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

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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