Stereotaxic head frame

Experimental cICH model in mice was induced by intrastriatal injection of collagenase as described previously^{47 (link)}. Briefly, mice were anesthetized with 10% chloral hydrate (500 mg/kg, intraperitoneal injection) and positioned prone in a stereotaxic head frame (Stoelting, Wood Dale, IL). The calvarium was exposed by a midline scalp incision from the nasion to the superior nuchal line to retract the skin. A burr hole (0.2 mm posterior to bregma and 2.3 mm to the right of the midline) was made with a drill (Fine Scientific Tools, Foster City, CA). A 26-G needle on a Hamilton syringe was inserted with stereotaxic guidance 3.5 mm into the deep cortex/basal ganglia. The collagenase (0.05 units in 1 µl saline; Sigma, St Louis, MO) in the syringe was infused into the brain at a rate of 0.2 μl/min over 5 minutes. Saline with same volume was injected in Sham group. The needle was left in place for an additional 7 minutes after injection to prevent the possible leakage of collagenase solution. The craniotomy was then sealed with bone wax, and the scalp was closed with sutures. Body temperature was maintained at 37 °C by a heating pad throughout the procedure. After waking up, the mice were given free access to food and water. The mice without neurological deficit or the dead mice (~20–30% mortality) were excluded from the following analysis.

ICH was induced via a stereotactically guided injection of collagenase into right basal ganglia as previously described^{52 (link)}. Briefly, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg, intraperitoneal injection) and positioned prone in a stereotaxic head frame (Stoelting, Wood Dale, IL, USA). An electronic thermostat-controlled warming blanket was used to maintain the core temperature at 37 °C. The calvarium was exposed by a midline scalp incision from the nasion to the superior nuchal line, and the skin was retracted laterally. With a variable speed drill (Fine Scientific Tools, Foster City, CA, USA) a 1.0 mm burr hole was made 0.9 mm posterior to bregma and 1.45 mm to the right of the midline. A 26-G needle on a Hamilton syringe was inserted with stereotaxic guidance 4.0 mm into the right deep cortex/basal ganglia at a rate 1 mm/min. The collagenase (0.075 units in 0.5 µl saline, VII-S; Sigma, St Louis, MO, USA) in the syringe was infused into the brain at a rate of 0.25 µl/min over 2 minutes with an infusion pump (Stoelting, Wood Dale, IL, USA). The needle was left in place for an additional 10 minutes after injection to prevent the possible leakage of collagenase solution. After removal of the needle, the incision was closed and the mice were allowed to recover. Mice were subjected to sham operation received only needle insertion.

Animal studies were reviewed and approved by the Committee on Animal Use for Research and Education at Augusta University, in compliance with NIH and USDA guidelines. Male CD-1 mice (8–10 weeks old; Charles River) were anesthetized with an intraperitoneal injection of ketamine and xylazine and positioned prone in a stereotaxic head frame (Stoelting, WI, USA). A small animal temperature controller (David Kopf Instruments, USA) was used to maintain the body temperature at 37 ± 0.5 °C throughout surgery. With a high-speed dental drill (Dremel, USA), a 0.5-mm burr hole was made 2.2 mm lateral to the bregma, taking care not to damage the underlying dura. A 26-G Hamilton syringe containing 0.04 U of bacterial type IV collagenase (Sigma, St. Louis, MO, USA) in 0.5-μl saline was inserted with stereotaxic guidance 3.0 mm into the left striatum to induce spontaneous ICH [35 (link), 36 (link)]. After removal of the needle, the burr hole was sealed with bone wax and the incision was surgically stapled. Sham animals underwent the same surgical procedure, but only a saline injection (0.5 μl) was performed. Mice were maintained at 37 °C until recovery.