magnetic resonance imaging in experimental stroke and ... et al 2015 [stroke] mri.pdf · magnetic...

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Many drugs have been tested in animal studies of focal cerebral ischemia, but despite this few interventions

of proven efficacy are available and tissue-type plasminogen activator is the only approved drug for clinical use during the acute phase.1,2 Three major factors are likely to have contrib-uted to this translational roadblock. First, weaknesses in the design of animal studies, such as threats to their internal valid-ity by the lack of blinding and randomization, and low sta-tistical power, may have caused interventions to seem falsely efficacious. Second, clinical trials may have been designed to test efficacy in conditions where efficacy had not been pre-viously demonstrated in preclinical studies, for example, in comorbid patients or substantially longer time to treatment than used in the animal studies. Third, pathophysiological differences between animal and human stroke may lead to a lack of fidelity of the stroke models themselves limiting their usefulness.3–5 Multicenter preclinical trials could increase the validity and generalizability of experimental findings by combining measurements from different centers,6,7 but the

inherent variability in methodological approaches should first be addressed before implementing such studies. The recent publication of guidelines for the reporting of animal research is a step forward8; however, little consideration has been given to the use and reporting of imaging in animal stroke studies.

Magnetic Resonance Imaging (MRI) is an invaluable and ver-satile imaging tool used in both clinical and preclinical research. In stroke, diffusion-weighted imaging (DWI) and perfusion imaging are sensitive to the consequences of early ischemia by detecting subtle changes in the diffusion of water or the hemo-dynamic status of injured brain, respectively. This information can be used in the laboratory for confirmation of occlusion, localization and tracking of the evolving ischemia, or progno-sis of late outcome.9–12 Conventional MRI, such as T

2-weighted

imaging (T2WI), is less sensitive to acute changes in the isch-emic brain and is usually performed late to identify the extent of neuronal death (infarct).9,10,13 Traditionally, experimental stroke studies have used ex vivo histological analysis of the latter.14,15 This approach requires that a subgroup of animals be killed at an

Background and Purpose—Because the new era of preclinical stroke research demands improvements in validity and generalizability of findings, moving from single site to multicenter studies could be pivotal. However, the conduct of magnetic resonance imaging (MRI) in stroke remains ill-defined. We sought to assess the variability in the use of MRI for evaluating lesions post stroke and to examine the possibility as an alternative to gold standard histology for measuring the infarct size.

Methods—We identified animal studies of ischemic stroke reporting lesion sizes using MRI. We assessed the degree of heterogeneity and reporting of scanning protocols, postprocessing methods, study design characteristics, and study quality. Studies performing histological evaluation of infarct size were further selected to compare with corresponding MRI using meta-regression.

Results—Fifty-four articles undertaking a total of 78 different MRI scanning protocols met the inclusion criteria. T2-weighted

imaging was most frequently used (83% of the studies), followed by diffusion-weighted imaging (43%). Reporting of the imaging parameters was adequate, but heterogeneity between studies was high. Twelve studies assessed the infarct size using both MRI and histology at corresponding time points, with T

2-weighted imaging–based treatment effect having a

significant positive correlation with histology ( R2

0 699= . ; P<0.001).Conclusions—Guidelines for standardized use and reporting of MRI in preclinical stroke are urgently needed. T

2-weighted

imaging could be used as an effective in vivo alternative to histology for estimating treatment effects based on the extent of infarction; however, additional studies are needed to explore the effect of individual parameters. (Stroke. 2015;46:843-851. DOI: 10.1161/STROKEAHA.114.007560.)

Key Words: brain ischemia ◼ magnetic resonance imaging ◼ models, animal

Magnetic Resonance Imaging in Experimental Stroke and Comparison With HistologySystematic Review and Meta-Analysis

Xenios Milidonis, MSc; Ian Marshall, PhD; Malcolm R. Macleod, PhD; Emily S. Sena, PhD

Received September 24, 2014; final revision received December 9, 2014; accepted January 7, 2015.From the Centre for Clinical Brain Sciences, The University of Edinburgh, Edinburgh, United Kingdom.Guest Editor for this article was Miguel A. Perez-Pinzon, PhD.The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.

114.007560/-/DC1.Correspondence to Emily S. Sena, PhD, Centre for Clinical Brain Sciences (CCBS), Chancellor’s Bldg, The University of Edinburgh, 49 Little France

Crescent, Edinburgh EH16 4SB, United Kingdom. E-mail [email protected]© 2015 American Heart Association, Inc.

Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.114.007560

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844 Stroke March 2015

arbitrary time point after the induction of ischemia; the choice of this time point is critical because brain injury post stroke is a spatiotemporally evolving process.13 MRI, in contrast, allows longitudinal assessment of the developing lesion in vivo and estimation of treatment effect in the same cohort of animals.

With the advent of high-resolution scanners for small animal imaging, MRI has now become a routine modality in many pre-clinical centers and the establishment of a standard approach to its use seems more imperative than ever. In addition, although several studies reported equivalence between MRI and histologi-cal infarct size measurements in rodents post stroke,16–18 a more robust assessment by comparing lesion-based treatment effects from multiple studies could be useful. The principal aim of this study was to review variability in the use of MRI in studies of focal cerebral ischemia and to identify the different postprocess-ing methods for the quantification of visible lesion sizes post stroke. We also set out to examine the association between histo-logical outcome, the current gold standard for assessing infarct size, and the different MRI techniques using meta-regression, to assess the performance of the modality as an infarct size bio-marker in animal studies of stroke.

MethodsIdentification of Relevant StudiesSince 2004, the Collaborative Approach to Meta-Analysis and Review of Animal Data in Experimental Studies (CAMARADES) have been conducting systematic reviews of interventions tested in animal models of stroke. The CAMARADES database holds details of 1824 publi-cations describing 5329 experiments testing the efficacy of candidate drugs in focal cerebral ischemia. For systematic review, we identified (April 30, 2014) studies that used MRI to quantify tissue injury and screened the full text to select controlled animal studies of focal ce-rebral ischemia, which determined the lesion size in vivo using MRI and reported measurements in text or graphically. We included studies published in any language. To examine how MRI outcome compares with histologically measured outcome by means of meta-analysis, we selected only the studies that reported infarct sizes using histology in the same cohort of animals where MRI was used.

Data ExtractionWe recorded details related to animal handling for MRI (position-ing method in scanner, anesthetic used, and physiological parameters monitored), scanning protocol (scanner field strength and manufac-turer, type of radiofrequency coil, pulse sequence type and param-eters, and scan duration), and postprocessing method for lesion size measurement (software and method used, blinding, and analysis by multiple observers) for all identified studies. We also extracted the animal species, the type of ischemia, the intervention tested, the isch-emic side of brain and the respective artery, which was occluded. Study quality was assessed using a 10-item checklist.19

For meta-analysis, we recorded infarct size measurements (mean and a measure of variance, number of animals per group) for both MRI and histology measures for both treatment and control groups. Where data were not given in the text, we used values previously re-corded in the database (based on requests to authors or measurement from figures if contact with authors was unsuccessful). Where studies used multiple treatment groups, we divided the number of animals in the control group by the number of treatment groups served for the purposes of meta-analysis.

Data AnalysisWe categorized experiments by individual imaging and study design parameters and calculated the percentage of studies reporting each.

Because certain aspects of MRI are influenced by the animal species used, we calculated the range and the median value of continuous im-aging variables separately for each species. Where parameters were reported in different units, we converted them to the same scale.

For meta-analysis, we calculated treatment effects measured using MRI and histology as a normalized mean difference,20 the percent-age improvement in the treated cohort. We used the DerSimonian and Laird random effects meta-analysis21 to pool treatment effects because we anticipated substantial heterogeneity between studies. The significance of heterogeneity between k groups/comparisons was assessed using the χ2 distribution with k−1 degrees of freedom.20 We used meta-regression (function metareg in Stata version 13; StataCorp®, College Station, TX) to assess the relationship between effect sizes determined histologically and by MRI.22 The proportion of the variance in the dependent variable (histological outcome) that is explained by variation in the independent variable (MRI) is indicat-ed by an adjusted coefficient of determination, R

2. We then included

the time of outcome assessment as a regression covariate. Statistical significance was set at P<0.05.

ResultsWe identified 57 studies using MRI, of which 2 did not report measurement of lesion size and 1 performed ex vivo MRI; therefore, we extracted data from 54 studies, pub-lished between 1994 and 2010 (Figure 1; included articles are listed in Table I in the online-only Data Supplement). One study (2%) was published in Chinese and was translated into English. Two (4%) were published in abstract only and were cross-checked to verify that the data were not later published in full to ensure that we did not include duplicate data. Most studies used rats (42; 78%), 11 used mice (20%), and 1 used baboons (2%). Forty-five studies used T2WI (83%), 23 used diffusion-weighted imaging (43%), 4 used T1WI (7%), 3 used perfusion imaging (6%), 2 used plasma volume imaging (4%), and 1 did not state the weighting applied (2%); overall there were 78 different scanning protocols, with 19 studies (35%) reporting >1 method for assessing lesion size.

Study QualityThe median number of study quality checklist items scored was 4 (range, 0–6; Figure 2). Few studies reported blind-ing the induction of ischemia (4%), a statement of potential conflicts of interest (6%) or the use of animals with comor-bidities (4%), and no study reported a sample size calcula-tion. Random allocation to group was reported in 18 studies (33%) and the blinded assessment of outcome in just 9 studies (17%), despite the fact that a few other studies reported blind-ing of the assessment of outcomes other than MRI. Reporting of control of animals’ temperature during surgery, compliance with animal welfare regulations, and the use of an anesthetic without known neuroprotective activity before or during out-come assessment were prevalent in the majority of reviewed articles (76%, 70%, and 63%, respectively).

MRI Acquisition and Postprocessing ParametersFigure 3 shows the level of reporting of various MRI items in reviewed articles. Reporting was adequate for most items, particularly those relevant to the scanning protocol. However, parameters indicative of the quality of imaging methodol-ogy (monitoring of animals’ physiological parameters during scanning and analysis of data by multiple observers) were the

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Milidonis et al MRI in Experimental Stroke 845

least commonly reported in examined articles. Heterogeneity in the design of MRI between studies was high, with a wide range of anesthetics, scanner equipment manufacturers, scan-ner field strengths, and radiofrequency coil types recorded (Table II in the online-only Data Supplement). Studies were performed on systems with field strengths ranging from 1.5 to 9.4 Tesla, with 4.7 and 7 Tesla animal scanners being the most prevalent (29% each). Volume coils were preferred for signal transmission and reception (55%), followed by surface coil or a combination of both types (23% each). Several studies reported the use of anesthetic or radiofrequency coil without providing details. Of 54 studies, only 16 stated monitoring of physiological parameters during scanning, with temperature having the highest prevalence among reported parameters (82%). In 12 studies, the side of the stroke was not reported, and in many others it had to be derived indirectly. In some studies, the stroke side on exemplar images conflicted with that reported in the text, introducing confusion in the interpre-tation of results.

There was a large range of acquisition parameters for the 2 most common MRI signal-weightings (T2WI, DWI; Table 1). For example, for T2WI of rats, echo time ranged from 30 to

120 ms, the number of slices from 6 to 35, the slice thickness from 0.35 to 3 mm, the field of view from 22×22 to 120×120 mm2, and the matrix size from 64×64 to 512×512. For mouse imaging, the range was usually narrower although median val-ues were similar. For DWI, details of the applied diffusion-sen-sitizing gradients were seldom reported, except for the b-factor, which ranged from 20 to 1898 s/mm2 for rat imaging and 70 to 1224 s/mm2 for mouse imaging. Unexpectedly, the type of pulse sequence was reported for only slightly over half of recorded protocols (29 T2WI protocols, 64%; 12 DWI protocols, 52%). The most commonly used types for T2WI were conventional spin echo and fast spin echo with 14 protocols each; 1 study used fluid-attenuated inversion recovery spin echo imaging. For DWI, conventional spin echo (6 protocols), spin echo echo-planar imaging (4 protocols), stimulated echo (1 protocol), and fast spin echo sequences (1 protocol) were reported. Despite the variety of pulse sequences, there was a similar prevalence of reporting and range of parameter values for these techniques. For larger scanner field strengths, echo time tended to be shorter and acquired slices thinner, but the majority of recorded parameters were not found to be dependent on field strength. Finally, the duration of the scanning protocol per animal was

1824 articles in CAMARADES database describing experiments

in focal cerebral ischemia

57 articles initially selected

1767 articles excluded for not reporting the use of MRI for the assessment

of lesion size

54 articles met inclusion criteriafor systematic review

3 articles excluded:No lesion size measurementsEx vivo MRI

21

12 articles (46 comparisons) metinclusion criteria for meta-analysis

42 articles excluded:No assessment of infarct sizeusing histology No corresponding MRI-histologytime-pointsNo measurements for control group

39

21

Publication type:

Journal articleAbstract

522

Species:

RatMouseBaboon

42111

Type of ischemia:

TransientPermanentThrombotic

27243

78 scanning protocols for the assessment of lesion size:T2-weighted imagingDiffusion-weighted imagingT1-weighted imagingPerfusion imagingPlasma volume imagingUnknown

45234321

Figure 1. Flow chart demonstrating the selection process for relevant articles in the systematic review and meta-analysis. Details for included and excluded articles are given. CAMARADES indicates Collaborative Approach to Meta-Analysis and Review of Animal Data in Experimental Studies; and MRI, magnetic resonance imaging.




reported in a small number of studies (7 T2WI protocols, 16%; 2 DWI protocols, 9%). The median duration for T2WI in rats was 7.5 (range, 1.1–9) minutes and in mice was 5 minutes (no range recorded), whereas DWI usually took longer (median, 11 [range, 7–15] minutes in rats; no data given for mice).

Lesions in MRI images were assessed using a range of com-mercially available (83%) or home-made (17%) tools. Methods for determining the extent of the lesions were recorded sepa-rately for each scanning protocol; semiautomated analysis using thresholds was performed for the majority of protocols (76%), followed by manual delineation (20%) and other automated

methods (4%). Of 45 recorded T2WI protocols, 44 were applied during the first few days after ischemia when brain edema sig-nificantly contributes to the visible hyperintensity in acquired images; only 11 of these (25%) used a method for taking into account its presence. Nine of the postprocessing protocols that applied an edema correction for estimating the infarct size used the method proposed by Swanson et al,23 whereas 2 studies nor-malized the observed lesion size by the ratio of the hemispheric volumes. Lesion size was presented in published reports using a variety of measures, with volume in cubic millimeters being the most frequent.

Scanner field strength

Scanner equipment manufacturer

Ischemic brain side

Software for image analysis

Anesthetic used during scanning

Type of radiofrequency coil

Animal positioning method in scanner

Monitoring of physiological parameters during scanning

Analysis by multiple observers

0 10 20 30 40 50 60 70 80 90 100

Slice thickness

Repetition time (TR)

Echo time (TE)

Field of view

Matrix size

Method of lesion size determination

Type of sequence

Number of slices

Number of signal averages

Scan duration

Prevalence of item (%)

A

B

Figure 3. Prevalence of imaging information, sorted by overall prevalence. A, Items assessed for reporting in each of the 54 included articles and (B) for each of the 78 different scanning protocols. TE indicates echo time; and TR, repetition time.

0 10 20 30 40 50 60 70 80 90 100

Peer-reviewed publication

Control of temperature

Compliance with animal welfare regulations

Use of anaesthetic without marked intrinsicneuroprotective activity

Random allocation to groups

Blinded assessment of outcome

Statement of potential conflicts of interest

Blinded induction of ischemia

Use of comorbid animals

Sample size calculation

Percentage of articles satisfying quality criterion (%)

Figure 2. Study quality criteria met by studies, sorted by overall prevalence.




Table 1. MRI Acquisition Parameters for 45 T2-Weighted and 23 Diffusion-Weighted Imaging Protocols

T2-Weighted Imaging Diffusion-Weighted Imaging

ParameterTotal Reported,

n/N (%)

Reported per Animal Species, Species: n/N

(%)Details per Animal

Species, Range (median)Total Reported,

n/N (%)

Reported per Animal Species, Species: n/N

(%)Details per Animal

Species, Range (Median)

Type of sequence 29/45 (64) Rat: 19/34 (56) – (–) 12/23 (52) Rat: 11/22 (50) – (–)

Mouse: 9/10 (90) – (–) Mouse: 1/1 (100) – (–)

Baboon: 1/1 (100) – (–) … …

Scan duration, min 7/45 (16) Rat: 5/34 (15) 1.1–9 (7.5) 2/23 (9) Rat: 2/22 (9) 7–15 (11)

Mouse: 2/10 (20) 5 (5) Mouse: 0/1 (0) N/G (N/G)

Baboon: 0/1 (0) N/G (N/G)

Echo time, ms 43/45 (96) Rat: 32/34 (94) 30–120 (60)* 17/23 (74) Rat: 16/22 (73) 33–100 (40)

Mouse: 10/10 (100) 30–120 (66) Mouse: 1/1 (100) 30 (30)

Baboon: 1/1 (100) 105 (105) … …

Repetition time, ms 43/45 (96) Rat: 32/34 (94) 1750–8000 (3000) 18/23 (78) Rat: 17/22 (77) 1000–4000 (3000)

Mouse: 10/10 (100) 1000–3000 (2550) Mouse: 1/1 (100) 1000–1000 (1000)

Baboon: 1/1 (100) 3000 (3000) … …

No. of slices 27/45 (60) Rat: 18/34 (53) 6–35 (14)* 12/23 (52) Rat: 11/22 (50) 4–13 (8)*

Mouse: 9/10 (90) 5–25 (13) Mouse: 1/1 (100) 5 (5)

Baboon: 0/1 (0) N/G (N/G) … …

Slice thickness, mm 44/45 (98) Rat: 33/34 (97) 0.35–3 (1.5) 19/23 (83) Rat: 18/22 (82) 1–2 (1.5)

Mouse: 10/10 (100) 0.5–1 (1) Mouse: 1/1 (100) 0.5 (0.5)

Baboon: 1/1 (100) 3 (3) … …

Field of view,† mm×mm 38/45 (84) Rat: 30/34 (88) 22×22–120×120 (30×30)

17/23 (74) Rat: 16/22 (73) 22×22–80×80 (30×30)

Mouse: 7/10 (70) 25.6×12.8–20×20 (20×20)

Mouse: 1/1 (100) 22×16 (22×16)

Baboon: 1/1 (100) 200×150 (200×150) … …

Matrix size,† pixels×pixels

34/45 (76) Rat: 28/34 (82) 64×64–512×512 (256×256)

17/23 (74) Rat: 16/22 (73) 64×64–256×256 (128×128)

Mouse: 5/10 (50) 128×64–256×256 (256×128)

Mouse: 1/1 (100) 128×64 (128×64)

Baboon: 1/1 (100) 256×192 (256×192) … …

No. of signal averages, NSA

12/45 (27) Rat: 9/34 (26) 2–8 (4) 4/23 (17) Rat: 4/22 (18) 2–4 (3)

Mouse: 2/10 (20) 8–16 (12) Mouse: 0/1 (0) N/G (N/G)

Baboon: 1/1 (100) 1 (1) … …

Echo train length for FSE‡

5/14 (36) Rat: 3/8 (38) 8–16 (7) 1/1 (100) Mouse: 1/1 (100) 8 (8)

Mouse: 1/5 (20) 8 (8) … …

Baboon: 1/1 (100) 10 (10) … …

Inversion time, TI, ms, for FLAIR‡

0/1 (0) Rat: 0/1 (0) N/G (N/G) N/A N/A N/A

Diffusion sensitivity, b-factor, s/mm2

N/A N/A N/A 18/23 (78) Rat: 17/22 (77) 20–1898 (1000)*

Mouse: 1/1 (100) 70–1224 (647)*

Diffusion gradient strength, G, mT/m

N/A N/A N/A 6/23 (26) Rat: 5/22 (23) 0–152 (80)*

Mouse: 1/1 (100) 120 (120)

Diffusion gradient duration, δ, ms

N/A N/A N/A 8/23 (35) Rat: 7/22 (32) 3.15–10 (10)

Mouse: 1/1 (100) 5 (5)

Diffusion gradient separation, Δ, ms

N/A N/A N/A 6/23 (26) Rat: 5/22 (23) 13–50 (24.7)

Mouse: 1/1 (100) 17.5 (17.5)

– indicates that the value cannot be calculated; FLAIR, fluid-attenuated inversion recovery; FSE, fast spin echo; MRI, magnetic resonance imaging; N, total number of protocols for which the parameter is relevant; n, number of protocols for which the parameter is reported; N/A, not applicable; and N/G, not given.

*Multiple values have been used for these parameters in some protocols. If only the range was reported, the minimum and maximum values were incorporated into the calculated range but they were excluded from the calculation of the median value.

†A representative range was estimated by sorting the products of each pair of values. The most frequently reported value is given instead of the median.‡Parameters applicable only to specific types of sequences.




Meta-AnalysisFifteen publications assessed infarct size post treatment using both MRI and histology (28%). In 2 of these, there was a substan-tial delay between MRI scanning and histological assessment and in 1 study data for a control group were not provided (Figure 1). Of 12 remaining studies (46 comparisons, n(animals)

MRI=428,

n(animals)hist

=228), 11 performed T2WI (33 comparisons, n(animals)

T2WI=410, n(animals)

hist=210) and 6 performed DWI

(11 comparisons, n(animals)DWI

=147, n(animals)hist

=105) imme-diately before sacrifice. These comparisons were used for meta-regression. Only 1 study reported T1WI and perfusion imaging (1 comparison each, n(animals)

MRI=19, n(animals)

hist=19) along-

side histology24; therefore, we were unable to assess the rela-tionship between histology and these specific imaging methods. Histological assessment of infarct size was performed with 2,3,5-triphenyltetrazolium chloride staining in 10 studies, hae-matoxylin and eosin staining in 1 study, and cresyl violet stain-ing in another one. All included studies involved rats, except one that used mice.

Overall, all imaging findings explained 56.6% of the observed variation in histological outcome (P<0.001), as shown in Table 2. This relationship was stronger for T2WI ( R

20 699= . ; P<0.001; Figure 4), and there was no significant

relationship for DWI ( R2

0 433= . ; P=0.136). Inclusion of the time of outcome assessment as an additional independent variable improved the performance of both models (T2WI: R

20 724= . ; P<0.001; coefficient, 1.13; 95% confidence inter-

val [CI], 0.83 to 1.44; DWI: R2

0 534= . ; P=0.079; coefficient, 0.36; 95% CI, −0.05 to 0.78). Overall, T2WI estimated an improvement in lesion size of 22.2% (95% CI, 17.9%–26.6%) in the treatment group when compared with that of 25.5% (95% CI, 17.5–33.5) when outcome was measured histologi-cally in the same cohorts of animals (Table 2). The improve-ment in lesion size for DWI was 42.4% (95% CI, 30.9–54.0) when compared with 31.8% (95% CI, 24.0–39.7) when mea-sured histologically.

DiscussionWe have shown substantial variability in the selection of scan-ning parameters and postprocessing methods used to assess lesion size in experimental focal cerebral ischemia and in the quality of published reports. Reporting of MRI method-ology was assessed against several important items covering

the animal handling procedures for imaging, the scanning protocols and the postprocessing methods for estimation of lesion sizes. We think that these constitute the minimum set of information that should be present in reports of experimental studies of stroke using MRI; yet, no study fulfilled this duty to the maximum extent. There was generally adequate report-ing of scanner equipment manufacturer, scanner field strength, TR, echo time, and slice thickness, but not of the method of animal positioning in the scanner, monitoring of physiological parameters, methods for improving analysis validity (blinding and multiple observers), scan duration, or pulse sequence–specific parameters. The diverse range of scanner vendors, scanner field strengths, coils, and postprocessing software is not in itself a cause for criticism, but given that in clinical research such variability can have profound effects on mea-sured outcome,25–27 it seems reasonable to assume that it might also affect lesion size measurement in animals. The signifi-cant variability in MRI protocols and postprocessing methods raises questions about which approach is best for each con-trast technique, and whether results from different studies are comparable. Furthermore, in our review, we identified only 1 study reporting that image analysis was performed by 2 inde-pendent observers, whereas in only 9 of 54 studies did the

−100

−50

0

50

100

Effe

ct s

ize

base

d on

his

tolo

gy (

%)

−20 0 20 40 60 80

Effect size based on T2−weighted imaging (%)

Figure 4. Meta-regression of the effect size estimated from his-tological and T2-weighted imaging infarct size measurements at corresponding time points. The size of the circles reflects the precision of each estimate; large circles mean small SE and thus larger weight in the regression (y=1.08x+2.10; R

–2=0.699).

Table 2. Effect Sizes Based on Infarct Size Measurements From MRI and Histology and Meta-Regression Analysis of Corresponding Comparisons

No. of Comparisons No. of AnimalsCombined NMD

(95% CI), % P Value

Meta-Regression

Coefficient (95% CI) P Value R2

All MRI 46 428 26.7 (22.6–30.8) <0.001 0.76 (0.52–1.00) <0.001 0.566

Histology 46 228 27.5 (21.3–33.7) <0.001 … … …

T2WI 33 410 22.2 (17.9–26.5) <0.001 1.08 (0.77–1.38) <0.001 0.699

Histology 33 210 25.5 (17.5–33.5) <0.001 … … …

DWI 11 147 42.4 (30.9–54.0) <0.001 0.31 (-0.12–0.73) 0.136 0.433

Histology 11 105 31.8 (24.0–39.7) 0.002 … … …

Histology was used as the dependent variable in meta-regression. CI indicates confidence interval; DWI, diffusion-weighted imaging; MRI, magnetic resonance imaging; NMD, normalized mean difference; R

2, adjusted coefficient of determination; and T2WI, T

2-weighted imaging.




authors describe blinding of this procedure (1 of 12 used in meta-analysis28), raising concerns about the presence of detec-tion bias and introducing dubiety to reported outcomes. On the basis of these observations, we are unable to make rec-ommendations for the best neuroimaging method to assess lesion size for each MRI technique. It should be acknowl-edged, however, that the optimal scanning protocol must be a compromise between resolution, anatomic coverage, contrast between normal and injured tissue, and speed, to allow accu-rate quantification of lesion size, while minimizing the dura-tion of anesthesia received by animals for MRI. The selection of parameters’ values, in turn, might depend on the animal species, the scanner field strength and the imaging technique used, as found in this review.

We assessed reported study quality using our standard approach19 and found poor reporting of items, despite several contemporary recommendations including Stroke Therapy Academic Industry Roundtable-I2,29; experimental stroke stud-ies that do not report such measures may overstate treatment effects.4 Unfortunately, no standard criteria for assessing the quality of imaging in animal studies exist in the literature. The effect of individual parameters to the quantification of lesion sizes and subsequently treatment effects should first be explored before checklists for robust MRI quality assessment are formed. The diversity of experimental protocols (different species, stroke models, drugs, and treatment regimens) identified here pre-cluded analysis of the effect of study design items or of any basic imaging parameters; segregation of studies by various criteria sometimes led to groups consisting of a single study.

In contrast, in our analysis of alternative approaches to measuring the infarct size, the observed heterogeneity is not a concern because study characteristics and design were matched across the 2 outcome measures. We accounted for heterogeneity between studies in our calculation of summary estimates by using random-effects meta-analysis. The inclu-sion of measurements from a range of time points post stroke (48–336 hours for T2WI and 21–174 hours for DWI) is useful in capturing evolving changes in outcome, but may have been misleading if the relationship between measurements at dif-ferent time points was influenced by different pathophysiolog-ical indicators. Indeed, the inclusion of time in the regression model improved its performance, suggesting that this may well be the case. Such observation is in agreement with stud-ies reporting that neuronal death is an evolving process,13,30 indicating that the selection of a time point for estimation of treatment effects based on infarct size is crucial. Although it is more convenient for studies to use earlier time points to avoid sample size shrinking because of animal death and reduce study costs, treatment effects might become less useful when measured early. The more pronounced effect on histology-DWI regression could imply that the use of this MRI technique during the considered time period is an unreliable indicator of the effectiveness of tested interventions. However, the small number of comparisons included in this analysis precludes the extraction of safe conclusions. Furthermore, we assumed equivalence of the different histological measures of infarct size,16,31,32 and this may also have confounded our findings.

Notwithstanding these concerns, our findings suggest that T2WI can be used effectively as an in vivo noninvasive alternative assessment of infarct size, at least ≤2 weeks after the onset of ischemia. Indeed, the confidence limits of the summary assessments suggest that this approach may have greater statistical power when compared with histology (and hence require fewer animals). This does not imply that T2WI provides an equivalent estimate of infarct size, and in fact reported infarct volumes (as distinct from treatment-related effect sizes) were larger for T2WI than for histology in both treatment and control groups. This may be because none of the included studies applied a correction to take into account the accumulated vasogenic edema on T2WI, which slightly decreases in histological slices because fixation may shrink the lesioned tissue.16,18

DWI hyperintensity did not correlate well with the his-tological lesion, giving larger estimates of effect size. This may be because DWI is sensitive to the restricted diffusion of extracellular water because of the abnormal swelling of cells (cytotoxic edema) at the early stages of stroke progression9–12; diffusion lesions were larger than postmortem stained lesions at 21 hours (1 comparison) and smaller between 168 and 174 hours after stroke (9 comparisons), suggesting an early effect of cytotoxic edema.

To our knowledge, this is the first systematic review and meta-analysis of MRI in experimental stroke. It lacks, how-ever, specificity by including studies using any MRI technique for assessing lesions at any time point. Our primary objective was to provide an overview of the use of the modality in pre-clinical stroke research and examine whether this could be a limiting factor in data pooling in meta-analyses or multicenter animal studies; our findings clearly support this hypothesis. Additional studies should focus on individual MRI tech-niques or outcomes. Rivers and Wardlaw30 reviewed the use of DWI in animal studies of focal cerebral ischemia and identi-fied incomplete reporting of experimental details. They also examined whether DWI could provide information on tissue viability by comparison with various histopathologic mark-ers, but their assessment was limited in description of findings reported in included articles. Dani et al33 assessed the report-ing of perfusion imaging in human stroke and found a similar poor consistency between studies. Perfusion imaging coreg-istered with diffusion imaging is a technique for identifying the salvageable tissue (represented by the mismatch of the 2 images), which provides a hypothetical target for reperfusion therapies. The concept of an ischemic penumbra has been subject to significant scrutiny in both clinical and preclini-cal research but no widely accepted definition.34 This could explain, in part, the small number of perfusion imaging proto-cols recorded in our review. There have been calls for consen-sus in the use of perfusion–diffusion imaging for defining the penumbra in humans and in animals,11,30 which we support. However, there is a more pressing case, demonstrated here, for consensus in the use of more routine MRI techniques.

A further weakness of this study is that we included data from a single database comprising a nonexhaustive list of animal studies limited to interventions, which had been assessed using systematic review. However, contributing




studies were identified using systematic search strategies, collected without consideration of the imaging methodology used. Therefore, the articles examined in this review can be considered representative of experimental stroke imaging studies in the literature. Few experiments assessed infarct size using both histology and MRI (46 of 5329 experiments), and this did not allow exploration of any effect imaging param-eters have on the relationship between MRI and histological outcome measures. A systematic review seeking to identify all such studies would enable such an analysis, but it is likely to require screening of a large number of publications.

ConclusionsWe show substantial variability between studies in the use of MRI for the assessment of lesion size. Strong correlation between histology and T2WI suggests that optimization of a T2WI scanning and postprocessing method for quantifica-tion of final infarct size is feasible. Guidelines for the use and reporting of methodology are needed, similar to those devel-oped for the conduct of human stroke imaging.35,36 This would permit exact replication of the experiments and analyses and would support the use of imaging end points in multicenter animal studies.

Sources of FundingThis study was partially supported by the Multicenter Preclinical Animal Research Team (http://www.multi-part.org). XM holds a University of Edinburgh Centre for Clinical Brain Sciences (http://www.ccbs.ed.ac.uk) PhD studentship.

DisclosuresNone.

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1

SUPPLEMENTAL MATERIAL

MRI in Experimental Stroke and Comparison with Histology: A Systematic Review and

Meta-analysis

Xenios Milidonis, MSc; Ian Marshall, PhD; Malcolm R. Macleod, PhD; Emily S. Sena, PhD

Centre for Clinical Brain Sciences, The University of Edinburgh

2

Supplemental Tables

Table I. List of papers included in the systematic review

*Studies that measured infarct size with both MRI and histology and were included in meta-analysis.

First Author Year Journal Volume (Issue) Pages

Umemura1 1994 European Journal of Pharmacology 251 (1) 69-74

Quast2 1995 Brain Research 677 (2) 204-212

Tatlisumak3 1996 Stroke 27 (12) 2292-2298

Wei4 1998 Brain Research 791 (1-2) 146-156

Wiessner5 1999 Neuroscience Letters 268 (3) 119-122

Mancuso6 2000 Brain Research 887 (1) 34-45

Saarelainen7 2000 Molecular and Cellular Neuroscience 16 (2) 87-96

Yenari8 2000 Brain Research 885 (2) 208-219

Zhang9 2000 Journal of Clinical Investigation 106 (7) 829-838

Cash10

2001 Brain Research 905 (1-2) 91-103

Lee11

2001 Journal of Cerebral Blood Flow and Metabolism 21 (Suppl 1) S130

Schabitz12

* 2001 Stroke 32 (5) 1226-1233

Shi13

2001 Stroke 32 (4) 987-992

Sinha14

2001 European Journal of Pharmacology 428 (2) 185-192

Wiessner15

2001 Journal of Cerebral Blood Flow and Metabolism 21 (7) 857-864

Goto16

* 2002 Stroke 33 (4) 1101-1106

Hughes17


Koistinaho18

2002 Proceedings of the National Academy of Sciences 99 (3) 1610-1615

Mack19

2003 Stroke 34 (8) 1994-1999

Sironi20

2003 Arteriosclerosis, Thrombosis, and Vascular Biology 23 (2) 322-327

Veldhuis21


Wagner22

2003 Brain Research 984 (1-2) 63-75

Kurozumi23

2004 Molecular Therapy 9 (2) 189-197

Shyu24

2004 Circulation 110 (13) 1847-1854

Cimino25

2005 NeuroToxicology 26 (5) 929-933

Koistinaho26


Kurozumi27

2005 Molecular Therapy 11 (1) 96-104

Nomura28

* 2005 Neuroscience 136 (1) 161-169

van der Weerd29

2005 Experimental Neurology 195 (1) 257-266

Yrjänheikki30

2005 Brain Research 1052 (2) 174-179

Boltze31

2006 Artificial Organs 30 (10) 756-763

Ding32

* 2006 Journal of the Neurological Sciences 246 (1-2) 139-147

Honma33

* 2006 Experimental Neurology 199 (1) 56-66

Horita34

* 2006 Journal of Neuroscience Research 84 (7) 1495-1504

Jiang35

2006 NeuroImage 32 (3) 1080-1089

Liu36

* 2006 Brain 129 (Pt 10) 2734-2745

Kameda37

2007 European Journal of Neuroscience 26 (6) 1462-1478

Pialat38

2007 NMR in Biomedicine 20 (3) 335-342

Shimamura39

2007 Stroke 38 (12) 3251-3258

Ukai40

* 2007 Journal of Neurotrauma 24 (3) 508-520

Uno41

2007 Journal of Cerebral Blood Flow and Metabolism BP10 05H

Wei42

2007 Acta Academiae Medicinae Sinicae 29 (1) 73-77

Esneault43


Kim44

2008 Stem Cells 26 (9) 2217-2228

Koh45

2008 Brain Research 1229 233-248

Li46

* 2008 Cell transplantation 17 (9) 1045-1059

Omori47

* 2008 Brain Research 1236 30-38

Onda48

* 2008 Journal of Cerebral Blood Flow and Metabolism 28 (2) 329-340

Schmerbach49

2008 Brain Research 1208 225-233

Yoo50

2008 Experimental and Molecular Medicine 40 (4) 387-397

Li51

2009 Stroke 40 (3) 936-941

Modo52

2009 NeuroImage 47 (Suppl 2) T133-T142

Toyama53

* 2009 Experimental Neurology 216 (1) 47-55

van der Weerd54


3

Table II. Reporting and details of basic characteristics of the imaging procedure

n indicates the number of protocols for which the parameter is reported; N, total number of protocols for which the parameter is

relevant; T, tesla; 𝐶, size of contralateral hemisphere; 𝐼, size of ipsilateral hemisphere; 𝐿, size of visible lesion in 𝐼. *Post-processing parameters, applicable to each of the 78 imaging protocols.

†Applied on T2-weighted images acquired at the acute to subacute stages of stroke (relevant to 44 out of 45 protocols).

Parameter Reported,

n/N (%)

Not Reported,

n/N (%)

Details of reported parameters,

n/N (%)

Ischemic brain side 42/54 (78) 12/54 (22) Left: 12/42 (29)

Right: 30/42 (71)

Animal positioning method

in scanner

22/54 (41) 32/54 (59) Carriage/cradle: 15/22 (68)

Stereotaxic: 7/22 (32)

Anesthetic used during

scanning

36/54 (67) 18/54 (33) Fentanyl-Fluanisone-Midazolame:

1/36 (3)

Halothane: 10/36 (28)

Isoflurane: 10/36 (28)

Ketamine: 12/36 (33)

Pentobarbital: 3/36 (8)

Monitoring of physiological

parameters during scanning

16/54 (30) 38/54 (70) Temperature only: 7/16 (44)

Temperature & other: 6/16 (38)

Other: 3/16 (19)

Scanner equipment

manufacturer

51/54 (94) 3/54 (6) Bruker: 14/51 (27)

General Electric: 9/51 (18)

Medinus: 2/51 (4)

Otsuka Electronics: 1/51 (2)

Philips: 1/51 (2)

Spectroscopy Imaging Systems: 1/51 (2)

Varian: 1/51 (2)

Multiple: 22/51 (43)

Scanner field strength 51/54 (94) 3/54 (6) 1.5T: 5/51 (10)

2T: 4/51 (8)

2.35T: 4/51 (8)

3T: 7/51 (14)

4.7T: 15/51 (29)

7T: 15/51 (29)

9.4T: 1/51 (2)

Type of radiofrequency coil 31/54 (57) 23/54 (43) Volume: 17/31 (55)

Surface: 7/31 (23)

Volume and surface: 7/31 (23)

Software for image analysis 40/54 (74) 14/54 (26) Commercial: 33/40 (83)

Home-made: 7/40 (17)

Method of lesion size

determination*

51/78 (65) 27/78 (35) Manual tracing: 10/51 (20)

Thresholding: 39/51 (76)

Automated algorithm: 2/51 (4)

How the lesion size is

presented*

77/78 (99) 1/78 (1) Lesion volume: 45/77 (58)

Lesion area at one slice: 1/77 (1)

Change from baseline: 7/77 (9)

Fraction/percent of ipsilateral side: 14/77 (18)

Fraction/percent of contralateral side: 6/77 (8)

Percent of brain: 4/77 (5)

Edema correction method

for infarct size†

11/44 (25) 34/44 (75) 𝐶 − (𝐼 − 𝐿): 9/11 (82)

𝐿 × (𝐶/𝐼): 2/11 (18)

4

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5

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Xenios Milidonis, Ian Marshall, Malcolm R. Macleod and Emily S. SenaSystematic Review and Meta-Analysis

Magnetic Resonance Imaging in Experimental Stroke and Comparison With Histology:

Print ISSN: 0039-2499. Online ISSN: 1524-4628 Copyright © 2015 American Heart Association, Inc. All rights reserved.

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