1. A Murine Model of Multiple Mild Concussive Brain Injuries and the Effects of Recovery Time on Cognitive Outcome Bill Meehan, MD Sports Concussion Clinic Children’s Hospital Boston Neuroscience Center Massachusetts General Hospital Support NIH: T32 HD040128-06A1 National Football League Charities Foundation

9. Nathan Horton was concussed.

10. Mechanism and Definition Rotational Acceleration Denny-Brown & Russell, Brain, 1941 Ommaya & Genarelli, Brain, 1974 Concussion Definition A disturbance of neurological function rather than a structural injury International Consensus on Concussion in Sports 2009

11. Extent of the problem CDC: As many as 3.8 million sport-related TBIs per year in US (MMWR 2007;56(29):733-737) Acutely: cognitive dysfunction, headaches, insomnia, depression After years: cognitive dysfunction, dementia, psychological disorders, increased risk of suicide Chronic Traumatic Encephalopathy

12. Research questions Are the cognitive effects of multiple concussions cumulative? Can the cognitive effects of multiple concussions be alleviated by increasing the time interval between injuries?

13. Criteria for model development Rotational acceleration Cognitive dysfunction without gross structural damage No cell death, edema, hematoma; dissociate effects of concussion from structural lesions Cumulative effects of multiple, mild injuries Explore the concept of recovery period

14. Methods Adult (12-16 weeks) male C57BL/6 mice Modified weight drop model (DeFord, et al. J Neurotrauma, 2002) Sham injured mice anesthetized only 54 g weight, variable drop height (42in, 38in) Variable time between injuries (Daily, Weekly, or Monthly)

15. Methods Gross examination of skull and brain Edema assessment: wet-dry weight method Immunohistochemistry: Cell death Axonal injury BBB compromise

16. Methods Morris water maze hidden platform trials 5 trials, each consisting of 4 sub-trials, 1 per quadrant Outcome = Latency to the platform 1 month or 1 year after final injury, platform location changed

18. No intracranial hemorrhage

19. No contusion

20. No cerebral edema (77.5 ± 0.1 vs. 77.3 ± 0.4 %)

22. Hippocampus

23. Thalamus

24. Corpus callosum

26. Research Question 1 Are the cognitive effects of multiple concussions cumulative? YES

27. 5 Weekly Concussions 5 Daily Concussions Trial # Trial # 5 Monthly Concussions Trial # (p=0.66, 8 mice per group) What is the optimal recovery time between concussions? Trial # (p=0.07, 7 mice per group) (p=0.01, 5 mice per group)

28. Research Question 2 Can the cognitive effects of multiple concussions be diminished by increasing the time interval between injuries? YES

29. 5 Daily Concussions One Month Later Trial # Trial # (p=0.01, 5 mice per group) (p=0.01, 5 mice per group) Do cognitive deficits after concussion resolve with time? One Month After Final Injury: Trial #

30. 5 Weekly Concussions Trial # One Month After Final Injury One Month Later Trial # (p=0.07, 7 mice per group) (p=0.02, 7 mice per group) Do cognitive deficits after concussion resolve with time? One Month After Final Injury * Suggests that the appropriate rest interval for sparing acute cognitive function may not be long enough to spare later cognitive effects.

31. One Month Later One Month Later Trial # Trial # (p=0.21, 5 sham, 4 from 42”, 8 from 38”) 5 Monthly Concussions Trial # (p=0.66, 8 mice per group) Do cognitive deficits after concussion resolve with time? One Month After Final Injury Trial #

32. One Year Later 5 Daily Concussions One Year Later One Year Later Trial # Trial # Trial # (p=0.01, 5 mice per group) (p=0.02, 4 mice per group) One Year Later One Year After Final Injury One Month Later Trial # Trial # Trial #

33. Conclusions Repetitive, mild concussions produce cognitive deficits in the absence of significant structural injury. The effects are cumulative Acute cognitive outcomes improve as the interval between injuries is increased. However, the rest interval required to reduce long-term effects may be longer than that required to diminish short-term effects.

34. Future Studies Currently repeating weekly Further characterize model: silver staining, electron microscopy, long-term atrophy Mechanism of cognitive dysfunction

35. Acknowledgments Michael J. Whalen, MD Jimmy Zhang, Jugta Khuman, Chris Lee NIH T32HD040128-06 National Football League Charities Foundation

37. Microgliosis Our model Sham IBA 1 staining in cortex and hippocampus and periventricular areas