2.2. Controlled Cortical Impact (CCI) Injury Model

To prepare the mice for CCI injury or sham craniectomy surgery, young adult mice (1–2 month-old) were anesthetized with 4–5% of isoflurane in 100% O₂ and received buprenorphine (0.1 mg/kg) intraperitoneally as preemptive analgesia. The mice were then placed in a stereotaxis frame equipped with a micromanipulator (Kopf Instruments, Tujunga, CA, USA), the isoflurane flow was maintained at 2%, and the animals were monitored during the entire procedure. An incision was made in the middle from the eyes to the neck and a topical anesthetic was applied on the skull (bupivacaine, 0.025% in saline). A craniectomy was made above the right hemisphere, halfway between bregma and lambda, with a 2.7 mm diameter trephine to remove a piece of the skull just above the parietal cerebral cortex. The dura was kept intact and animals which showed herniation or dura damage were discarded. Under microscopic control, the PinPoint Precision Cortical Impactor, Model PCI3000 (Hatteras Instruments, Cary, NC, USA) was positioned over the exposed dura, tilted at a 4–10° angle to ensure that the entire surface of the probe was in contact with the dura mater. The CCI injury was delivered with the following parameters: 1.5 mm depth, 3 m/s velocity and 500 ms contusion time. Using these parameters, the impactor penetrates the cerebral cortex, causing extensive structural damage in the surrounding region, but does not penetrate or cause apparent tissue damage in the underlying hippocampal formation. After delivering the brain injury the skin was closed with Vetbond tissue adhesive (3M) and the mice received an intraperitoneal saline injection before being allowed to recover in their home cage.