKF-96365 was used to confirm channel conductance was of the SOCC nature. Results – 200uM GSNO treatment enhanced store operated calcium channel conductance in 66% of BV2 microglia, while 33% of cells showed a reduction in conductance using whole cell electrophysiology. Calcium imaging showed an increase in calcium entry for 56% of BV2 cells in response to 250uM SNAP. 24% of BV2 cells showed a decrease in calcium entry, while 20% of cells showed no change in calcium entry in response to 250uM SNAP. Conclusion – These results suggest that calcium entry in BV2 microglia is a dynamic process in response to exogenous NO, with the majority of responses enhancing calcium entry.

30

BRAIN CHANGES OBSERVED WITH MRI IN FEMALE ATHLETES AFTER ONE OR MORE CONCUSSION-FREE SEASONS

Kathryn Y. Manning1,2

, Kevin Blackney3, Arthur Brown

4, Lisa Fischer

5, Robert Bartha

1,2, Tim Doherty

6, Amy Schranz

1,2, Christy

Barreira3, Douglas Fraser

7, Jeff Holmes

8, Gregory A. Dekaban

3, and Ravi S. Menon

1,2

1Medical Biophysics, Western University;

2Centre for Functional and Metabolic Mapping, Robarts Research Institute;

3Department of Microbiology and Immunology, Western University;

4Department of Anatomy and Cell Biology, Western

University; 5Primary Care Sport Medicine, Fowler Kennedy Sport Medicine;

6Physical Medicine and Rehabilitation, Western

University; 7Paediatrics Critical Care Medicine, London Health Sciences Centre;

8School of Occupational Therapy, Western

University

Rugby players experience repetitive impacts during practice and play that don’t result in concussion symptoms or diagnosis. Recent studies suggest that these regular impacts could have a cumulative effect on brain health. Here we aim to determine the changes in MRI parameters, clinical cognitive measures and blood markers pre- and postseason in unconcussed athletes participating in a contact sport. We follow a female university-level rugby team (n = 54, age 18-23) over a three-year period. Players are evaluated during intense pre-season tryouts and practices and after a period of relative rest 2-3 months post-season if they did not experience a diagnosed concussion. Diffusion tensor imaging (DTI) was used to quantify the structural integrity of major white matter tracts and resting state fMRI (RS-fMRI) was used to examine functional connectivity between 136 anatomical regions. Hematology, flow cytometry and standard clinical measures were used to mechanistically interpret imaging changes. After 2-3 months of post-season recovery, there were significant increases in fractional anisotropy and decreases in mean and radial diffusivity in a number of white matter tracts along with decreases in RS-fMRI functional connectivity between brain regions involving those same tracts. Total leukocyte populations decreased and lymphocytes increases, while verbal, visual and immediate memory composites improved following the 2-3 months of recovery. Together, this could indicate transient, neuroprotective changes in myelin over one or more concussion-free seasons.

PTHR175

TAU IS ASSOCIATED WITH TAU PATHOLOGY IN A SPECTRUM OF TAUOPATHIES

Moszczynski AJ1,2

, Yang W 1,3

, Strong MJ1,2,3

Robarts Research Institute1, Schulich School of Medicine and Dentistry

2 Department of Clinical Neurological Sciences

3,

Western University Microtubule associated protein tau (tau) deposition is associated with a spectrum of neurodegenerative diseases (tauopathies). Amyotrophic lateral sclerosis (ALS) with cognitive impairment (ALSci) is associated with tau phosphorylation at Thr

175 which leads to pathological tau fibril formation through a GSK3β mediated pathway resulting in pThr

231. To

determine the involvement of this pathway in multiple tauopathies we have examined the extent of pThr175

expression across a range of frontotemporal degenerations. Sections from the superior frontal cortex, anterior cingulate cortex, amygdala, hippocampus, basal ganglia, and substantia nigra were selected from neuropathologically confirmed cases of Alzheimer’s disease, vascular dementia, frontotemporal lobar degeneration, ALS, ALSci, ALS-FTD, Parkinson’s disease, corticobasal degeneration, diffuse Lewy body dementia, mixed DLBD, multisystem atrophy and Pick’s disease. Sections were stained by immunohistochemistry using a panel of phospho-tau antibodies (pSer

208,210, pThr

217, pThr

175, and

pSer202

,pThr231

and T22). Semiquantitation was used to score pathological load. Across diseases, tau load was heaviest in layers II/III of the entorhinal cortex, amygdala and CA1/2 of hippocampus with all antibodies. With the exception of Lewy body dementia, we observed pThr

175 co-localizing with pThr

231, suggesting that this pathway may be a common mechanism

of toxicity across the tauopathies.

31

NEUROPLASTICITY AND LOCOMOTOR RECOVERY IN THE INJURED MOUSE SPINAL CORD FOLLOWING SOX9 ABLATION AT THREE

WEEKS POST INJURY

Natalie M. Ossowski, Nicole M. Geremia, Todd Hryciw, Kathy Xu, Arthur Brown

Robarts Research Institute, Neuroscience Graduate Program, The University of Western Ontario, London, ON, Canada

Recovery after spinal cord injury (SCI) is limited, as neuronal connections lost due to trauma are never fully restored. This poor recovery is due to the molecular environment of the spinal cord that limits neuroplasticity. Perineuronal nets are matrix structures that pose a major impediment to reactive sprouting owing to their high content of chondroitin sulfate proteoglycans (CSPGs). Our laboratory has shown that conditional Sox9 ablation prior to spinal cord injury effectively decreases CSPG levels in the perineuronal nets distal to the lesion. We have also demonstrated that the decrease in perineuronal net CSPG levels increases neuroplasticity and improves locomotor recovery after spinal cord injury. In the present study we have set out to determine whether conditional ablation of Sox9 in the subacute period after spinal cord injury also yields increased neuroplasticity compared to controls. In this study, Sox9 was ablated in mice 3 weeks after spinal cord injury. Using the Basso Mouse Scale (BMS), there were no differences in hindlimb function detected between Sox9 knockout and control mice throughout the 14-week recovery period. However, this may be due to the SCI lesion being located directly on the lumbar enlargement where hindlimb motoneurons are located. Immunohistochemistry and neuronal tracing to assess neuroplasticity and levels of perineuronal nets are currently being completed.

REDUCED BRAIN GLUTAMINE IN FEMALE VARSITY RUGBY ATHLETES AFTER CONCUSSION

Schranz A1, Blackney K

1, Brown A

1, Fischer L

2, Manning K

1, Menon R

1, Barreira C

1, Doherty T

3, Fraser D

4, Dekaban G

1,

Bartha R1

Robarts Research Institute1, Primary Care Sport Medicine, Fowler Kennedy Sport Medicine

2, Physical Medicine and

Rehabilitation3, University of Western Ontario, Paediatrics Critical Care Medicine, London Health Sciences Centre

4

A concussion is a brain injury caused by a blow to the head or another part of the body that causes the brain to experience rapid rotational and translational accelerations. Due to the lack of proper diagnosis and prognosis of a concussion, athletes often return to play before the concussion has fully resolved, putting them at an increased risk of more serious injury. Biomarkers must be found that identify concussion as well as recovery. Non-invasive imaging biomarkers in particular could be used to directly measure brain changes following concussion.

Magnetic resonance spectroscopy was used to quantify brain metabolite levels (N-acetyl aspartate, choline, creatine, glutamate, glutamine, and myo-inositol), as potential biomarkers, in female athletes over the course of a rugby season. Players were subdivided into two groups; those that were diagnosed with a single concussion and players that were not concussed. Each athlete had an MRI at the beginning and end of season (~6 months apart). Concussed athletes were additionally evaluated 24-72 hours post-concussion. It was hypothesized that N-acetyl aspartate would decrease, and myo-inositol would increase after concussion.

In the concussed group, a 42% reduction (p<0.05) in glutamine was found post-concussion compared to baseline. Interestingly, in the non-concussed group a 22% reduction (p<0.05) in glutamine was found at the end of the season, compared to baseline. No other metabolite changes were observed in either group. The reduced glutamine may be the result of reduced oxidative metabolism.

32

CEREBRAL VASCULATURE

NETWORK-WIDE FUNCTIONAL CONNECTIVITY CHANGES DURING UNILATERAL FRONTAL STROKE RECOVERY IN NONHUMAN

PRIMATES

Adam R1,2

, Johnston K1,3

, Hutchison RM4, Everling S

1,2,3

Robarts Research Institute1, Departments of Neuroscience

2 and Physiology and Pharmacology

3, Western University; Centre

for Brain Science, Harvard University4

Spatial extinction is an attention deficit commonly seen following stroke in humans, typically in the right hemisphere, and nonhuman primates. It is characterized by impaired detection of a contralesional stimulus when two stimuli are presented simultaneously in both the ipsilesional and contralesional hemifield. This results in a disabling ipsilesional saccade selection bias that often recovers over time. Here, we used resting-state fMRI at 7T to investigate the functional connectivity (FC) changes following a right frontal stroke while measuring recovery of the saccade selection bias on a free-choice saccade task. We created a nonhuman primate model of ischemic stroke using macaque monkeys by injecting endothelin-1, a potent vasoconstrictor, in the right dorsolateral prefrontal cortex and frontal eye field. One week post-stroke, when the saccade selection bias was most severe, ipsilesional premotor cortex and bilateral parietal and occipital areas showed decreased network-wide FC while contralesional premotor cortex showed increased FC with bilateral temporal areas. By 2-4 months post-stroke, when the saccade selection bias had recovered, ipsilesional caudate nucleus and putamen showed increased contralesional network-wide FC and contralesional premotor cortex showed increased FC with surrounding contralesional frontal areas. Meanwhile, bilateral occipital areas showed decreased network-wide FC. Our preliminary findings suggest a pattern of FC changes following unilateral stroke that may underlie behavioural recovery of the saccade selection bias in spatial extinction.

CYCLIC CONTINUOUS MAX-FLOW MRI PHASE PROCESSING

Baxter JSH1,2

, Hosseini Z1,2

, Liu J1, Drangova M

1,2,3, Peters TM

1,2,3

Robarts Research Institute1, Biomedical Engineering Graduate Program, Department Medical Biophysics

3, Western

University

Tissue susceptibility differences manifest in MR phase images as high-frequency changes in an otherwise smooth phase

background. Two paradigms currently exist for isolating these changes: one involves phase unwrapping followed by high-

pass filtering; the other involves homodyne high-pass filtering (HHPF) on the complex signal directly. Both rely on a

Euclidean topology, which can result in artifacts such as phase wraps and shadowing, as phase is inherently cyclic. This work

presents cyclic continuous max-flow (CCMF), a novel non-linear phase filtering approach based on variational optimization,

which processes phase using a cylindrical manifold expressing possible phase images, rather a Euclidean topology. The

individual channel phase data were processed with both CCMF and HHPF (filter size equal to 30% of FOV). The resulting

images were then combined using the inter-echo variance channel combination technique. The raw phase data were also

combined, unwrapped using the PUROR algorithm and high-pass filtered. By processing phase information using its

inherent topology, higher quality high-pass phase maps can be reconstructed in a relatively short amount of time. This

improves the visualization of structures such as veins in channel-combined phase images at the periphery and around the

sinuses where additional phase artifacts are often present as a result of linearizing the phase topology.

33

SMOOTH MUSCLE/ENDOTHELIAL KIR CHANNELS MODULATE CELL-TO-CELL COMMUNICATION IN CEREBRAL ARTERIES

Maria Sancho1,2

, Nina C. Samson1, Bjørn O. Hald

3, Ahmed Hashad

1,2, Sean Marrelli

4, Suzanne E. Brett

1,2, Donald G. Welsh

1,2

1Robarts Research Institute. Dept of Physiology & Pharmacology, Schulich School of Medicine, Western University, Canada.

2Dept of Physiology & Pharmacology, University of Calgary, Canada.

3Dept of Biomedical Sciences, University of

Copenhagen. 4Dept. of Anesthesiology, Baylor College of Medicine, Houston Texas

Global blood flow control is enabled by the conduction of charge along arteries. The distance over which electrical phenomena spread is governed by 3 factors, one being the ionic properties of vascular cells. In this study, we determined the role of inward rectifying K+ channels (KIR) in setting membrane conductance and charge spread along the arterial wall. Small middle cerebral arteries (~100 μm diameter) from hamster were probed with a standard conduction protocol. A focal KCl stimulus elicited a constrictor response that conducted robustly, with a decay constant of ~0.4 μm/100μm vessel length. Selective inhibition each K+ channel class had no effect on conduction, the exception being Ba2+ blockade of KIR, which facilitated decay (~1.6 μm/100μm vessel length). Patch clamp electrophysiology and Q-PCR highlighted the presence of a KIR current in smooth muscle comprised of KIR2.1/2.2 subunits. The incorporation of this current into an electrical model revealed that it was too small to account for the change in conduction decay; consequently another KIR current must be present. Electrophysiology and Q-PCR confirmed a second KIR current in the endothelium. Computational modeling subsequently confirmed that inhibiting both currents had a greater effect on conduction decay, the result of a sizable voltage shift increasing feedback from voltage dependent- and Ca2+ activated K+ channels. In summary, our observations indicate that KIR channels are present in endothelial and smooth muscle cells, and together these K+ conductances can tune electrical communication.

ANGIOTENSIN II INHIBITS T-TYPE CA2+ CHANNELS IN CEREBRAL ARTERIAL SMOOTH MUSCLE CELLS

Ahmed M. Hashad, Maria Sancho, Donald G. Welsh Department of Physiology and Pharmacology, University of Calgary, Canada. Robarts Research Institute, Department of Physiology and Pharmacology, University of Western Ontario, Canada. T-type Ca2+ channels (CaV3.1 and CaV3.2) are expressed in cerebral arterial smooth muscle cells and they play a key role in regulating arterial tone. In this study, we investigated whether and by what mechanism Angiotensin II (Ang II), a vasoactive peptide produced within the cerebral arterial wall, influences T-type Ca2+ channels. Using patch clamp electrophysiology and rat cerebral arterial myocytes, Ang II (100 nM) was shown to inhibit T-type Ca2+ currents in a time-dependent manner (~ 40% after 15 min); it also shifted the voltage-dependency of activation by 7 mV. Ang II induced inhibition was mediated through the AT1 receptor, as preincubation with Losartan (1 μM) abolished the effect. Protein kinase C (PKC) blockade by GF 109203X (100 nM) did not eliminate Ang II-mediated inhibition, demonstrating that this signaling protein was not involved. In contrast, NADPH oxidase inhibitors abolished the inhibitory effect of Ang II revealing a role for reactive oxygen species (ROS) on T-type Ca2+ channels. To delineate if Ang II is targeting a particular T-type channel, Ni2+ (50 μM) was added to selectively block CaV3.2. In the presence of Ni2+, Ang II did not induce further inhibition of the residual current, indicating that Ang II was selectively targeting CaV3.2. In summary, Ang II inhibits CaV3.2 channels in cerebral myocytes through the generation of ROS. These findings suggest that T-type channels are a regulatory target of vasoactive stimuli, known to facilitate the “constrictive” phenotype prevalent with cerebrovascular disease.

34

LOCAL FREQUENCY SHIFT MAPS GENERATED BY PROCESSING SINGLE CHANNEL MR PHASE IMAGES RESULT IN IMPROVED

CONSPICUITY OF CEREBRAL STRUCTURES

Hosseini, Z1,2

, Liu, J2, Drangova M

1,2,3

Biomedical Engineering Graduate Program1, Imaging Laboratories, Robarts Research Institute

2 and Department of Medical

Biophysics3, Western University

Magnetic resonance imaging plays a key role in imaging of the brain thereby enabling the monitoring of cerebral health, diagnosis as well as staging of numerous neurological diseases. Particularly, phase component of MR images can be used to generate a local frequency shift (LFS) map, which allows for simultaneous visualization of cerebrals structures (vessels, deep nuclei of the brain, gray/white matter) and quantification of biomarkers, such as iron. Recently, image acquisition using multiple transmit/receive coils has become commonly used in order to enable for shorter scan times and higher signal-to-noise ratio. With the advent of this practice, the post-processing of phase images acquired from multiple coils has introduced a new challenge. Conventional multi-channel LFS mapping involves application of a channel combination followed by high-pass filtering to obtain local frequency shift maps. Appropriate channel combination technique is important in order to prevent phase cancellation, and therefore loss of information, during the combination process. In this work we present a novel approach to multi-channel phase imaging, through which we unwrap the individual channel phase images and subsequently apply a high-pass filter to them prior to application of channel combination. Our results, compared to conventional processing pipelines, suggest that the proposed technique results in LFS maps with reduced background noise/artifacts, and outstanding structural contrast including white/gray matter, deep nuclei, and venous vasculature.

COIL COMBINATION USING WHOLE TIME COURSE SINGULAR VALUE DECOMPOSITION TO IMPROVE PHASE SIGNAL-TO-NOISE

RATIOS IN FUNCTIONAL MRI

O Stanley1,2,3

, M Klassen1,2,3

, T Whelan2 and R Menon

1,2,3

Centre for Functional and Metabolic Mapping1, Robarts Research Institute

2, Department of Medical Biophysics

3, University

of Western Ontario

Functional magnetic resonance imaging experiments can output two images per single scan (magnitude and phase). Although magnitude images are used in clinical fMRI applications, phase images can also be used to obtain the blood oxygenation level dependent (BOLD) contrast used in fMRI. This can be used as a filter in order to suppress extraneous BOLD signal from large draining veins in the brain. A current issue in phase based fMRI applications is that phase images are conventionally combined by calculating the phase of the complex sum of the channels. This assumes all channels to be equally sensitive to all positions in space which is untrue and results in low phase SNR near the center of the image. Phase SNR can be restored by using a fixed, singular value decomposition (SVD) combination of magnitude and phase over the entire time course of an experiment. This is because the SVD method requires that the channel weights across time be unique to each voxel’s position in space opposed to constant. In order to validate this, an oil phantom was scanned using an echo planar imaging (EPI) sequence with 3 different acceleration factors. Each scan had a 150 volumes with a repetition time of 1.25s. Coil combination was completed using the conventional combination and the fixed SVD algorithm. The temporal standard deviation of the phase images at the highest acceleration factor using conventional combination was 0.0341± 0.0131 radians (mean±std); using fixed SVD combination, the temporal standard deviation was 0.0268±0.0138 radians. This decrease illustrates that fixed SVD combination of the whole time course increases the phase SNR in accelerated EPIs and should be used when completing fMRI studies requiring phase data.

35

IMPLICATIONS OF ELECTRICAL COMMUNICATION IN BRAIN BLOOD FLOW REGULATION AND STROKE INJURY

Anil Zechariah1, Cam Ha T. Tran

2, Bjorn O. Hald

3, Ursula I. Tuor

2, Grant Gordon

2, Donald G. Welsh

1

Robarts Research Institute1, Department of Physiology and Pharmacology, University of Western Ontario, Ontario, Canada;

2Department of Physiology and Pharmacology, University of Calgary, Alberta, Canada;

3Department of Biomedical Sciences,

University of Copenhagen, Copenhagen, Denmark An integrated network of cerebral arteries actively coordinates brain blood flow in accordance with metabolic demands. Large regional changes require synchronized vasomotor responses across vessel segments starting at the origin of a stimulus and spreading to the major upstream resistors. The coordination of vasomotor tone is intimately tied to the spread of charge through gap junctions, a process that equalizes membrane potential, [Ca2+] and myosin light chain phosphorylation among thousands of smooth muscle cells. Coordinated responses are critically important during cerebrovascular accidents (i.e. stroke) where the synchronized dilation of collateral vessels diverts flow into ischemic tissue. We hypothesized that electrical communication, via gap junctions, plays a central role and that dysfunctional communication between vascular cells impairs collateral responses, increasing stroke injury. Using isolated cerebral arteries from mouse, hamster and resected human tissue, we show that electrical stimuli can effectively spread along vascular cells in an endothelium dependent manner. Genetic deletion of Connexin40 (Cx40-/-), a component protein of gap junctions, compromised electrical communication in the cerebral arteries. Subjecting Cx40-/- mice to focal cerebral ischemia induced greater deficit in cerebral blood flow than in wild type controls, during stroke and the subsequent reperfusion period. This attenuation of blood flow translated to augmented stroke injury in the Cx40-/- mice. In closing, our study illustrates the presence of robust electrical communication and that compromising this integrated process restricts the ability of the cerebral circulation to respond to, and moderate injury from vascular accidents, including stroke.

36

IMAGING SAFETY, QUALITY & EFFICIENCY

PERSEUS: ENHANCING ULTRASOUND IMAGING WITH PATTERN RECOGNITION CAPABILITIES

Cantor D1,2

, Ameri G1,2

, Baxter J1,2

, McLeod J1,2

, Elvis C1,2

, Daley M3, Peters T

1,2

Robarts Research Institute1, Biomedical Engineering Graduate Program

2 and Brain and Mind Institute


Ultrasound imaging (US) stands out as one of the most cost-efficient, and therefore, ubiquitous diagnostic technologies in the medical domain. Nonetheless, US diagnostic value is determined by both image quality and the expertise of the clinician performing the scan. As image quality deteriorates due to constraints such as hardware power, distance of the probe to the organ of interest, and the accuracy of hardware and software elements, clinical interpretation becomes a challenging cognitive task which increases the time and amount of training required by clinicians to become proficient

Current research focuses on the employment of machine learning methods to assist medical pattern recognition in US. However, computational requirements associated with these methods (data transformation, feature extraction, employment of GPUs, etc.) reduce the scope of their applicability in the clinical practice, particularly in low-power, mobile scanners.

Our work investigates the role of machine learning in ultrasound image interpretation. Our goal is to enhance ultrasound scanners with pattern recognition capabilities to perform well-defined perceptual tasks of clinical relevance.

EVOLUTIONARY MAGNETIC RESONANCE IMAGING ANTENNA DESIGN

Ian R.O. Connell1,2

& Ravi S. Menon1,2

1Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada

2Centre for Functional and Metabolic Mapping (CFMM), Robarts Research Institute, London, Ontario, Canada

The two greatest difficulties in the routine application of Magnetic Resonance Imaging are increased power deposition inside the patient during a scan (Specific Absorption Rate or SAR), and increased tissue-independent inhomogeneity of the reconstructed image. An increase in SAR correlates with a subsequent rise in tissue temperature, which leads to biological damage if left unmitigated. Conversely, inhomogeneity of the reconstructed image is manifested in tissue-independent contrast and decreasing radiological confidence in the images obtained. These difficulties remain the most significant limitations in moving MRI-based neuroscience research to the clinic and are the foci of the developments presented here. We present advancements in the field of biomedical engineering that overcome the aforementioned limitations to performing MRI on the human brain. These developments are focused on a novel design of an essential component of MRI hardware – the radio frequency (RF) coil. This novel RF coil is designed to minimize patient safety concerns while simultaneously increasing the quality of the final reconstructed image. The RF coil was designed utilizing an evolutionary computer algorithm in conjunction with a full-wave electromagnetic solver. Through an iterative process, the final design was selected based on two objective functions: (1) increased image quality, and (2) minimal SAR. In comparison to the clinical research standard RF coil, our new design reduced the standard deviation of the tissue-independent contrast across the entire human brain by 10%. The mean reconstructed image signal per unit input power was increased by 12%. Similarly, our RF coil reduced the total power deposition occurring in all human tissue by 39%. These engineering advancements ensure the ability to obtain higher quality functional and anatomical images of the human brain, while ensuring patient safety is maintained for any routine neurological investigation. The two major barriers to clinical translation of state-of-the art research MRI scanners have been addressed by this study.

37

DETECTIVE QUANTUM EFFICIENCY: ENSURING HIGH QUALITY X-RAY IMAGES USING LOW PATIENT EXPOSURES

Terenz Escartin, Tomi Nano & Ian A. Cunningham Imaging Research Laboratories, Robarts Research Institute, Department of Medical Biophysics, Western University In x-ray imaging, there is always a trade-off between the level of radiation exposure used and the quality of images acquired. The detective quantum efficiency (DQE) describes the ability of a x-ray detector to produce high signal-to-noise (SNR) images using low radiation exposures. It is the effective fraction of x-ray quanta contributing to the image SNR and can be considered a surrogate measure of the “dose efficiency” of a detector. A recent clinical study by the Ontario Breast Screening Program showed that direct radiography (DR) systems had a 50% higher cancer detection rate than computed radiography (CR) systems, and attributed this difference to a difference in SNR and DQE. As a result, the low-DQE systems were removed and replaced with high-DQE systems in Ontario. While DQE is used by the research community to optimize detector design, it is rarely used by end users in radiology facilities in purchasing decisions and to ensure their systems continue to produce the best possible images for low patient exposures, due to a lack of awareness and expertise in DQE concepts. Our lab is developing the science and instrumentation required to bring practical DQE testing into clinical environments. An early prototype was evaluated on five new clinical x-ray systems. We showed that the DQE at low spatial frequencies varied between systems by a factor of 1.5x, indicating that some systems require 50% more radiation than others for the visualization of large patient structures. Of greater concern is that high-frequency DQE values varied by 10x, indicating some systems require 10x more radiation than others for the visualization of fine detail. This means that not all patients who need diagnostic x-ray imaging are receiving the best possible images for low exposures, due to differences in system performance. While many considerations are part of any purchase decision, it is critically important that awareness of DQE concepts become more readily available to the end user and decision maker.

AUTOMATIC DETECTION OF LOCAL GEOMETRIC DISTORTION IN ULTRA-HIGH-FIELD MRI FOR STEREOTACTIC

INTERVENTIONS

Lau JC1,2

, Khan AR1, MacDougall K

2, Parrent AG

2, Peters TM

1

Robarts Research Institute1, Department of Clinical Neurological Sciences


Ultra-high-field magnetic resonance images (MRI) provide improved visualization of subcortical structures that may be useful for surgical planning in stereotactic procedures. However, the increase in field strength can result in geometric distortion. We propose a workflow for automatic local geometric distortion detection using computational morphometry. 24 subjects obtained structural MRI scans on both the 3T and 7T scanners at Robarts. An image processing pipeline was developed to align 7T to 3T images for each subject using an iterative linear and nonlinear registration workflow. Voxel-based displacement was calculated from the deformation fields used for nonlinear registration. Group analysis was performed in standard MNI152 space. Nonlinear registration improved our ability to align 7T to 3T images. Using group and subject-specific analysis, we were able to identify regions systematically affected by geometric distortion. Average displacements in the parasagittal cortex were 1.5-3.5 mm. Our automated pipeline provides a framework for assessing the extent of geometric distortion resulting from ultra-high-field structural MRI in an exploratory fashion. The derived displacement maps can be used to prospectively evaluate subject-specific distortions that should be taken into account in stereotactic planning.

38

IMPROVED DETECTABILITY IN X-RAY IMAGES USING THE APODIZED APERTURE PIXEL (AAP) DESIGN.

Tomi Nano1, Terenz Escartin

1, Karim S. Karim

2, and Ian A. Cunningham

1

Imaging Robarts Research Institute, Departments of Medical Biophysics, Western University1; Department of Computer

Engineering, University of Waterloo2

Imaging procedures using ionizing radiation provide high resolution information and should be used when high signal-to-noise ratio (SNR) images are obtained with low radiation exposures. The detective quantum efficiency (DQE) as a function of spatial-frequency quantifies an x-ray detector’s ability to produce high quality images. The ability to visualize structural information requires high DQE values across all spatial frequencies, especially at high frequencies which are critical for the detection of fine detail and small structures. Current x-ray system DQE values are much lower at high spatial-frequencies and can be attributed to aliasing image artifacts. The apodized aperture pixel (AAP) design improves high-frequency DQE by eliminating noise aliasing using detector sensor-elements of size 10-25 m to optimally synthesize typical 100-200 m pixel images. The AAP design improves the DQE of different converter material types. Previous proof-of-concept experiments show DQE values are doubled near the sampling cut-off frequency in detectors that have converter materials such as amorphous selenium. Simulations of sinusoidal pattern images have twice the contrast at high frequencies and show removal of aliasing artifacts. Optimum high-frequency DQE values achievable with the AAP design result in improved image contrast and can be used to obtain more patient information during imaging procedures which is critical for early detection of disease.

39

MOVEMENT & MOBILITY

PERFORMANCE EVALUATION OF A PERIPHERAL CONE-BEAM CT SCANNER WITH WEIGHT-BEARING CAPABILITIES

Rudy Baronette1,2,3

, Yuan X2, Pollmann SI

2, Teeter MG

1,3,4,5, Holdsworth DW

1,2,3,4

1Western Bone and Joint Institute;

2Imaging Research Laboratories, Robarts Research Institute, Depts. of Medical

Biophysics3 and Surgery,

4 Western University;

5Lawson Health Research Institute, London ON, Canada

Joint space narrowing can be caused by inflammatory or degenerative musculoskeletal conditions such as rheumatoid arthritis or osteoarthritis. Studies have shown that weight-bearing images improve detection of joint space narrowing more reliably than supine views. Furthermore, weight-bearing imaging provides functional information about joint biomechanics. Weight-bearing imaging of the lower extremity was limited to the supine position by scanner design in conventional clinical CT scanners. Due to recent advances in cone beam computed tomography (CBCT), true 3D weight-bearing CT is available for clinical evaluations of patients in a health care setting. The Verity CT scanner (Planmed Oy) is a CBCT imaging system optimized for imaging upper and lower extremities. The system acquires high-resolution volumetric images of the target and includes a motorized gantry that allows for weight-bearing CBCT imaging of the knee, ankle, and foot. The purpose of this study is to independently evaluate performance results of the peripheral cone-beam CT scanner using phantoms representing the size of an average knee or elbow. Performance of the Verity CBCT system was evaluated using two custom-built quality-control phantoms which were used to assess the noise, resolution, and uniformity. The performance evaluation of the peripheral CBCT scanner shows the Verity meets the manufacturer’s specifications and demonstrates the potential for use in future clinical studies to understand how various musculoskeletal treatments impact joint biomechanics.

MICRO-CT COMPATIBLE LOAD CONTROLLED KNEE JOINT MOTION SIMULATOR

Blokker A1,2

, Burkhart T2,4

, Getgood A3,4

, Holdsworth D1,2,3


1, Departments of Biomedical Engineering

2 and Surgery and Medical Biophysics

3, Fowler Kennedy

Sports Medicine Clinic4, Western University

Over 100,000 anterior cruciate ligament (ACL) reconstructions are performed annually with upwards of 14% failing due to non-anatomic graft placement and incorporation failure. To date, there is no experimental method in the literature to non-invasively image and measure ACL and graft kinematics under varying fixations and loading protocols; this has contributed to sub-optimal reconstruction methods. The purpose of this research was therefore to develop and validate a novel knee joint motion simulator that actively manipulates human cadaveric knee joint specimens while micro-CT imaging non-invasively captures the kinematics of the in-tact joint capsule. The simulator consists of an actuating platform that fixes the tibia to a 6 degree-of-freedom load cell while a flexion jig, rigidly mounted to the CT-scanner bed, immobilizes the femur and centers the knee in the scanner’s 10cm axial field of view. This design facilitates 30˚ of static flexion in addition to active control over anterior translation, valgus rotation, internal rotation, and axial compression. Physiologically relevant loads were applied to a knee joint surrogate (4.85cm diameter solid rubber tubing) while micro-CT scans were taken with a standard imaging protocol (16s anatomical scan, 120 kVp, 20mA). Each axis was tested separately with increasing load targets repeated five times. The average percent difference between the actual and target loads was less than 5%, while coefficients of variations (CV) were less than 10% over the five trials. The motion simulator then loaded the surrogate while high quality micro-CT images were acquired. The simulator will be further validated with cadaveric specimens. Independent axis actuation facilitates a wide range of loading patterns and future investigations will determine the effect of different ACL reconstruction characteristics on soft tissue and bone responses.

40

THE SENSORIMOTOR PROPERTIES OF THE FAST VISUOMOTOR SYSTEM

Gu C1-3

, Wood DK5, Pruszynski JA

1-4, Gribble PL

2-4, Corneil BD

1-4

Robarts Research Institute1, Departments of Psychology

2 and Physiology and Pharmacology

4, Brain and Mind Institute

3,

Western University, Department of Neurobiology5, Northwestern University

When a novel visual stimulus appears, a transient signal sweeps through the neuraxis; it also generates a rapid stimulus-locked response (SLR) within 100 ms of stimulus onset on upper limb muscles in humans. Here we investigate the sensorimotor properties of the SLR in different 4 reaching paradigms. Subjects performed reach movements with their right arm while we measured EMG activity from their right pectoralis major muscle. 1st, we investigated if the SLR was a visual or a preparatory motor response, by having subjects reach either toward or 180° away from a visual stimulus. The SLR was modulated based on the visual stimulus location regardless of the ensuing reaching movement. 2nd, we had subjects make reaching movements with different initial hand and eye positions to determine whether the SLR represented the visual stimulus in an eye- or hand-centric reference frame. The SLR was modulated based on stimulus location relative to the hand and not the eye and thus was in a hand-centric reference frame. 3rd, we investigated if hand trajectory altered the SLR by having subjects reach to the same stimulus around different obstacles. We found that the SLR was influenced based on the preplanned hand trajectory. 4th, we had subjects learn to reach in a novel visuomotor environment, which required subjects to learn a new motor command for the same visual stimulus. The SLR adapted in a similar fashion as the voluntary movements, however the adaptation was incomplete to the new sensorimotor environment. Taken these results together, despite the short-latency, the SLR shares many characteristics that have been classically associated with voluntary motor control. We propose that the SLR reflects a fast visuomotor system, which enables human for rapid feed-forward response to unexpected changes to a visual stimulus.

THE CONNECTION BETWEEN MICRORNAS, RNA-BINDING PROTEINS AND INTERMEDIATE FILAMENTS DYSREGULATION IN ALS

Hawley Z1,2

, Campos-Melo D1, Volkening K

1, Strong M

1,3

Robarts Research Institute1, Departments of Neuroscience

2 and Pathology


Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease that results in loss of motor function and is fatal within 3-5 years of diagnosis. Common pathological features of ALS include changes to the mRNA and protein levels of RNA-binding proteins (RBPs) - TDP-43 and RGNEF - and intermediate filaments (IFs) - NEFL, INA and PRPH. Interestingly, microRNAs (miRNAs) – small RNA molecules that alter mRNA metabolism – were observed to be massively dysregulated in the spinal cord tissue of ALS patients, providing a possible explanation for the changes we see in RBPs and IFs. I hypothesize the dysregulation of specific groups of miRNAs in ALS leads to alterations in the mRNA and protein levels of IFs (NEFL, INA and PRPH) and RBPs (TDP-43, FUS and RGNEF) linked to ALS pathology. Bioinformatic analysis provided 8 miRNA candidates (miR-105, 140-5p, 185, 1179, 1297, 3120, 4306 and b4335) and four miRNA candidates (miR-194, 548x, sb659 and b2122) that may regulate the expression of IFs and RBPs, respectively. Real time PCR analysis showed a significant downregulation of only miR-194, b2122, 105 and 140-5p in sALS patients compared to control groups. Currently, in vitro studies within HEK293T cells are being executed, which include transfection of our miRNAs and genes of interest to perform gene reporter, RT-qPCR and site-directed mutagenesis assays to determine the effect and specificity of these miRNA candidates on RBP and IF expression. This experimental design will determine the miRNAs that participate in the aberrant regulation and expression of RBPs and IFs, and provide a potential pathogenic mechanism for several ALS pathologies.

41

GEOMETRIC DISTORTION PHANTOM FOR MRI

Gregory Hong,1,3,4

Matthew G Teeter,1,2,3,4

Jaques S Milner,3 Steven I Pollmann,

3 David W Holdsworth,

1,2,3,4 Maria

Drangova1,3,4

1Western Bone and Joint Institute,

2Department of Surgery,



Western University The conditions leading to hip implant failure would ideally be imaged with MRI, since it provides excellent soft tissue contrast and shows bone lesions displaying bright signal due to fluid. However, metal artifacts obscure diagnosis, which arise from the large susceptibility difference between tissue and metal. In order to characterize the severe geometric distortion caused by these artifacts, we have designed a phantom based on a 3D-printed grid containing spherical markers with fixed, regular spacing. 3D printing provides the means to fabricate these grids in a way that secures and encapsulates implants to generate MRI images that can be quantitatively assessed. We hypothesize that an accurate, cost-effective distortion phantom can be fabricated from polylactic acid (PLA), a common 3D printing material. The proposed structure is comprised of a series of small marker spheres, supported by a grid of thin walls. To test this hypothesis, we must fabricate a prototype phantom without the hip and evaluate its suitability for MR imaging. In particular, for quantitative analysis we require that the susceptibility difference between PLA and surrounding liquid be negligible. The device was fabricated by fused-deposition printing using a commercially available 3D printer (Dremel® Idea Builder). In order to accurately portray the conductivity and other MRI properties of soft tissues, the grid is immersed in a solution of copper sulfate in saline. The phantom was scanned in a knee coil on a GE 3T scanner using a dual echo acquisition with different echo times; we then mapped the frequency shifts, which arise from susceptibility differences, by using the phase difference between the echoes. Finally, to evaluate the geometric accuracy of the device, it was scanned in a calibrated micro-CT scanner, with isotropic resolution of 0.15 mm.

GAIT DISTURBANCES IN THE 5XFAD TRANSGENIC MOUSE MODEL OF ALZHEIMER’S DISEASE

Lee WV1,2

, Beraldo FH1, Cowan MF

1, Winters B

5, Prado VF

1,2,3,4, Prado AM

1,2,3,4

Robarts Research Institute1, Graduate Program in Neuroscience

2, and Departments of Physiology and Pharmacology

3,

Anatomy and Cell Biology4, Schulich School of Medicine and Dentistry, University of Western Ontario. Department of

Psychology5, University of Guelph.

Alzheimer’s Disease (AD) is a disabling chronic disorder characterized by progressive cognitive impairment. However, patients may also experience gait disturbances. Given the high prevalence and poor prognosis of AD, the characterization of animal models for AD with face validity reproducing cognitive and non-cognitive disturbances of AD is important. However, whether gait disturbance is reproduced in a mouse model of AD amyloidosis is not clear. We hypothesize that similar to AD affected individuals, the pathological aggressive 5xFAD mouse line will develop an age-dependent motor phenotype – particularly disturbances in gait parameters. Fourteen month old male 5xFAD mice and wild-type controls with the same genetic background were subjected to grip and locomotor tests. They were also subjected to gait assessments using the CatWalk automated gait analysis system. 5xFAD mice demonstrate deficits in motor coordination and speed. In addition to previously reported cognitive impairments, 5xFAD mice also demonstrate disturbances in gait, which is observed in patients throughout all stages of AD. The assessment of gait impairments in mouse models is highly relevant, as it can influence the results of behavioural analyses, and it could also serve as an endpoint for testing potential therapeutics for non-cognitive symptoms.

42

DEVELOPMENT AND VALIDATION OF A SYSTEM FOR HIGH-FREQUENCY VIBRATION OF LIVE CELLS DURING REAL-TIME

MICROSCOPY

Lorusso, D. 1234

, Nikolov, H.N. 3

, Chmiel, T.3, Ochotny, N.M.

14, Sims, S.M.

14, Dixon, S. J.

14, Holdsworth, D.W.

1356

1Bone and Joint Institute;

2Collaborative Training Program in Musculoskeletal Health Research;

3Imaging Research

Laboratories; Robarts Research Institute; 4

Department of Physiology & Pharmacology; 5

Department of Medical Biophysics; 6Department of Surgery, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada

Introduction: Mechanotransduction is the process by which cells sense and respond to the local mechanical environment and is a critical component of mammalian; yet, the underlying mechanisms are still poorly understood. Our goal is to observe the immediate responses of cells to high-frequency vibrations, a form of mechanical stimulus. Here, we describe the development and validation of an integrated motion-control system for vibrating live cells at frequencies up to 500 Hz and accelerations up to 1 g, which is compatible with real-time optical microscopy.

Materials and Methods: The motion-control system was mounted on an inverted microscope and the moving parts were suspended on a linear air bushing system which was actuated by a voice-coil. Accelerations were measured via an on-board calibrated accelerometer. To ensure vibrations were transferred effectively to the cell culture dish, motion waveforms were imaged with a high-speed camera at 1200 FPS. MC3T3-E1 and UMR 106 cells were then seeded onto compatible dishes and imaged. In addition, cells were transfected with GFP-actin and exposed to vibration during photometry.

Results: During operation between 15 – 500 Hz and 0.1 – 1 g, sinusoidal motion of waveforms were observed from both optical and accelerometer-derived measurements, with displacements ranging from the nanometer to millimeter range. Cultured osteoblast-like cells were vibrated 0.3 g at 45 Hz during microscopy and remained adherent and viable. Changes in the actin cytoskeleton appear to have been observed during vibration.

Discussion and Summary: We have developed, fabricated, and tested a motion-control system capable of – for the first time – delivering physiologically relevant vibrations to live-cells during real-time microscopy.

ENHANCED UBIQUITINATION AND PROTEASOMAL-DEGRADATION OF CATALYTICALLY-DEFICIENT HUMAN CHOLINE

ACETYLTRANSFERASE MUTANTS

Trevor M. Morey1,2

, Shawn Albers1,2

and R. Jane Rylett1,2

1Molecular Medicine Research Group, Robarts Research Institute.

2Department of Physiology and Pharmacology, Schulich

School Medicine & Dentistry, University of Western Ontario. Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine required for cholinergic neurotransmission. ChAT mutations are linked to congenital myasthenic syndrome (CMS), a rare neuromuscular disorder. One CMS-related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is located within a highly-conserved proline-rich motif at residues 14-PKLPVPP-20 that shares homology with SH3-binding motifs. In this study we demonstrate that disruption of this proline-rich motif by N-terminal truncation or Pro→Ala mutation reduces steady-state ChAT protein levels and cellular ChAT activity in cholinergic SN56 neural cells, with P17A/P19A-ChAT yielding the greatest effects comparable to N-terminal truncation. Proteasome inhibition by MG132 increases protein levels of P17A/P19A-ChAT, though failed to recover cellular activity. The reduction in protein levels of P17A/P19A-ChAT appears to be due to enhanced ubiquitination, resulting in increased proteasomal degradation. CMS-related V18M and A513T-ChAT also demonstrate decreased protein levels and enhanced ubiquitination in SN56 cells, and treatment with MG132 can partially restore both protein levels and cellular activity of CMS-mutant ChAT. By performing proximity-dependent biotin identification (BioID) we have identified HSP70 and HSP90, members of the heat-shock family of molecular chaperones, as novel ChAT protein interactors that are enriched in HEK293 cells expressing P17A/P19A-ChAT. Lastly, by co-immunoprecipitation we have confirmed these interactions in HEK293 and SN56 cells and show that they are enhanced with P17A/P19A-ChAT. Heat shock proteins are well characterized for their role during nascent protein folding and proteasomal degradation of misfolded proteins. Our results support further research into the regulation of ChAT ubiquitination and the role that ubiquitination may play in relation to ChAT function during cellular stress and disease.

43

TOWARDS OPTIMIZING A CUSTOM SMALL ANIMAL MODEL OF PARTIAL JOINT REPLACEMENT SYSTEM CREATED VIA ADDITIVE

MANUFACTURING

Adam DM Paish1,2

, Hristo N Nikolov1, Alexander O El-Warrak

3, Ian Welch

4, Matthew G Teeter

2,5, Douglas D Naudie

2,5, David

W Holdsworth1,2,5

. 1Imaging Research Laboratories, Robarts Research Institute,

2Medical Biophysics, Schulich School of Medicine & Dentistry,

3Animal Care and Veterinary Services,

4Animal Care Services, University of British Columbia,

5Division of Orthopaedic

Surgery, Department of Surgery, Schulich School of Medicine & Dentsitry, Western University. Optimization of the integration of metal with bone is important in a number of clinical applications, including joint replacements and fracture repair. In some situations, it can be difficult to develop a strong bond between bone and metal (i.e. osseointegration), leading to the need for revision surgery. During research to improve osseointegration, it is useful to test new procedures in animal models, and small animals (such as the rat) are ideal in the initial stages of research. In previous works we describe the first rat-specific load-bearing hip joint replacements, created through the use of three-dimensional image analysis, computer-aided design (CAD), and selective laser melting. Although we have our implants to be feasible, several areas for optimization have been identified. Thus, we describe recent advances to our prototypes, particularly in implant stability following installation. A set of eight broaches was created in CAD software (SolidWorks) to allow for incremental widening of the inner femoral cortex. A collar was added to CAD files of our prototype components to improve implant stability post-operatively. New components were printed in 316L stainless steel on a commercial system for 3D additive manufacturing in metal (AM 125, Renishaw plc). A dental hand tool was used with several polishing stones, rubber wheels and buffing cloths to remove the inherent surface texturing in specific regions of components. Broaches were selectively polished to leave a rough surface at the bone-metal interface, which can allow for better cutting through the bone. Polishing of the articulating surface of components was improved substantially using dental hand tools over previous hand-polishing methods. These components are the first of their kind and represent another step towards an optimized small animal joint replacement system for preclinical orthopaedic research.

ENHANCING MANUAL DEXTERITY IN OLDER ADULTS WITH TRANSCRANIAL DIRECT CURRENT STIMULATION

Ryan, K, Bartha, R, Duggal N

Background: Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that has been shown to increase cortical activity when applied to certain areas of the brain. Rubber electrodes sit on the scalp and pass a low, continuous current to selected brain regions, resulting in depolarization of the resting membrane potential allowing for increased cortical activity. tDCS has become popular for the treatment of many psychological disorders, such as depression and schizophrenia, etc., and as recently been shown to improve motor function in healthy and neurologically injured individuals. Purpose: The current study aims to determine if tDCS can improve performance of manual dexterity after one stimulation session in healthy older adults. Methods: 14 healthy older adults, aged 67-84 participated in a double-blind, randomized, cross over design. Participants were randomized to receive stimulation or sham on their first visit and received the contrary on their second visit, 7 days later. Participants performed the Complete Minnesota Dexterity Task, a motor test of 5 tasks to assess unilateral and bilateral dexterity while receiving 2mA of bilateral tDCS to the motor areas of the brain for 20 minutes. The total time it took the participant to complete three trials of each of the 5 tasks was recorded. Results: Performance times were significantly improved in the tDCS condition compared to sham in a unilateral task of dexterity. The average time of completion of the three trials during sham condition was 292 seconds, compared to 264 seconds for the stimulation session, a 2.5% improvement in motor performance. Discussion: Application of tDCS to bilateral motor areas resulted in improvement of performance on a complex, one handed task. It is likely that increasing excitability of motor regions raises the probability of forming stronger and more effective synaptic connections between activated neurons. Additionally, bilateral tDCS may have strengthened interhemispheric connections between the two regions, leading to recruitment of additional interconnected brain regions.

44

IDENTIFICATION OF A NUCLEAR LOCALIZATION SIGNAL (NLS) WITHIN THE PH DOMAIN OF RGNEF

Michael Tavolieri1, Cristian A. Droppelmann

2, Kathryn Volkening

2,3 and Michael J. Strong

3

1Department of Pathology and Laboratory Medicine,

2Molecular Medicine Group, Robarts Research Institute, and

3Department of Clinical Neurological Sciences.

Rho Guanine Nucleotide Exchange factor (RGNEF), encoded by the gene ARHGEF28, is a 190kDA protein. In the context of amyotrophic lateral sclerosis (ALS) RGNEF is a particularly interesting candidate. The mRNA target species destabilized by RGNEF, NEFL, encodes for low-molecular weight neurofilament, a protein which has long been implicated in ALS pathology. As well, histological study of the anterior horn of ALS tissue has shown RGNEF within pathological neuronal cytoplasmic inclusions and an ALS-specific mutation in ARHGEF28 has been identified. Given this evidence, our lab has endeavoured to elucidate the function of RGNEF. RGNEF bears a Pleckstrin Homology (PH) domain, a domain widely conserved domain thought to localize the protein to substrate rich membranes. We identified a nuclear localization signal (NLS) embedded within this region of RGNEF. RGNEF has previously been shown in the nucleus, though until now no NLS has been described. Mutagenesis was perform to create a PH Domain (1083-1201) deleted construct (RGNEF-ΔPH) and an NLS mutated (RGNEF-ΔNLS) construct. These constructs along with full-length RGNEF were transfected in HEK293T cells using Lipofectamine 2000 (Invitrogen). Levels of nuclear localization were determined by two methods: cells were stained by immunocytochemistry and confocal microscopy; or cells fractionation and immunoblotting. cNLS Mapper, an online software, was used to identify the NLS and I-Tasser was used to determine 3-D configuration of the PH.

Both RGNEF-ΔPH and RGNEF-ΔNLS show reduced levels of localization to the nucleus compared to full-length. Cells transfected with full-length showed localization to the nucleus, while RGNEF-ΔPH and RGNEF-ΔNLS showed significantly less expression in the nucleus when examined by confocal microscopy. Fractionation of cellular compartments and immunoblotting showed similar results.

This is the first study to identify a possible mechanism of RGNEF nuclear localization. We have identified a putative NLS located within the PH domain. Modeling software confirms that this region is accessible for interaction with the Importin αβ Pathway.

45

NEURODEGENERATION, MEMORY & ATTENTION

THE FUNCTIONAL ROLE OF NUCLEAR 82-KDA CHAT AND ITS POTENTIAL NEUROPROTECTIVE SIGNIFICANCE

AlQot HE, Rylett RJ Molecular Medicine Research Group, Robarts Research Institute, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada Choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, is a fundamental component of the cholinergic system whose levels and activity are reduced in AD. In humans, ChAT mRNA can translate to 69-kDa ChAT and 82-kDa ChAT, the former favoring cytoplasmic accumulation while the latter primarily resides within the nucleus. Interestingly, this unique nuclear confinement of 82-kDa ChAT is altered in aging, mild cognitive impairment (MCI) and AD, shifting to a more diffuse cytoplasmic distribution. In addition, a recent study from our laboratory demonstrated a gene-regulatory function for nuclear 82-kDa ChAT, influencing genes related to APP metabolism, oxidative stress and inflammation. Collectively, these findings suggest a potential role for nuclear ChAT in neuronal vulnerability and neuroprotection. A comprehensive understanding of the functions of nuclear 82-kDa ChAT is not fully developed. In this regards, this study aims to understand the roles of nuclear ChAT and the physiologic consequences on behavior and neuronal viability/activity in relation to its subcellular distribution. We hypothesize that the expression of nuclear 82-kDa ChAT will alter genes to reduce the amyloidogenic processing of APP and improve cognition and cholinergic function accordingly. Since, 82-kDa ChAT is not endogenously expressed in mice, these studies will use transgenic mouse models expressing human 82-kDa ChAT that have been successfully generated by our laboratory. Behavioral, imaging, biochemical and neurochemical techniques will be implemented to assess cognitive function, subcellular distribution, gene/protein expression and neuronal activity/viability accordingly. If data validate our hypotheses, this can indicate a neuroprotective role for nuclear 82-kDa ChAT thus opening new therapeutic horizons to promote/sustain its role and potentially halt the progression of AD and related dementias.

SPIKE-FIELD COUPLING OF THE ANTERIOR CINGULATE CORTEX AND FRONTAL EYE FIELD IN GOAL-DIRECTED EYE

MOVEMENTS.

Babapoor-Farrokhran S1, Vinck M

2, Womelsdorf T

3, Everling S

1, 4

Neuroscience graduate program, Western University, Canada1; Department of Neurobiology, Yale University, USA

2;

Department of Biology, York University, Canada3; Robarts Research Institute, Canada

4;

Although numerous functional imaging studies in human subjects have shown that the frontal eye fields (FEF) and anterior cingulate cortex (ACC) are co-activated during cognitively demanding saccade tasks, single unit and local field potential (LFP) studies in nonhuman primates have shown important differences in the activity between these two areas. Little is known about the coupling of spiking activity and local field potentials in these areas. Here we investigated the spike-field synchrony of ACC and FEF neurons with the corresponding LFPs recorded at the same area while monkeys performed a memory-guided saccade task and a pro/anti-saccade task. During the delay period of the memory-guided saccade task, ACC units exhibited increased low gamma (30-60Hz) coupling prior to ipsiversive saccades whereas FEF units displayed increased high gamma (60-100Hz) synchrony prior to contraversive saccades. This finding supports the saccade-generating role of the FEF for contraversive saccades and suggests a potential inhibitory role of the ACC. Furthermore, ACC and FEF units showed a different pattern of coupling during the preparatory period of the pro/anti-saccade task. ACC units exhibited an increased theta band (3-8Hz) coupling in pro- vs. anti-saccade trials and no difference across gamma band coupling. Similar to the ACC units, FEF units exhibited increased coupling to the theta band for pro-saccades and an increased gamma band (60-100Hz) coupling of FEF units for anti-saccades compared to pro-saccades. These findings support a role of the theta band in rule maintenance, whereas the gamma band may be more involved in motor-related functions. Taken together, our results indicate that theta and gamma oscillations have different roles in cognitive processes and these frequency bands are modulated differently across ACC and FEF.

46

IDENTIFYING THE GENETIC BASIS OF NEURODEGENERATION IN 75 PATIENTS USING A TARGETED RESEQUENCING PANEL

Dilliott AD1,2

, Farhan SMK1,2

, Liang E, McIntyre AD1, Cao H

1, Robinson JF

1, Bulman DE

3, Rogaeva E

4, St. George-Hyslop P

4,

Strong MJ1,5

, and Hegele RA1,2

for the ONDRI Investigators Robarts Research Institute, Western University

1, Department of Biochemistry, Western University

2, CHEO Research

Institute, Faculty of Medicine, University of Ottawa3, Tanz Centre for Research in Neurodegenerative Diseases, The

University of Toronto4, Department of Clinical Neurological Sciences, Western University

5.

The Ontario Neurodegenerative Disease Research Initiative (ONDRI) is a multidisciplinary study aimed to characterize five neurodegenerative diseases: 1) Alzheimer’s disease and/or mild cognitive impairment (AD/MCI); 2) amyotrophic lateral sclerosis (ALS); 3) frontotemporal dementia (FTD); 4) Parkinson’s disease (PD); and 5) vascular cognitive impairment (VCI). We used a custom-designed, next-generation sequencing based disease-specific panel to elucidate the genetic basis of the diseases in 75 ONDRI participants. DNA from participants was extracted from blood and sequenced using a next-generation sequencing based panel, ONDRISeq. A bioinformatics workflow was then used to obtain non-synonymous, rare variants. Over 70% of participants carried at least one genetic variant, with a total of 91 unique non-synonymous, rare variants identified in 44 of 80 genes represented on ONDRISeq. Three particular genes were more frequently mutated, namely: 1) LRRK2, 2) NOTCH3, and 3) DNAJC13. Additionally, four ONDRI participants were homozygous for the APOE E4/E4 genotype, which confers risk for late-onset AD. The APOE genotypes of the 75 individuals were determined using ONDRISeq and were independently validated using a TaqMan allelic discrimination assay. To test whether candidate variants observed are disease causing, they will need to be functionally validated. Methods described in this paper will also be reproduced on all 600 participants of the ONDRI study. Novel variants and genes with multiple disease associations allow us to further understand mechanisms of these diseases and allow for the development of new therapeutic targets.

VAST MAJORITY OF FOREBRAIN CHOLINERGIC NEURONS IS ABLE TO RELEASE GLUTAMATE AS ITS SECOND EUROTRANSMITTER

DURING DEVELOPMENT BUT ONLY SOME MAINTAIN THIS ABILITY IN ADULT BRAIN

Janickova H, Guzman MS, Al-Onaizi MA, Rosborough K, Goncalves DF, Rapps J, Prado VF, Prado MAM. Robarts Research Institute, Graduate Program in Neuroscience, Department of Physiology and Pharmacology, Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON Cholinergic interneurons (CINs) in the striatum and cholinergic neurons in the basal forebrain (BF) are the main sources of cholinergic signaling in the forebrain. While virtually all striatal CINs release also glutamate thanks to their expression of vesicular glutamate transporter 3 (Vglut3), this ability is thought to be more limited in the cholinergic neurons in the BF. We hypothesized that deletion of vesicular acetylcholine transporter (VAChT) from neurons expressing Vglut3 will result in specific deletion of acetylcholine release in the striatum while the BF cholinergic neurons remain intact. We investigated expression of Vglut3 in cholinergic neurons since early development in mice expressing Cre under Vglut3 promoter. We also measured VAChT levels in the striatum and in the cortex, hippocampus and amygdala in Vglut3-Cre-VAChTfx/fx mice to investigate the specificity of VAChT deletion. Finally, we characterized Vglut3-Cre-VAChTfx/fx mice behaviourally. Contrary to our hypothesis, the Vglut3 expression was high in the BF and we found significant decrease of VAChT protein levels in all investigated brain areas. In addition, the behavioural deficit of Vglut3-Cre-VAChTfx/fx mice was more profound than expected and included alterations of locomotion, sensitivity to psychostimulants and antipsychotics, weight and sleep pattern, cognitive impairment and impulsivity. We conclude that expression of Vglut3 in forebrain cholinergic neurons is more widespread than previously thought and dysfunction of Vglut3 expressing cholinergic neurons leads to significant impairment of several behavioural and cognitive domains.

47

STRIATAL REGULATION BY ACETYLCHOLINE AND GLUTAMATE CO-TRANSMISSION

Kljakic O1,2

, Janickova H1, Sakae DY

4, Favier M

4, Mestikawy SE

4, Prado MAM

1,2,3, Prado VF

1,2,3

Robarts Research Institute1, Departments of Anatomy and Cell Biology2, and Physiology and Pharmacology3, Western University; Department of Psychiatry4, McGill University Cholinergic tonically active neurons (TANs) are neurons thought to be critical for information processing and modulation of the striatum. TANs co-express vesicular acetylcholine transporter (VAChT) and vesicular glutamate transporter 3 (VGLUT3) and thus can store and release acetylcholine (ACh) and glutamate (Glut). Recent studies suggest that the balance between ACh and Glut is critical for controlling striatal-dependent behaviour. We hypothesize that ACh and Glut differentially regulate striatal function, where a balance favouring ACh may facilitate cognition processing and a balance favouring Glut may control reward behaviour. We selectively eliminated VAChT or VGLUT3 in TANs to generate mice with an altered striatal balance of ACh and Glut (D2CreVAChTfx2, D2CreVGLUT3fx2). These mouse lines were tested for behaviours regulated by the striatum and we found that D2CreVGLUT3fx2, but not D2CreVAChTfx2 mice were hyperactive and D2CreVAChTfx2, but not D2CreVGLUT3fx2 mice had an anti-depressive-like phenotype and required more sessions to reach criteria during the reversal phase of the pairwise discrimination and reversal task. These results suggest that ACh and Glut have different impacts on key behaviours regulated by the striatum. Mice unable to release ACh from TANs display reduced depressive-like behaviours and lack cognitive flexibility whereas mice unable to release Glut were hyperactive. Ultimately, our experiments indicate that the ability of TANs to co-release neurotransmitters is critical to regulating the complex and diverse behavioural functions of the striatum. Acknowledgements: Supported by CIHR, Brain Canada Project

THE ROLE OF NEUROINFLAMMATION IN A MOUSE MODEL OF ALZHEIMER’S DISEASE

Natalie Kozyrev1, Shawn Albers

1-2, Shuxin Zhang

1, Vania F. Prado

1-3, R. Jane Rylett

1-2, Gregory A. Dekaban

1,4

1Molecular Medicine Research Group, Robarts Research Institute,

2Departments of Physiology and Pharmacology,

3Anatomy and Cell Biology,

4Microbiology and Immunology, University of Western Ontario, London, ON, Canada.

Neuroinflammation in the course of Alzheimer’s disease (AD) serves to exacerbate AD and promote disease progression. Aβ peptides and plaques are highly cytotoxic and trigger chronic inflammation in the cellular environment, contributing to the infiltration of macrophages within the brain. Upon activation, macrophages release pro-inflammatory cytokines that promote further neurotoxicity and apoptosis associated with AD. A major impediment to investigating neuroinflammation involving macrophage activity is the inability to discriminate between CNS-resident microglia-derived macrophages (mMΦ) and hematogenous monocyte-derived macrophages (hMΦ) in a temporal and spatial manner as these two populations of macrophages are morphologically and phenotypically similar in the activated state. Therefore, the overall goal of this research is to distinguish between hMΦ and mMΦ, and determine their respective roles in chronic inflammation associated with AD progression. The lys-EGFP-ki transgenic mouse line that enables the discrimination between mMΦ and hMΦ was crossed with the 5xFAD transgenic mouse line. The F1 offspring (LysFAD+) mice exhibited aggressive AD pathology of 5xFAD mice while enabling the spatial and temporal discrimination of mMΦ and hMΦ in the brain. Plaque load was measured using ELISA for Aβ42 in the cortex and hippocampus and spatial learning and memory was assessed using the Morris Water Maze (MWM). Levels of Aβ42 were significantly higher in the brains of male and female LysFAD + compared to littermate controls at 1.5-7 months. Additionally, LysFAD+ mice demonstrated spatial learning and memory deficits as measured by the MWM, at 5 and 7 months of age with deficits more pronounced in females. Based on these results, the time of onset of the behavioral deficits and accumulation of Aβ42 in the brains of LysFAD+ mice was identified to be 5-7 months. Therefore, immunostaining with specific antibodies for GFP, Iba1 (marker of microglia/macrophages) and Aβ plaques will be performed on frozen brain sections of these 5-7 month old mice in order to determine whether the macrophages surrounding Aβ plaques are hematogenous in origin or microglia-derived.

48

THE ROLE OF THE TPR1 DOMAIN FROM STRESS-INDUCIBLE PHOSPHOPROTEIN 1 (STIP1) IN CELLULAR RESILIENCE

Rachel Lackie1,4

, Jose Lopes1,4

, Flavio H. Beraldo4, Jue Fan

4, Vilma R. Martins

5, Vania F. Prado

1,2,3,4, Marco. A.M. Prado

1,2,3,4

Program in Neuroscience1, Department of Physiology and Pharmacology

2, and Department of Anatomy & Cell Biology

3,

Schulich School of Medicine and Dentistry, Molecular Medicine4, Robarts Research Institute, University of Western Ontario,

London ON. Department of Molecular and Cell Biology, International Research Center, A. C. Camargo Cancer Center and National Institute for Translational Neuroscience, São Paulo, Brazil

5.

Stress-inducible phosphoprotein-1 (STIP1) has two major functions; it works as an extracellular signalling molecule by interacting with the prion protein (PrPC), and is also a critical co-chaperone that regulates the activities of the Hsp70/Hsp90 chaperone machinery. STIP1 contains three tetratricopeptide repeat domains (TPR1, TPR2A and TPR2B) and two aspartate proline rich domains (DP1 and DP2). The TPR2A domain forms the major binding site for Hsp90, whereas Hsp70 can bind to both TPR1 and TPR2B. In yeast, TPR2A and TPR2B seem essential for STIP1 cochaperone functions, but much less is known about the roles of TPR1. In addition to modulating Hsp70 binding, recent results from our laboratory suggest that both TPR2A and TRP1 can bind to PrPC. Here we investigated the role of TPR1 in vivo by generating a mouse line lacking this domain (STIP1ΔTPR1). Mouse embryonic fibroblasts (MEFs) were isolated from wild-type (WT), heterozygous, and homozygous STIP1ΔTPR1 pups. In order to understand the role of TPR1 in proper development and growth, cell viability was assessed using the Live/Dead Kit, BrdU immuno-labeling to quantify proliferative activity, and genotyping and counting of pups. Our preliminary results indicate a role for TPR1 in development. Reduced cellular viability and resilience may be a result of a reduction in PrPC-STIP1 interaction or ternary complex formation between Hsp70-Hsp90 and chaperone activity. Further experiments will be conducted to elucidate the functional role of TPR1 in STIP1 co-chaperone function, stress response, and PrPC-dependent protective effects.

KETAMINE-INDUCED CHANGES IN PREFRONTAL CORTICAL OSCILLATORY ACTIVITIES DURING A WORKING MEMORY TASK IN

MONKEYS

Ma L2, Skoblenick K

3, Everling S

1,2,3,4

Robarts Research Institute1, Departments of Physiology and Pharmacology

2 and Anatomy and Cell Biology

3, Brain and Mind

Institute4, Western University

Acute injection of subanesthetic dose of ketamine in non-human primate is an effective model for creating schizophrenia-like symptoms, such as deficits in working memory. We trained three rhesus monkeys to perform a rule-based working memory task involving pro- and anti-saccades, and recorded both spiking activity and local field potentials from the dorsolateral prefrontal cortex both before and after ketamine injection. Previously we reported that ketamine injection negatively impacted the maintenance and the application of the correct task rule. In patients with schizophrenia, either abnormal enhancement or reduction in gamma band activity depending on the working memory load was correlated with impaired performance. Meanwhile, low frequency oscillations are often attenuated in patients. Here we report enhanced delay-related low gamma activities (30 to 60Hz) accompanied by reduced power in the beta band (13 to 30Hz) during the delay period preceding correct responses following ketamine treatment. To understand how ketamine affects the rule information encoded in oscillatory activity, we calculated the task selectivity, defined as the difference between oscillatory amplitudes associated with the two rules, divided by the sum of these amplitudes. In two of the three animals, task selectivity was significantly reduced in both beta and low gamma frequency ranges following ketamine injection. These results support an important role of prefrontal oscillatory activities in information processing in working memory.

49

IONTOPHORESIS OF MUSCARINIC AGONISTS ONTO PREFRONTAL CORTICAL NEURONS ENGAGED IN A RULE-CONTINGENT

SACCADIC TASK

Alex J. Major1, Susheel Vijayraghavan

2, and Stefan Everling

2,3

Graduate Program in Neuroscience1, Department of Physiology and Pharmacology

2, Robarts Research Institute

3, Western

University The dorsolateral prefrontal cortex (DLPFC) has a well-studied role in executive function, including attention, working memory (WM), and rule-contingent processing. These functions are modulated by the ascending cholinergic neuromodulatory system. Cholinergic dysfunction is thought to be a contributing factor in the cognitive deficits of Alzheimer’s disease and schizophrenia. Further, cholinergic denervation of the DLPFC in macaques or systemic pharmacological blockade of muscarinic receptors disrupts WM processing. Local muscarinic modulation of rule-contingent mnemonic processing in DLPFC has not been examined in detail hitherto. We have previously shown that the muscarinic antagonist scopolamine suppresses rule-memory activity in prefrontal neurons during a rule-cued pro- and antisaccade task. Two receptor classes mediate muscarinic actions: the M1 receptor family, predominantly expressed on pyramidal spines, and the M2 receptor family, located presynaptically. In this study, we examined the in vivo effects of three iontophoretically applied cholinergic agonists on macaque DLPFC neurons: nonspecific cholinergic agonists carbachol and Oxotremorine-M, and M1-preferring agonist McN-A-343. Preliminary data indicate that all three agonists had both excitatory and inhibitory effects on DLPFC neuronsencoding rule selectivity. These are the first results to report the effect of local muscarinic stimulation in primate DLPFC rule-contingent WM circuits, and support muscarinic receptors as pharmacological targets for treatment of cognitive deficits.

ASSESSING LONGITUDINAL COGNITIVE DEFICITS BETWEEN SEXES IN A MOUSE MODEL OF ALZHEIMER’S

DISEASE USING TOUCHSCREEN TASKS

Justin Mels1,2

, Flavio H. Beraldo1, Daniel Palmer

5, David Wasserman

5, Matthew F. Cowan

1, Vania F. Prado

1,2,3,4, Boyer D.

Winters5, Marco A. M. Prado

1,2,3,4


2Graduate Program in Neuroscience,


4Department of Anatomy and Cell Biology, Schulich School of Medicine, University of Western Ontario, London, ON,

N6A5K8, 5Department of Psychology and Neuroscience Program, University of Guelph, Guelph, ON, N1G 2W1

Rapid onset of amyloidosis is a hallmark of several mouse models of Alzheimer’s Disease (AD). At the behavioural level, disruption of memory and attention has been shown in some mouse models of AD, but whether attention deficits are observed in all mouse models of AD and how early in the disease is unclear. The APP/PS1 model of AD exhibits a slower progression of amyloidosis with Aβ deposition being reported to initiate at 3 months of age, whereas plaques are detected after 6-7 months. However, the initial onset of behavioural deficits in this model has been widely variable and inconsistent in different laboratories. The earliest cognitive deficits have been reported as early as 6 months in spatial memory. However, these findings have failed to be consistently replicated. Furthermore, it is unclear if there are sex differences in the rate of cognitive decline in this mouse model.

This study focuses on testing the cognitive function of both male and female APP/PS1 mice using automated touchscreen tasks. We used the 5- Choice Serial Reaction Time Task (5-CSRTT) and a Pairwise Discrimination (PD) task, which assess attention and visuomotor learning and reversal learning, respectively, to test the mice at 4, 7, 10 and 15 months of age. To address the reproducibility of the results, different cohorts of mice were tested at two locations (University of Western Ontario and University of Guelph). At the first three time points, APP/PS1 male or female mice did not show cognitive deficits compared to wild-type mice in both test locations. These results point to attention and cognitive flexibility being intact at 10 months. Future work will test cognitive function of 15 month-old mice and whether the onset of behavioral deficits occurs at this age.

Acknowledgements: Supported by the Weston Brain Institute

50

82-KDA CHOLINE ACETYLTRANSFERASE ALTERS THE EXPRESSION OF SYNAPSE FORMATION RELATED GENES

Warren Winick-Ng1,2

and R. Jane Rylett1,2

1Department of Physiology and Pharmacology, and

2Molecular Medicine Research Group, Robarts Research Group,

University of Western Ontario, London, Ontario, Canada The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that localizes to nuclei of cholinergic neurons, with this localization reduced in elderly individuals and Alzheimer’s disease (AD) subjects. We previously showed that 82-kDa ChAT associates with chromatin at synapse and cell membrane related genes by chromatin immunoprecipitation (ChIP)-sequencing. Using this dataset in the present study, we determined that 82-kDa ChAT ChIP-associations with synapse-related genes were enriched with binding-motifs for the nuclear factor of activated T-cells (NFAT) transcription factor, which targets synapse and membrane related genes and has an altered cellular distribution in AD. For several of these genes, we found significantly higher mRNA and protein steady-state levels in human SH-SY5Y cells stably expressing 82-kDa ChAT, compared to cells expressing an empty vector. Two of these genes encoded proteins related to synaptogenesis in cholinergic neurons, neuregulin 1 (NRG1) and the metabotropic glutamate receptor 5 (mGlu5). Long-term mGlu5-related signal transduction is associated with cholinergic synapse formation, while NRG1 cleaved within the cell membrane to produce a small 6 kDa extracellular peptide that also promotes long-term synapse formation and maintenance. In cells expressing 82-kDa ChAT, we found higher levels of this NRG1 extracellular peptide released into cell media samples, compared to empty vector. We are currently using conditioned media from 82-kDa ChAT expressing cells to assess synapse development and cell morphology-related changes in vector expressing cells. We are also using transgenic mice expressing 82-kDa ChAT to assess changes to cholinergic synapse formation in vivo. These experiments suggest that 82-kDa ChAT may play a developmental role in synapse formation and maintenance of human cholinergic neurons, with implications for the etiology of AD when cholinergic synapses degenerate, and the localization of 82-kDa ChAT is altered.

82-KDA CHOLINE ACETYLTRANSFERASE EXPRESSION ALTERS LEVEL OF FE65 AFTER Β-AMYLOID EXPOSURE

Shuxin Zhang1,3

, Warren Winick-Ng2,3

, and R. Jane Rylett2,3

1West China School of Medicine, Sichuan University, Chengdu, China;


3Molecular Medicine Research Group, Robarts Research Group, University of Western Ontario, London, Ontario, Canada

The 82-kDa choline acetyltransferase (82-kDa ChAT) protein is localized in the nuclei of cholinergic neurons of human and non-human primates. This localization has been shown to decrease in aging individuals and Alzheimer’s disease (AD) patients. We previously found that 82-kDa ChAT is associated with multiple genomic DNA sites in human SH-SY5Y cells using chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). In the ChIP-seq data, we identified that 82-kDa ChAT was associated with the amyloid beta (A4) precursor protein-binding, family B, member 1 (APBB1) gene, but this association was lost after acute treatment of 100 nM oligomeric β-amyloid1-42 (Aβ1-42) for 4 hours. APBB1 encodes the Fe65 protein, which was shown previously to promote Aβ production in SH-SY5Y cells. In the present study, we investigated the effect of Aβ1-42 exposure on the mRNA steady-state level of the APBB1 gene in SH-SY5Y cells using real-time PCR. After 24 hours of exposure to 100 nM Aβ1-42, we found that the mRNA steady-state level of APBB1 was increased in cells expressing an empty vector, but that this increase was prevented in cells expressing 82-kDa ChAT. We are currently determining the effect of Aβ1-42-exposure on Fe65 protein level in SH-SY5Y cells expressing empty vector or 82-kDa ChAT using western immunoblotting. In cells expressing 82-kDa ChAT, there was a trend towards a reduction in Fe65 protein levels after 48-hour exposure of 100 nM Aβ1-42, with a trend towards increased Fe65 levels in vector-expressing cells. Our current data suggests that 82-kDa ChAT alters the cellular response to Aβ1-42 exposure, by mediating the levels of APBB1 and Fe65. Future studies will be important to provide further evidence for the role of Fe65 in the cellular response to Aβ1-42 exposure and elucidate the mechanism in the effect of 82-kDa ChAT on the APBB1 gene.

51

NEUROLOGICAL DISORDERS & EPILEPSY

FUNCTIONAL CONNECTIVITY OF THE SUPERIOR COLLICULUS IN MACAQUES AND MARMOSETS INVESTIGATED WITH RESTING-STATE ULTRAHIGH-FIELD FMRI

Maryam Ghahremani1,2

, R. Matthew Hutchison3, Joe S. Gati

2, Kyle Gilbert

2, Ravi S. Menon

1,2, Stefan Everling

1,2,4

1Graduate Program in Neuroscience, University of Western Ontario,

2Robarts Research Institute, University of Western

Ontario, London, Ontario, Canada, 3Center for Brain Science, Harvard University, Cambridge, MSA, USA,

4Department of

Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada Interest in common marmoset monkeys is growing rapidly as it is poised to become the leading candidate transgenic primate model. In contrast to the established Old World macaque, little is known about functional organization of the saccade circuitry in these New World primates. Here we used resting-state fMRI from 12 macaques and 4 marmosets, while anesthetized, to examine functional connectivity of the superior colliculus(SC), a major node in the neural network underlying primate saccade control. Macaque data were obtained on a 7T MR scanner and marmoset data on 9.4T, both with custom-built transmit/receive coils. In both species, the seed region analysis revealed functional connectivity of a frontoparietal network with the SC. In macaques, the network overlapped with previously described frontal eye fields connectivity patterns which included the intraparietal sulcus, dorsolateral prefrontal cortex, anterior cingulate cortex, and supplementary eye fields. Visualization of marmosets' cortical functional connectivity on a surface-based registration revealed the strongest connectivity in frontal areas 6DC, 6DR, 8C, 8B, 8aV, 8aD, 46D, parietal areas PG, PGM, PO, MIP, LIP, and temporal areas MT, MST, FST. In addition, the independent component analysis successfully extracted eight resting state networks in marmosets, greatly overlapping with corresponding networks in macaques. Frontoparietal network was among those identified, coinciding with acquired SC-connectivity maps. The results support an evolutionarily preserved frontoparietal system providing a starting point for invasive neurophysiological studies in marmosets.

EFFECTS OF PHOSPHORYLATION ON NEUROSTEROID-INDUCED MODULATION OF GABAA RECEPTOR CURRENTS

Jeong J1 and Poulter MO

1,2

1Departments of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University

2Molecular Medicine Research Group, Robarts Research Institute, Western University

Neurosteroids are well known modulators of GABAA receptor activity. GABA receptor mediated mIPSCs recorded from pyramidal neurons cultured for >14 days decay in two phases, one lasting about 10 ms (τ1) and second lasting about 50 ms (τ2). Neurosteroids like THDOC affect mIPSCs usually by altering (both shortening and prolonging) these decay rates. However, previous studies have reported highly variable effects of neurosteroids on the decay and amplitude of mIPSCs. We have hypothesized this variability may be dependent on phosphorylation state of GABAA receptors. Here we have examined the activity of the neurosteroid THDOC on GABA mediated mIPSCs after treatment with compounds that activate various kinases. In general, we found that kinase activity altered the efficacy of neurosteroid activity although each kinase had differing and specific effects. Activation of PKC using phorbol ester PMA prolonged τ2 to a less extent than in untreated recordings (300 % versus 225%). However, the effect on τ1 was smaller in THDOC/PMA treated cells resulting in about the same change of charge transfer in both conditions. THDOC, after activation of PKA activity, increased the amplitude of mIPSCs and prolonged the duration of τ2, increasing transfer by about 300%. Finally, THDOC after activation of the TrkB by DHF reduced mIPSCs amplitude, increased the rate of τ1 while prolonging the τ2. The combination of these three outcomes resulted in no change in the charge transfer. These data show that kinase activity greatly determines the activity of THDOC on GABAA receptor mediated mIPSCs.

52

THE COMMON MARMOSET AS A MODEL ANIMAL FOR INVESTIGATIONS OF OCULOMOTOR NEUROPHYSIOLOGY

Johnston K1, Everling S

1,2,

Department of Physiology and Pharmacology1, Western University, Robarts Research Institute

2

The common marmoset (Callithrix jacchus) is a New World primate that shows considerable promise as a model animal for neuroscience research. The first primate transgenic lines have been developed in this species, making possible the integration of genetic and neurophysiological tools in primates. Practical advantages, such as a lissencephalic cortex that allows recording with linear and planar electrode arrays, and a clear extant knowledge of the anatomy and physiology of the visual system in this species further support its value. The oculomotor system is the most thoroughly understood sensorimotor system in the brain, and provides a window into neurodegenerative and neuropsychiatric disorders. The current gold standard model for electrophysiological and fMRI investigations of this system is the rhesus macaque, however few studies have evaluated the behaviour of the common marmoset in oculomotor tasks. To address this, we compared the behaviour of the common marmoset and rhesus macaque on a well-established oculomotor phenomenon - the gap effect. This effect refers to the decrease in saccadic reaction times observed when the fixation spot is extinguished prior to the appearance of a target in saccade tasks. We trained, under identical viewing conditions, a common marmoset and two rhesus macaques to make saccades to peripheral targets for liquid reward. In a “step” condition, an initial fixation spot was extinguished simultaneously with the appearance of the target. In a “gap” condition, the fixation spot was extinguished 200ms prior to target appearance. Consistent with previous observations in both humans and macaques, we observed reductions in reaction times for both the marmoset and macaques in the gap as compared to step condition. These reductions were of similar magnitude, suggesting that conserved oculomotor circuits mediate this effect in both primate species, and supporting the use of the common marmoset as a model for investigations of oculomotor neurophysiology.

INVESTIGATING SPATIAL PATTERNS OF WHITE MATTER INTEGRITY IN TEMPORAL LOBE EPILEPSY USING BUNDLE-SPECIFIC

DIFFUSION MRI AND HISTOLOGY

Loxlan W Kasa a, b

, Terry Peters a, b, c

, Ali Khan a, b, c

a Imaging Research Laboratories, Robarts Research Institute, London, Ontario, Canada

b Biomedical Engineering, Western University, London, Ontario, Canada

c Department of Medical Biophysics, Western University, London, Ontario, Canada

Temporal lobe epilepsy (TLE) is the most common form of drug-resistant epilepsy, affecting approximately 60,000 Canadians, and is often characterized by seizures originating from the hippocampus. Diagnosing epilepsy using electroencephalography (EEG) is the traditional approach, but can be very invasive. MR imaging is an important non-invasive neuroimaging test for these patients, but unfortunately about 30% of patients have normal MRI, making it very difficult to diagnose and plan epilepsy surgery, resulting in poor outcomes. Diffusion tensor imaging (DTI) techniques have shown increased sensitivity in identifying epileptogenic tissue or networks, with TLE patients displaying water diffusion abnormalities (increases in mean diffusivity) in specific white matter structures connected within and beyond the epileptogenic temporal lobe. However, the neurobiological substrate for the changes in the diffusivity in and around epileptogenic tissue are not known, and could be related to demyelination, gliosis, or neuronal integrity. The aim of this project will be to investigate white matter (WM) bundles connected to the hippocampus using advanced diffusion MR imaging and image analysis techniques, and study the variation in diffusion using profiles along the bundles. First we will segment out the two fiber bundles of interest (cingulum and fornix) from the tractography data and calculate variations in diffusion parameters through the length of each bundles. Secondly we will validate our results using the corresponding patient’s histology samples retrieved from lobectomy and spatially-registered using an existing MRI-histology framework. The expected results will aid our understanding of the neurobiological changes that take place which influence brain microstructure, and potentially provide histological validation of a biomarker for TLE based on diffusion profiling in WM bundles.

53

PGE2 DRIVES NEUROENDOCRINE STRESS RESPONSE THROUGH ATTENUATION OF THE GABA RELEASE

Zahra Khazaeipool2, and Wataru Inoue

1,2

1 Robarts Research Institute,

2Department of Physiology and Pharmacology, University of Western Ontario

Immune-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to a rise of circulating glucocoriticoids, powerful anti-inflammatory hormone. This negative-feedback immune regulation is mediated by prostaglandin E2 (PGE2) produced in the brain during inflammation. However, how PGE2 elicits neuronal mechanisms leading to the HPA axis activation remains unclear. The initial step of the HPA axis activation is the release of corticotropin releasing hormone from parvocellular neuroendocrine cells (PNCs) in the paraventricular nucleus of the hypothalamus (PVN). Here, we report that PGE2 decreases GABA-mediated inhibitory synaptic transmission to PNCs, by using whole cell patch clamp recordings in brain slices in rats. Bath application of the PGE2 (0.01−100 μM) dose-dependently decreased the amplitude of the evoked inhibitory post synaptic currents (eIPSCs) with maximum effect achieved at 10 μM. The effect of PGE2 was long lasting (> 25 min after wash out) and was also accompanied by an increase in paired pulse ratio. Moreover, PGE2 decreased the frequency of the spontaneous IPSCs, without showing any effect on the amplitude. This is consistent with the model that PGE2 decreases the probability of the GABA release from presynaptic termini. Finally, application of the EP3 agonist (sulprostone, 1 μM) mimicked the effects of the PGE2, whereas EP3 antagonist (L798,106) blocked PGE2 effects. Taken together, we propose that PGE2-EP3 signaling decreases GABA release onto PNCs, and thereby disinhibits (i.e. excites) PNCs. This data could provide a possible mechanism for the HPA axis activation during inflammation.

EFFECTS OF TRANSCRANIAL MAGNETIC STIMULATION OF FEF ON NEUROPHYSIOLOGICAL ACTIVITY IN CONTRALATERAL FEF

Sebastian J. Lehmann1, 2, 3

, Brian D. Corneil1, 2, 3


1, The Brain and Mind Institute

2, Department of Physiology and Pharmacology

3

Transcranial magnetic stimulation (TMS) allows non-invasive perturbation of neural activity, induced by a rapidly changing magnetic field. Both single pulse and repetitive TMS (rTMS) have been shown to modulate behavioural output. Despite being considered an important methodology in cognitive neuroscience and offering a potential treatment for neurological disorders, a precise understanding of the effect of TMS on neural activity in an interconnected brain network, and how such effects influence behavior, is largely lacking. To overcome this gap, we are developing an animal model of TMS, focusing on the oculomotor network in non-human primates (NHPs). Previous work has shown that delivering single-pulse TMS to the frontal eye fields (FEF) evokes feed-forward neck muscle responses, likely through the downstream superior colliculus, that can be used to localize optimal TMS position over the frontal cortex. Now having a reliable means of delivering TMS to the FEF, we are able to apply TMS either in a rapid pattern (e.g., single- or double-pulse TMS delivered at a precise time during a behavioural task), or in a repetitive pattern (e.g., 1-Hz TMS for 10-15 minutes). Our current focus is to examine the effects of TMS of one FEF on spiking and local field potential activity in the contra-lateral FEF. NHPs are performing either an intermixed pro- and anti-saccade task, or a memory-guided saccade task. In several recording sessions, we tested the effects of TMS on neurophysiological activity and behaviour after-single pulse TMS at various times of task execution, or by investigating the effects of rTMS in a block design. We present preliminary results from the intermixed pro- and anti-saccade task, which revealed a diversity of effects on single unit spiking activity, further contributing to optimization of our experimental approach.

54

NEURONAL CORRELATES FOR NEUROENDOCRINE ADAPTATION TO REPEATED STRESS.

Sara Matovic1,2

, Eric Salter2, and Wataru Inoue

1,2

Graduate Program in Neuroscience1, Robarts Research Institute


Exposure to a stressor activates the hypothalamic-pituitary-adrenal (HPA) axis resulting in a surge of circulating glucocorticoids. This generalized neuroendocrine response can diminish (i.e. adapt) when a same stressor is repeatedly presented. Here, we report neuronal correlates for this physiological learning. Neuroendocrine neurons that synthesize corticotropin-releasing-hormone (CRH) in the paraventricular nucleus of the hypothalamus (PVN) form the apex of the HPA axis; these neurons releases CRH in an activity-dependent manner. By using patch clamp electrophysiology in brain slices, we found that the intrinsic excitability of PVN CRH neurons was decreased following repeated exposures to restraint stress in mice (1 h/day, 21 consecutive days). Specifically, we found that the time to elicit an action potential in response to depolarizing current injections was significantly delayed in repeatedly stressed mice compared to naïve controls: this stress-induced change was reversed by a potassium channel blocker 4-aminopyridine (4-AP). Furthermore, the repeated stress decreased the frequency of tonic action potential firing during a large current injection. Interestingly, this stress-induced change was insensitive to 4-AP but was completely reversed by blocking Ca2+ signaling in the postsynaptic neurons by including a Ca2+ chelator BAPTA in the recording solution. Thus, at least two dissociable mechanisms were involved to decrease the intrinsic excitability of CRH neurons following repeated restraint stress. Hyper-activity of the HPA axis contributes to stress-induced psychiatric disorders such as depression; therefore, understanding proper HPA axis adaptation can help us understand the etiology of such disorders.

THALAMIC STRUCTURAL PARCELLATION IN THE QUANTITATIVE INVESTIGATION OF TLE PATIENTS.

Brendan Santyr1, Jonathan Lau

2, Jorge Burneo

2, Seyed Mirsattari

2, Sandrine de Ribaupierre

2, David Steven

2, Andrew

Parrent2, Keith MacDougall

2, Ali Khan

1,3

Schulich School of Medicine and Dentistry1, Departments of Clinical Neurological Sciences


3,

Western University Introduction: Morphometry and connectivity studies targeting the thalamus have revealed specific patterns of atrophy and deafferentiation in patients with temporal lobe epilepsy (TLE). These studies relied on thalamic parcellation using probabilistic tractography. Investigating thalamic connectivity to smaller or less-connected regions could allow for more precise localization of abnormalities. In this work we propose a connectivity map normalization procedure, allowing us to compare thalamic connected volumes for a wider range of cortical and limbic structures. Methods: Patients (N=23) with drug-resistant TLE (9 LTLE, 14 RTLE) were scanned using MRI and compared with 34 healthy controls. The Harvard-Oxford atlas was modified to create 14 target volumes in the temporal lobe. Probabilistic tractography (FSL) was used to generate thalamic connectivity maps for each target segmentation. A normalization procedure was devised based upon the observed power-law relationship in connectivity map histograms. This allows estimating the extent of connectivity while removing the overall magnitude of connectivity. These masks were used to obtain mean T1, T2, FA and MD. Results/Conclusions: Our exploratory analysis revealed multiple volumetric and quantitative T1 related differences between TLE patients and controls. Structural aberrancies found in thalamotemporal networks could eventually be useful biomarkers of disease state.

55

PULMONARY HEALTH

MEASUREMENT OF ASTHMA TREATMENT RESPONSE USING FREE-BREATHING 1H VENTILATION MRI

Capaldi DPI1,2

, Sheikh K1,2

, Svenningsen S1,2

, Kirby M3, Coxson HO

3, McCormack DG

4, Parraga G

1,2


2 and Department of Medicine - Division of Respirology

4,

Western University; UBC Centre for Heart Lung Innovation 3, University of British Columbia

Pulmonary functional imaging using inhaled hyperpolarized gas MRI has been used to evaluate asthma following methacholine challenge (MCh), showing ventilation defects that respond to MCh and salbutamol. Fourier decomposition (FDMRI) of conventional anatomical 1H MRI acquired during free-tidal breathing patients over the course of a few minutes has emerged as a non-contrast MRI method to generate ventilation and perfusion images by exploiting non-rigid registration and free-breathing pulmonary MRI. Here our objective was to evaluate asthmatics using FDMRI with direct comparison to hyperpolarized noble gas MRI following methacholine and salbutamol. We hypothesized that the ventilation defects measured using FDMRI would increase after a methacholine challenge and would decrease after salbutamol administration. We also thought that FDMRI ventilation defects would be quantitatively and spatially related to inhaled-gas-MRI ventilation abnormalities. In this proof-of-concept study, three poorly-controlled asthmatics provided written informed consent to pulmonary function tests, hyperpolarized 3He MRI, and dynamic free tidal-breathing 1H MRI. Ventilation defects for both 3He MRI and FDMRI significantly increased following MCh (n=3, FEV1=92±12%pred; baseline: VDPHe=3.6±1.5%, VDPFD=0.7±0.5%; methacholine: VDPHe=9.0±5.7%, VDPFD=5.0±3.6%) and responded post-salbutamol (salbutamol: VDPHe=3.7±2.9%, VDPFD=1.2±0.8%). In this proof-of-concept study, we showed that FDMRI can be safely and rapidly acquired in poorly-controlled severe asthmatics to directly quantify response to methacholine and salbutamol. This is important because for the first time we show that conventional MRI can be used to generate ventilation maps that identify the abnormal lung functioning segments that contribute to or explain poor control.

TESTING THE FLETCHER-PETO ASSUMPTIONS USING PULMONARY IMAGING BIOMARKER LONGITUDINAL MEASUREMENTS

Eddy RL1,2

, Kirby M3, Pike D

1,2, Sheikh K

1,2, Paulin GA

1, McCormack DG

4 and Parraga G

1,2

Robarts Research Institute1, Departments of Medical Biophysics, Western University, London

2 UBC Centre for Heart Lung

Innovation, St. Paul’s Hospital, University of British Columbia, Vancouver3, Division of Respirology, Department of Medicine,

Western University, London4

The change in the forced expiratory volume in one second (FEV1) is universally-accepted as a way to measure lung function decline since the landmark findings of Fletcher and Peto. Inhaled-gas magnetic resonance imaging (MRI) provides a direct way to measure both emphysema using the apparent diffusion coefficient (ADC) and airways disease using ventilation defect percent (VDP) that reflects the underlying heterogeneity of lung structural and functional abnormalities in chronic obstructive pulmonary disease (COPD). We have previously shown that lung MRI measurements significantly worsened in a small group of COPD ex-smokers who remained FEV1 stable over two years, and here we extend this work in a large group of never- and ex-smokers in a longitudinal investigation. We evaluated 42 never-smokers, 33 ex-smokers without COPD and 49 ex-smokers with COPD using spirometry, plethysmography and inhaled gas MRI at baseline and 28±7 months later. As predicted by the Fletcher-Peto curves, FEV1%pred did not significantly change in any subgroups and their rates of change were not significantly different. However, VDP significantly worsened in all ex-smokers and the change in VDP was significantly greater in ex-smokers compared to never-smokers. While consistent with Fletcher-Peto prediction for FEV1, MRI data indicate that ventilation declined while FEV1 did not. Moreover, the ventilation decline in ex-smokers with and without COPD was greater than in never-smokers, which does not agree with predictions base on the natural history of FEV1 decline.

56

TOWARDS IMAGE-GUIDED ASTHMA THERAPY: A CLINICAL PIPELINE TO GENERATE MRI SEGMENTAL VENTILATION

MEASUREMENTS

F Guo1,2

, S Svenningsen1,3

, RL Eddy1,3

, M Fennema1, DG McCormack

4, G Parraga

1-3

Robarts Research Institute1, Graduate Program in Biomedical Engineering

2, Department of Medical Biophysics

3, Division of

Respirology, Department of Medicine4, The University of Western Ontario, London, Canada

RATIONALE: In asthma, airway abnormalities can be identified and quantified by combining anatomical and functional pulmonary information. Our group and others have shown that airway and functional abnormalities are heterogeneously distributed and reproducible over long time-periods. We think it is now possible to specifically target dysfunctional or morphologically abnormal airways as intermediate endpoints to guide therapy and maximize treatment efficiency. Therefore our objective was to develop an automated and clinically-practical image analysis pipeline to regionally identify and measure airway and ventilation abnormalities for image-guided asthma therapy. METHODS: Anatomical 1H and functional 3He MR images were acquired under breath-hold conditions at functional residual capacity plus 1.0 L. Thoracic CT was performed 10 minutes after MRI with the same breath-hold maneuver. 3He MRI was rigidly registered to 1H MRI and CT-1H MRI image-pair was initialized using affine registration. Next, CT was simultaneously registered to pre-aligned 3He and 1H using a deformable registration approach. In parallel, 3He ventilation defects were segmented using a hierarchical K-means method and CT lung/airways were segmented (VIDA Diagnosis Inc., Coralville, Iowa, USA), and used to generate 19 segments. Finally, the CT airways and segments were deformed using the transformation and deformation fields. Segmental ventilation defects percent (VDP) was calculated by normalizing the 3He ventilation defects to each segment. RESULTS: The proposed approach is fully automated and requires less than 10 minutes to generate a 3D spatially oriented display of: 1) CT airway-3He ventilation spatial relationships, 2) segmental VDP measurements, and 3) CT airway wall and lumen measurements proximal to the functional abnormalities with clinically-acceptable image registration accuracy that was improved compared to previous approaches. CONCLUSIONS: We developed a fully automated approach to determine the spatial relationship between ventilation defects and airway abnormalities. The quantitative measurements of segmental VDP may be used to facilitate decision making for image-guided asthma therapy.

LONGITUDINAL FIVE-YEAR DECLINE IN ALPHA-1 ANTITRYPSIN DEFICIENCY: REGIONAL WORSENING IN EMPHYSEMA AND

VENTILATION

Eric Lessard1, 2

, Alexei Ouriadov1, Damien Pike

1, 2, David G. McCormack

3 and Grace Parraga

1, 2

1Robarts Research Institute;

2Department of Medical Biophysics;

3Division of Respirology, Department of Medicine, The

University of Western Ontario, London, Canada.

Augmentation therapy for Alpha-1 antitrypsin deficiency (AATD) is a costly and controversial treatment for which there are few ways to measure response locally in the lung. Newly emergent, non-invasive MRI measurements of the pulmonary parenchyma that do not pose a risk due to ionizing radiation may be used to monitor lung structure and function in AATD to better understand the natural history of disease progression. In a proof-of-concept study, we longitudinally evaluated an AATD patient undergoing augmentation therapy using spirometry, plethysmography and inhaled gas MRI over a period of 58 months. We hypothesized that measurements derived from MRI would sensitively detect lung structure-function decline. A 62 year-old male never-smoker with AATD provided written informed consent to a local ethics-board-approved protocol. He underwent five visits over five years, during which weekly augmentation therapy was administered – except for a seven-month period between V3 and V4. During 5 visits, the subject underwent spirometry, plethysmography, DLCO and MRI including anatomical 1H and 3He diffusion-weighted (apparent diffusion coefficients (ADC) and static ventilation imaging. Thoracic CT was acquired on V2 and V4. Ventilation defect percent (VDP) was computed as previously described, 3 as was the CT relative-area-of-the-lung-with-attenuation ≤ -950 Hounsfield Units (RA950) using Pulmonary Workstation 2.0 (VIDA Diagnostics, Coralville, IA). Over the course of the augmentation therapy, ventilation (VDP) and emphysema (MRI and CT) worsened, whilst pulmonary function test measurements did not worsen until V4. This suggests that noble gas MRI provides complementary information for evaluating disease progression in AATD.

57

THE ROLE OF GABA SIGNALLING IN REGULATION OF ALVEOLAR MACROPHAGES’ IMMUNE RESPONSE

Jacob Poirier, Yun-Yan Xiang, Wei-Yang Lu Robarts Research Institute Molecular Medicine Research Group and Department of Physiology and Pharmacology, University of Western Ontario Alveolar macrophages (AMs) play a key role in pulmonary innate immunity. In order to adapt to the needs of the immediate pulmonary environment, AMs polarize in response to infectious pathogens as well as cytokines secreted from nearby T cells. Specifically, bacterial toxin lipopolysaccharide (LPS), and Th1 cytokines such as interferon gamma (IFNγ) stimulate AMs to shift to the pro-inflammatory M1 phenotype, of which the upregulated inducible Nitric Oxide Synthase (iNOS) is a typical marker. In response to Th2 cytokines such as interleukin (IL) 4 and 13, AMs shift toward the M2 phenotype that is marked by increased arginase-1 and involved in resolution of the immune response. Previous studies in our laboratory showed that both the gamma-aminobutyric acid (GABA) synthesizing enzyme glutamic acid decarboxylase (GAD) A-type GABA receptors (GABAAR) are expressed by murine AMs, suggesting that AMs are endowed with an autocrine GABA signaling system. Results from this study revealed that antagonizing the autocrine GABA signaling in primary AMs increased the secretion of the M1 cytokine tumor necrosis factor-alpha (TNFα), suggesting a potentially important role for GABA signaling in immune response. In both RAW 264.7 cell line and primary AMs LPS and IFNγ treatment resulted in increased iNOS expression paired with a decrease in GAD and GABAAR. Conversely, treatment with IL4/13 induced an upregulation of arginase-1, GAD, and GABAAR. However, treatment of primary AMs with IL4/13 and GABAAR antagonist picrotoxin resulted in decrease in arginase-1 and GAD expression, and increased iNOS levels. These results suggest that the GABA signaling system in AMs may function to limit the pro-inflammatory response, and that a change in the GABA signaling may alter the inflammatory response of these cells.

WHAT DOES MAGNETIC RESONANCE IMAGING SIGNAL-INTENSITY MEAN IN ASTHMA?

Khadija Sheikh1,2

, Fumin Guo1,3

, Sarah Svenningsen1,2

, Alexei Ouriadov1, Dante PI Capaldi

1,2, Rachel L Eddy

1,2, David G

McCormack4, and Grace Parraga

1,2

1Robarts Research Institute;

2Department of Medical Biophysics;

3Graduate Program in Biomedical Engineering;

4Division of

Respirology, Department of Medicine, The University of Western Ontario, London, Canada. Currently, imaging of asthma is confined to chest radiography and computed tomography (CT). Ultra-short echo-time (UTE) 1H MRI has recently emerged as a way to minimize MRI tissue signal decay in the lung for the quantification of lung tissue density without exposure of ionizing radiation. Here, our objective was to determine the underlying structural and clinical determinants of MRI signal-intensity in patients with asthma during a methacholine challenge (MCH). We hypothesized that lung tissue density measured using UTE MRI would be related to gas-trapping, measured using pulmonary function tests, inhaled-gas MRI and CT. Subjects with a clinical diagnosis of severe, uncontrolled asthma provided written informed consent and were evaluated using UTE and inhaled-gas MRI, spirometry, and plethysmography at baseline, post-methacholine at PC20, and post-salbutamol. Thoracic CT was acquired post-salbutamol. Whole lung UTE MRI was acquired during a 15s breath-hold at FRC+1L and signal-intensity was normalized and ventilation defect percent (VDP) was determined. Univariate Spearman correlation coefficients (ρ) were generated using SPSS 23.0 software (IBM, Armonk, NY). Six asthmatics (46±6yrs, 3M/3F) underwent tests at baseline and post-salbutamol. In the three subjects that underwent MCH, mean whole lung UTE signal-intensity/VDP at baseline, post-methacholine, and post-salbutamol were 38±2%/4±2%, 33±4%/9±6%, and 35±2%/4±3%, respectively. Mean whole lung MRI signal-intensity post-salbutamol was significantly correlated with CT radiodensity (ρ=0.97/p=.03), VDP (ρ=-0.81/p=.03), FEV1%pred (ρ=0.94/p=.02), FEV1/FVC (ρ=0.94/p=.02), RV/TLC (ρ=-0.82/p=.03), and FRC%pred (ρ=-0.82/p<.001). These results are consistent with the notion that UTE MRI signal-intensity may reflect gas-trapping and/or hypo-perfusion, similar to CT lucency findings in asthmatics. This is important because signal-intensity measurements derived from UTE MRI may identify the regional location of gas-trapping in severe asthma without the use of inhaled-gas contrast or CT making this approach amenable to immediate clinical use to help guide therapy.

58

AUTHOR INDEX Adam Ramina 32

Adeyanju Kemi 25

Albatany Mohammed 7

AlQot Hadir 45

Araya Yonathan 7

Arpino John-Michael 20

Babapoor-Farrokhran Sahand 45

Balint Brittany 17

Baronette Rudolph 39

Baxter John 17

32

Belliveau Jean-Guy 28

Blackney Kevin 28

Blokker Alexandria 39

Cantor Diego 36

Capaldi Dante 55

Christiansen Spencer 20

Connell Ian 36

Cooper Tyler 21

Cruje Charmaine 21

Dilliott Allison 46

Dron Jacqueline 22

Eddy Rachel 55

Elgamel Ruth 25

Escartin Terenze 36

Fink Corby 26

Gaudet Jeff 26

Gelman Daniel 18

Ghahremani Maryam 51

Ghoreshi Sina 22

Gillies Derek 8

Gonzalez Maria 33

Gu Chao 40

Guo Fumin 56

Hashad Ahmed 33

Hawley Zachary 40

Hong Gregory 41

Hosseini Zahra 34

Hrinivich Tom 8

Inoue Jiro 18

Janickova Helena 46

Jeong Jaymin 51

Johnston Kevin 52

Kara Jenna 12

Kasa Loxlan 52

Khazaei Zahra 53

Kljakic Ornela 47

59

Kowal Szymon 27

Kozyrev Natalie 47

Lackie Rachel 48

Lau Jonathan 29

36

Le Trung 12

Lee Wai-Jane Virginia 41

Lehmann Sebastian 53

Lessard Eric 56

Lim Patrick 9

Liu Junmin 19

Liu Shirley 9

Lorusso Daniel 42

Lowerison Matthew 13

Ma Liya 48

Maitland Matthew 13

Major Alex 49

Makela Ashley 14

Maksoud Matthew 29

Manning Kathryn 30

Matovic Sara 54

McTavish Christine 14

Mels John 49

Micheal Justin 10

Morey Trevor 42

Moszczynski Alexander 30

Murrell Donna 15

Nano Tomi 36

Ossowski Natalie 31

Paish Adam 43

Pardasani Utsav 10

Parkins Katie 15

Poirer Jacob 57

Rankin Adam 19

Rodgers Jessica 11

Ryan Kayla 43

Salemi Louisa 16

Santyr Brendan 54

Schranz Amy 31

Sheikh Khadija 57

Sherman Stephen 23

Smith Micheal 27

Stanley Olivia 34

Tavolieri Micheal 44

Walden Elizabeth 16

Watson Alanna 23

Winick-Ng Warren 50

Xu Yiwen 24

60

Yin Hao 24

Zechariah Anil 35

Zhang Shuxin 50

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