out of the foginduction, 25% mortality at peak 5 of 6 bl6 mice per sex relapsed sjl m were not...
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OUT OF THE FOG: Elucidating the Somatosensory and Behavioral Effects of Experimental Autoimmune Encephalitis on Mice
THE FOG Multiple Sclerosis (MS)
Autoimmune, demyelinating disease
400,000+ Americans - mostly young women
85% relapsing-remitting form
Fatigue, mood disorders, and pain are most debilitating symptoms (“MS Fog”)
Experimental Autoimmune Encephalitis (EAE) C57BL/6 mice - monophasic
SJL females - relapsing-remitting form
Used to explore motor deficits
Lack of research on sensory effects and mood disorders
Healthy nerve
damaged Myelin in MS
THE PROJECT GOAL: to adapt existing tests to the EAE model in order to assess somatosensory deficits in relation to motor signs AIM 1: Determine the effects of EAE on sensitivity to mechanical stimuli and the impact on species-
typical behaviors AIM 2: Assess the differences inherent to each sex and strain AIM 3: Chart the progression of motor and somatosensory deficits throughout the course of disease
HYPOTHESIS: EAE causes significant somatosensory deficits that precede motor dysfunction and negatively impact social behavior and quality of life
THE PLAN
12 females 12 males
SJL
12 females 12 males
C57BL/6 8 actively immunized with MOG in CFA + PTX
4 sham-immunized with CFA + injectable saline
8 actively immunized with PLP in CFA + PTX
4 sham-immunized with CFA + injectable saline
Daily Weight Clinical scoring
Periodic sensory, motor, and behavioral testing: Baseline (Day 0, CS 0) Preclinical (Day 6-7, CS 0) Clinical (CS 2-3) Recovery (CS 1-2.5) Relapse (CS 2-3) – SJL
females only Statistical analysis via 2-way
ANOVA
THE MOTOR TESTS
Weight Lift Test
Konziela Screen Test
Rope Hang Test
Clinical Scoring
5 Moribund or Dead
THE SOMATOSENSORY TESTS Von Frey: Mechanical Sensitivity
Crawley: Sociability, Ethogram
Behaviors Measured: Time in each chamber Encounters with novel mouse
vs. empty chamber Encounters with novel mouse
vs. novel object Rearing behavior Self-grooming behavior More with video analysis
THE RESULTS: CLINICAL & MOTOR DEFICITS
05
1015202530354045
Onset Peak Recovery Relapse
Day
s Po
st-Im
mun
izat
ion
-50-40-30-20-10
01020
Wei
ght C
hang
e fr
om B
asel
ine
(%) BL6 F EAE
BL6 F ShamBL6 M EAEBL6 M ShamSJL F EAESJL F Sham
BL6 F EAE: 75% induction; 0% mortality at peak
BL6 M and SJL F EAE: 100% induction, 25% mortality at peak
5 of 6 BL6 mice per sex relapsed SJL M were not immunized
STATISTICS: CLINICAL EFFECTS
Disease Progression
Naïve novel mice for Crawley
Weight Change from Baseline
Source of Variation
% of total variation P value
P value summary
EAE stage 9.531 0.7256 ns Sex & Strain 4.333 0.744 ns
Interaction 0.3702 > 0.9999 ns
Source of Variation
% of total variation P value
P value summary
EAE stage 26.49 < 0.0001 **** Sex & Strain 28.55 < 0.0001 ****
Interaction 8.631 0.0268 *
Sex/strain and EAE stage both affected changes in body weight Differences in disease progression trended towards significance
THE RESULTS: VON FREY TEST CS 2 Control
EAE ↑awareness & enhanced sex/strain differences Threshold was impacted more by sex/strain than EAE stage Threshold failed to ↓ as awareness ↑ due to motor
deficits
STATISTICS: VON FREY
Awareness
Source of Variation
% of total variation P value
P value summary
EAE stage 40.05 < 0.0001 **** Sex & Strain 16.8 < 0.0001 ****
Interaction 18.93 < 0.0001 ****
Threshold
Source of Variation
% of total variation P value
P value summary
EAE stage 4.054 0.0434 * Sex & Strain 9.933 0.0019 **
Interaction 35.39 < 0.0001 ****
THE RESULTS: TIME WITH NOVEL MOUSE
Sex/strain significantly affected behavior
EAE did not
THE RESULTS: ENCOUNTERS WITH NOVEL MOUSE
Sex/strain significantly affected sociability
Mice with EAE had significantly ↓ sociability prior to clinical signs
THE RESULTS: EXPLORATORY BEHAVIORS
Session 1 = encounters with empty cylinder
Session 2 = encounters with novel object
Sex/strain and EAE independently influenced exploratory behavior
STATISTICS: CRAWLEY Time with Novel Mouse Encounters with Novel Mouse
Source of Variation
% of total variation P value
P value summary
EAE stage 4.251 0.0951 ns Sex & Strain 5.193 0.0117 *
Interaction 16.36 0.0973 ns
Source of Variation
% of total variation P value
P value summary
EAE stage 20.93 < 0.0001 **** Sex & Strain 23.81 < 0.0001 ****
Interaction 13.48 0.0009 ***
Encounters with Empty Cylinder or Novel Object Source of Variation
% of total variation P value
P value summary
EAE stage 12.31 0.0762 ns Sex & Strain 17.4 < 0.0001 ****
Interaction 15.19 < 0.0001 ****
THE BOTTOM LINE Conclusions
Tests can be used with the EAE model – but with caveats
EAE enhanced sensory and behavioral differences between sex/strain groups
Somatosensory deficits preceded onset of motor signs
Severity of motor disability did not always correlate with behavior
Future Directions/Troubleshooting Training and reward system
Naïve novel mice for Crawley
Video analysis for Crawley
SJL male group
Histology
Other pain assessments
THE SHOULDERS OF GIANTS References Cunha, T.; Veril, W.; Vivancos, G.; Moreira, I.; Reis, S.; Parada, C.; Cunha, F.; Ferrerira, S. (2004) An electronic pressure-
meter nociception paw test for mice. Braz J Med Biol Res 37(3): 401-407. Baker, D.; Gerritsen, W.; Rundle, J.; Armor, S. (2011) Critical appraisal of animal models of multiple sclerosis. Multiple
Sclerosis Journal 17(6): 647-657. Bar, K.; Brehm, S.; Boettger, M.; Boettger, S.; Wagner, G.; Sauer, H. (2005) Pain perception in major depression depends
on pain modality. Pain 117: 97-103. Bittner, R. et al (1999) Dysferlin deletion in SJL mice (SJL-Dysf) defines a natural model for limb girdle musclular dystrophy
2B. Nature Genetics 23: 141-142. Cadden, M. and Arnett, P. (2015) Factors associated with unemployment status in individuals with multiple sclerosis. Int J
MS Case 17(6): 284-291. Deacon, R. (2013) Measuring the Strength of Mice. J. Vis. Exp. (76), e2610, doi:10.3791/2610 Kaidanovich-Beilin, O.; Lipina, T.; Vukobradovic, I.; Rodor, J.; Woodget, J. (2011) Assessment of social interaction
behaviors. J Vis Exp 48: 2473. Kudryavtseva, N.; Bakshtanovskaya, V.; Koryakina, A. (1991) Social model of depression in mice of C57BL/6J strain.
Pharmacology, Biochemistry, and Behavior. 38: 315-320. Maloni, H. (2016) Pain in MS. National Multiple Sclerosis Society Clinical Bulletin. Moy, S.; Nadler, J.; Perez, A.; Barbaro, R.; Johns, J.; Magnuson, T.; Piven, J.; Crawley, J. (2004) Sociability and preference
for social novelty in five inbred strains: An approach to assess autistic-like behavior in mice. Genes, Brain, and Behavior 3(5): 287-302. MS the disease. National multiple sclerosis society. http://www.nationalmssociety.org Multiple sclerosis information page. National Institute of Neurological Disorders and Stroke.
http://www.ninds.nih.gov/disorders/multiple_sclerosis/multiple_sclerosis.htm Peled-Kamar, M.; Lotem, J.; Wirguin, I.; Weiner, L.; Hermalin, A.; Groner, Y. (1997) Oxidative stress mediates impairment
of muscle function in transgenic mice with elevated level of wild type Cu/Zn dismutase. Proc. Natl. Acad. Sci. USA, 94: 3883-3887. Racke, M. (2001) Experimental autoimmune encephalomyelitis. Current Protocols in Neuroscience (J. Crawley, Ed.). New
York, NY: John Wiley & Sons, Inc. 9.7.1-9.7.11 Valzelli, L. (1973) The “isolation syndrome” in mice. Psychopharmacologia 31(4): 305-320.
Acknowledgements Mentorship: Dr. Lillian Cruz-Orengo
Crawley Test Assistance Aida Nasirishargh Lillian Cruz-Orengo Jennifer Yee Sarah Tirrell Rachel Smith
Equipment Construction Jedediah Roach and the Machine Shop staff Justin Klein
Funding: NIH
QUESTIONS? Katelynn Ondek kondek@ucdavis.edu
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