Bacterial collagenase 7

C57BL/6 mice were anesthetized by injecting pentobarbital (40 mg/kg) intraperitoneally and placed in a stereotaxic frame (Stoelting Stereotaxic instrument). A 1-mm burr hole in diameter was made in the skull (0.2 mm anterior to the bregma, 2.2 mm right lateral to the midline). Then, the experimental ICH model was induced by injecting bacterial collagenase VII (0.1 U in 0.5 μL, Sigma, St. Louis, MO) into the right basal ganglia (3.5 mm depth below the skull) using a micro-perfusion pump within 5 min. In case backflows occurred, the needle was not removed for an additional 10 min after the completion of the injection [2 (link), 25 (link)]. The burr hole was blocked with bone wax. In the sham group, C57BL/6 mice underwent the same procedures, but 0.5 μL saline was injected instead of collagenase. During the procedure, rectal temperature was maintained at 37.0 ± 0.5 °C using a temperature-regulated heating pad.

Mice were anaesthetized with 1% pentobarbital sodium (50 mg/kg) intraperitoneally. After being anaesthetized, mice were secured onto a stereotaxic instrument. Skull burr holes based on the stereotaxic coordinates of the Paxinos and Franklin mouse brain atlas (0.2 mm posterior, 2.8 mm ventral, and 2.2 mm lateral to the bregma) were drilled prior to insertion of a needle into the striatum. Bacterial collagenase VII (0.1 U in 0.4 μl; Sigma) was infused at a rate of 400 nl/min over 1 minute into the right striatum. After 10 minutes, the needles were gently removed. 15 minutes after this, either CLM diluted in 200 μl normal saline, or equal vehicle volume were given by intraperitoneal injection to the mice. All experiments were performed in threes.

A total of 180 eight to ten weeks old male C57BL/6 mice weighing 25-35 g (8-10 weeks old) were used in the experiments and the mice were provided by the Animal Center at Huafukang (Beijing, China). All animal procedures were conducted in compliance with the guidelines of the Army General Hospital's Committee on the Ethics of Animal Experiments, and reported using the ARRIVE criteria. Throughout the study, the mice had unrestricted access to food and water. ICH was induced with collagenase injection as reported previously. Briefly, the mice were anesthetized with isoflurane (3% for induction, 1% for maintenance) and ventilated with oxygen-enriched air (20%:80%) via a nasal cone. To induce bleeding, bacterial collagenase VII (0.1 U in 0.4μl; Sigma) was injected into the right caudate at the following stereotactic coordinates: 0.5 mm anterior and 2.0 mm lateral of the bregma, 2.8 mm in depth. Collagenase was administered over a 5-minute period, and the needle was kept in place for another 15 minutes to prevent reflux. The mice/10g were intraperitoneally injected with 100μL RA or an equal amount of vehicle (DMSO concentration <1%) 15 minutes after ICH.