The mouse rmTBI model was used as previously described with important modifications.12 (link) Briefly, mice (3-month-old males) were anesthetized for 45 seconds using 4.5% isoflurane in a 70:30 mixture of nitrous oxide and oxygen. Anesthetized mice were placed on a delicate task wiper (Kimwipe; Kimberly-Clark, Irving, TX) and grasped by the tail. The head was placed directly under a hollow guide tube 28 inches in length. A 54g metal bolt was used to deliver the impact to the dorsal aspect of the skull. At impact, the mouse head readily penetrated the Kimwipe, resulting in a rotational acceleration of the head. Sham-injured age-matched control mice underwent anesthesia but not concussive injury. All mice recovered in room air. LOC was defined as the time from removal of anesthesia to spontaneous righting. Anesthesia exposure for each mouse was strictly controlled to 45 seconds exposure. LOC times reflected the effects of anesthesia as well as the effects of rmTBI.
Mice were randomized to receive injury or sham injury. Mice randomized to injury underwent 1 of the following injury regimens: (1) 5 daily concussive injuries (n = 7–10/group), (2) 7 concussive injuries in 9 days (n = 8–10/group), (3) 1 concussive injury weekly for 5 weeks (n = 16), (4) 1 concussive injury biweekly for 10 weeks (n = 14), (5) 1 concussive injury monthly for 5 months (n = 16), and (6) 1 concussive injury (n = 15). Sham-injured controls underwent anesthesia exposures (n = 4–12/group) at the same time intervals as injured mice. For all behavioral testing, experimenters were blinded to injury and genotype status, using color coding stored in a password-protected computer. Details of the study flow are found in Figure 1.