Collagenase type 7

Rats were housed in a 12 h dark (7 p.m. to 7 a.m.) and 12 h light (7 a.m. to 7 p.m.) cycle. Animals were anesthetized and placed in a stereotaxic frame. Type VII collagenase (0.5 U/μL × 1.0 μL, C-0773, Sigma Aldrich, St. Louis, MO, USA) was stereotactically injected into the right striatum (coordinates: 0.0 mm rostral and 3.0 mm lateral to bregma, 5.5 mm below the skull) at 0.4 μL/min over 5 min on day 0. Then, 2FL (400 mg/kg/day × 5 days) or vehicle was administered i.p. from days 1 to day 5. Animals were sacrificed on day 5 for histological and PCR analysis.

Twelve‐weeks‐old’ C57BL/6J male mice (Charles River) were divided into four groups (5 males/group). The approaches to induce arthritis were described in previous study.^[43 (link)
^] After being anesthetized with 0.8% pentobarbital, the mice in sham group had the skin of the right knee joint cut without injecting collagenase. While for arthritis group, the knee joints were fully exposed and injected with 1 unit type VII collagenase (Sigma, C0773) in 10 µl CaCl₂ at day 1 and day 3, respectively. For transgenic mice, the same procedures were applied to experimental groups.
The cargoes for AnCar‐Exo^LaIMTS3 were loaded by using Exo‐Fect exosome transfection reagent (SBI, EXFT20A‐1). In brief, ≈1 × 10⁹ exosomes were transfected with 20 pmol siRNA using 10 µl Exo‐Fect exosome transfection reagent. After incubating the exosome transfection solution at 37 °C in a shaker for 10 min, the mixture was placed on ice immediately. Exosomes were injected into joint cavity at the dose of 1 × 10⁹ particles per mouse. According to the distribution of exosomes in vivo, exosomes were injected at days 4, 7, 10, and 13 after induction of arthritis by collagenase at day 1. Mice were sacrificed on day 14. The sequences of siRNA‐HIF‐1α were the following: siRNA‐HIF‐1α−1, GGAAAGAACUAAACACACATT; siRNA‐HIF‐1α−2, CUGAAGACACAGAGGCAAATT; siRNA‐HIF‐1α−3, AAACAGAGACGAAGGACAATT.

The ICH model of mice was established according to previously described methods [25 (link)]. Briefly, mice were anesthetized with 3% isoflurane for induction and placed in a stereotaxic instrument (RWD Life Science Co., Shenzhen, China). Then, 1.5% isoflurane was used for maintenance to minimize the suffering of the mice during surgery. Approximately 0.5 μL of 0.075 IU type VII collagenase (Sigma Aldrich) was injected at a rate of 0.0625 μL/min into the left striatum (anterior: 0.8 mm, lateral of bregma: 2 mm, depth: 3.5 mm) using a syringe pump (Hamilton, Bonaduz, AG). In contrast, 0.5 μL of saline was injected into the control (sham) group. During the surgery, the body temperature of the animals was maintained at 37°C. After surgery, the mice were monitored at least once a day, and no accidental deaths were observed. Three days after ICH, the mice were euthanized by cervical dislocation under deep isoflurane anesthesia. Then, perihematomal and contralateral brain tissues were obtained to perform the succinylproteome analysis.

The establishment of the ICH model followed a previously described protocol [20 (link)]. Briefly, mice were anesthetized by intraperitoneal administration of pentobarbital sodium (40 mg/kg, 1%) and collagenase (0.05 U type VII collagenase in 0.5 μl saline, from Clostridium histolyticum; Sigma–Aldrich) was stereotactically injected into the right basal ganglia (2.5 mm lateral to the bregma, 3 mm deep at a 5° angle). The rectal temperature of the mice was kept constant at 37.0 ± 0.5°C. The sham-operated group was treated similarly, but no collagenase was included in the injection.