Controlled cortical impact (CCI) animal model

Adult male Sprague-Dawley rats (∼275 g, Harlan Laboratories) were used after a week of acclimation under approval from the Virginia Commonwealth University Institutional Laboratory Animal Care and Use Committee in accordance with National Institutes of Health guidelines and the Guide for the Care and Use of Laboratory Animals (U.S. Department of Health and Human Services). Animal facilities were accredited fully by the Association for Assessment and Accreditation of Laboratory Animal Care International. Food and water were provided ad libitum in ventilated cages in a temperature and humidity-controlled environment on 12 h light/dark cycle.

The CCI was performed using a Leica Impact One Stereotaxic Impactor as described previously (Supplementary Fig. S1A).²⁹ Briefly, rats were anesthetized using 4% isoflurane and maintained at 2% throughout the surgical procedure. Rectal temperature was monitored to maintain 37°C via heating pad. Further, heart rate, respiratory rate, and arterial blood oxygenation were monitored via pulse oximetry (MouseOx, Starr Life Sciences) using a rat foot sensor to affirm physiological homeostasis (Supplementary Fig. S2A–E). A 5 mm craniotomy was centered 1.6 mm caudal from bregma and 3.4 mm lateral (Supplementary Fig. 1B). Focal injury was administered using a 3 mm flat-tip stainless steel impactor translating perpendicular to the brain surface at 4.0 m/sec to a depth of 2 mm with a dwell time of 0.5 sec. The scalp was sutured closed with Neosporin and 2% lidocaine applied topically. Anesthesia was discontinued and righting reflex was monitored as a correlate measure of injury severity, with TBI animals falling within the mild to moderate range (4.1 ± 0.94 min), which was significantly greater than sham (2.8 ± 0.56 min; p = 0.001).³⁰

Brains were collected at 1, 2, 4, 7, and 14 days post-injury by decapitation under isoflurane anesthesia. Brains were removed from the skull, rinsed with ice-cold phosphate buffered saline (PBS) and flash-frozen in liquid nitrogen. A 2 mm thick coronal frozen tissue block centered on the lesion was mounted on a ThermoFisher HM 525 cryostat. The block was sectioned interleaving two adjacent 150 μm sections for protein lysate with five 10 μm sections for imaging. Each 150 μm cut was dissected further by scalpel to extract perilesioned tissue between 0.5 mm and 1.5 mm lateral from the core boundary (Supplementary Fig. 1C), with pieces combined for protein analysis.