The Rat Collagenase-Induced ICH Model

All procedures involving animals were performed to minimize pain or discomfort in accordance with current protocols approved by the Animal Research Ethics Committee of The Chinese University of Hong Kong. A cohort of 21 male Sprague-Dawley rats with a mean age of 12 weeks and body weight of 220 g were used.

Subject rats were randomly separated into two groups, one injected with PBS (sham surgery control, n = 3) and the other injected with collagenase (ICH model, n = 18). These rats were first anesthetized by intraperitoneal injection of 30 mg/kg ketamine (Alfasan, FarmaVet SA, Bucharest, Romania) and 4 mg/kg xylazine (Alfasan) then fixed to a stereotactic frame (David Kopf Instruments, Tujunga, CA). A surgical incision was made in the midline of the skull. Using a microdrill, a puncture was made at 0.2 mm posterior to the bregma and 3 mm left lateral to the midline. A Hamilton syringe with a 26-gauge needle was inserted 6 mm below the skull surface, and 1.5 μL of PBS or 0.21 collagenase digestive units (Sigma) was slowly injected into the internal capsule over 5 min. The needle was allowed to remain in place for a further 5 min before gentle withdrawal. The burr hole was sealed with bone wax, and the skin incision was sutured. Rats were allowed access to food and water ad libitum in isolator cages at 25 °C under a 12-h light/dark cycle. This was basically a collagenase-induced ICH into the striatum based on Rosenberg et al. [17] and MacLellan et al. [18]. The sham surgery group was anesthetized and sacrificed at 1 day post-PBS injection, while the ICH model groups were operated at days 1, 2, 7, 14, 30, and 60 post-collagenase injection (n = 3 at each time point). The brain hematoma and the cardiac blood were harvested, and the blood was further separated into blood cells and plasma fractions.