Stanford University Department of Comparative Medicine

1. Stanford UniversityDepartment of Comparative Medicine Developing Best Care Practices and Endpoint Guidelines for Rodent Models of Neurologic Disease Lisa Heath, DVM, DACLAM

2. Rodent Models • Experimental Allergic Encephalomyelitis (EAE) • Amyotrophic Lateral Sclerosis (ALS) • Huntington’s Disease

3. Guidelines and Endpoints • Developing Guidelines • Resources • Working with Researchers

4. Multiple Sclerosis in People Fatigue Numbness Gait, balance, coordination problems • Bowel/bladder dysfunction • Cognitive dysfunction • Pain

5. Multiple Sclerosis Health.howstuffworks.com

6. Multiple Sclerosis • http://www.elements4health.com/images/stories/conditions/myelin-sheath.jpg

7. EAE Models Active EAE: immunized with myelin antigen or peptide emulsified in CFA Monophasic or relapsing-remitting disease Disease process commences 10-15 days post-immunization 75-80% incidence of disease

8. EAE Models Passive or adoptively transferred EAE: Recipient mice injected with activated, myelin-specific T cells (harvested from immunized donor mice) Relapsing-remitting disease

9. EAE Guidelines Benchmarking We benchmarked with other institutions to see if their IACUC had specific guidelines to cover EAE rodent models 64 institutions were surveyed regarding whether or not they had rodent EAE models 19 institutions responded Survey Results • Data and graphs courtesy of Sonja Wallace

10. EAE Benchmarking Survey

11. EAE Benchmarking Survey

12. EAE Benchmarking Survey

13. EAE Benchmarking Survey

14. Summary Majority of respondents had an IACUC guideline specifically for EAE models Some respondents used their humane endpoint guideline for EAE models Majority of respondents used 20% percent weight loss as an endpoint Majority of respondents separated symptomatic from asymptomatic animals

15. Refining Guidelines • Intervention/Monitoring: • When and how often • Nutrition and Hydration: • What, when and how often • Include the Researchers

16. EAE Scoring Score 0 = No overt signs of disease Requirements: Label Special Request Card with “EAE Model” on Experimental Day 1 Baseline Body Weight recorded on Day 1 Assess and record scoring observations at least 3 times per week.

17. EAE Scoring Score 1 = Limp tail, flaccidity of tail Requirements (same as for Score 0)

18. EAE Scoring Score 2 = Paraparesis; limp tail and hindlimb weakness, waddling gait. Provide gel pack and/or moistened food on cage floor, or other appropriate supplements. Weigh and/or assess body condition score at least 3 times per week.

19. Tail Suspension Test • Normal Plantar Reflex • Hindlimb Clasping Animal Welfare Information Center Bulletin, Winter 1999/2000, Vol. 10 No. 3-4

20. EAE Scoring Score 3 = Hindlimb paralysis. Total loss of moment in hindlimbs. Mouse moves itself with forelimbs. Daily Scoring and monitoring. Weigh and/or assess BCS at least once daily. Assess hydration status (body weight and skin turgor). Assess bladder for evidence of urinary retention. Express bladder as needed. Provide fluid supplementation, SC or oral gavage.

21. EAE Scoring Score 4: Subdivided into 4A and 4B (for guidelines only*) 4a = Hindlimb paralysis and mild forelimb weakness 4b. Hindlimb paralysis and moderate to severe forelimb paralysis; lateral recumbency.

22. EAE Scoring Score 4b. Hindlimb paralysis and moderate to severe forelimb paresis; lateral recumbency. Mice must be monitored at least twice daily for no more than 24 hours. Any animal that does not remit to score 3 within the time frame must be euthanized.

23. EAE Scoring Score 5 : Moribund, or complete hindlimb paralysis with moderate to severe forelimb paresis with no remittance after 24 hrs and intensive supportive care. Euthanize immediately.

24. EAE Scoring Any animal which loses >20% body weight or has a body condition score <2 must also be euthanized, regardless of EAE score.

25. A Sidenote on Body Weight Recording • Each mouse’s body weight data is unique • No averages! • Data recording: pre-disease weight and pre-calculated 20% loss cut-off

26. EAE Scoring: Mouse Posture 0 : Tail has tone. 1: Tail no longer stands up, loss of tail tone. Walks normally. 2: Limp tail, hindlimb dysfunction. 3: No longer holds onto underside of cagelid with hindlimbs. Moves hindlimbs. 4: Hindlimbs drag behind, not used for movement. Current Protocols in Neuroscience

27. EAE Scoring Models of Multiple Sclerosis. ACNR. Vol 6. No 6. 2007

28. Amyotrophic Lateral Sclerosis Fatal neurodegenerative disease Familial and Sporadic Loss of upper and lower motor neurons Paralysis of muscles Lou Gehrig’s Disease

29. www.alsa.org

31. ALS Models SOD1 mice

32. ALS Mouse Models Muscle atrophy Weight loss Limb weakness and paralysis Oral and pharyngeal dysphagia Respiratory weakness and paralysis

33. ALS Scoring System Working with ALS Mice: Guidelines for preclinical testing & colony management. The Jackson Laboratory

34. ALS Scoring System *If one hind leg is scored as 2, food pellets are left on bedding. If both hind legs are scored as 2, Nutra-Gel is provided as food in addition to food pellets on bedding and a long sipper tube is placed on the water bottle. Working with ALS Mice: Guidelines for preclinical testing & colony management. The Jackson Laboratory

35. ALS Scoring System:: 8 Point Scale Solomon, et.al. One Universal Common Endpoint in Mouse Models of Amyotrophic Lateral Sclerosis. June 2011, vol. 6, issue 6.

36. Endpoint Scoring for ALS: 8 Point Scale

37. Huntington’s Disease

38. Huntington’s Disease Scoring: Part 1 Auerbach, et.al. Human Molecular Genetics, 2001, Vol. 10, No. 22.

39. Huntington’s Disease Scoring: Movement Abnormalities Tail stiffness or paralysis Hind-limb stiffness or paralysis Reduced left-right alteration of hindlimbs (“hopping”) Resting tremors Difficulties walking after handling Seizure-like episodes

40. Huntington’s Disease Scoring: Part 2

41. Huntington’s Disease Scoring: Combining Part 1 and 2 Scores range from 0:0 (normal, no disease) to 3:3 (greatest expression of clinical signs) Choose score/score combinations for adding care : food and water accessibility, supplements heat source, nesting materials

42. Huntington’s Disease Reference: Guyenet, et.al. (2010). A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia. JoVE. 39. ID 1787. Tests: ledge test, hindlimb clasping, gait, kyphosis

43. Huntington’s Disease Noise sensitivity: construction, cage changes, hoods and ventilation Seizure-like activity and deaths related to noise and other disturbances Hypothermia in late stage disease: external heat source; nestlets vs. paper nesting material Enviro-dri® (Shepherd Specialty Papers) www.ssponline.com

44. Summary • Meet with investigators: look at their scoring and weight data • Provide specific solutions and options • Include investigators on guidelines development