After the rats were anesthetized by intramuscular injection with ketamine (60 mg/kg) and xylazine (6 mg/kg), 2 μL (0.2 μL/min) of saline containing 0.4 units of bacterial collagenase (type IV; Sigma-Aldrich, Germany) were infused via a 30-gauge needle into the striatum (1.0 mm posterior, 4.0 mm lateral, 6.0 mm ventral to the cortical surface) [44 (link)]. The sham group was infused with 2 μL of saline. The needle was left in place for an additional 10 minutes following infusion to prevent backflow. The craniotomies were sealed with bone wax. The rats were left to recover in individual cages in the animal center under a 12/12 h light/dark cycle with free access to food and water. Rats in the NBP group were treated with NBP sodium chloride 30 minutes after ICH induction (CSPC NBP Pharmaceutical Co., Ltd., China, 25 mg/kg, twice per day, intraperitoneally [18 (link), 31 (link)]), while rats in the sham and vehicle groups were given the same amount of normal saline by intraperitoneal injection.
Bacterial type 4 collagenase
Manufactured by Merck Group
Sourced in United States
Bacterial type IV collagenase is a hydrolytic enzyme that cleaves type IV collagen, a major structural component of basement membranes. It is commonly used in cell isolation and tissue dissociation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Small animal temperature controller, by Kopf Instruments (3 mentions)
Stereotaxic head frame, by Stoelting (3 mentions)
Temperature controller, by Kopf Instruments (2 mentions)
Dental drill, by Dremel (2 mentions)
Cd 1 mice, by Charles River Laboratories (2 mentions)
Stereotaxic frame, by Stoelting (2 mentions)
High speed drill, by Dremel (2 mentions)
Hamilton syringe 26 g, by Merck Group (2 mentions)
Stereotaxic frame, by Kopf Instruments (1 mentions)
Male cd 1 mice, by Charles River Laboratories (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Adult male cd1 mice, by Charles River Laboratories (1 mentions)
11 protocols using bacterial type 4 collagenase
1
Intracerebral Collagenase-Induced Hemorrhage in Rats
2
Intracerebral Hemorrhage Model in C57BL/6 Mice
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The adult male C57BL/6 mice (aged 8–10 weeks; weight, 25–30 g) used in this study were obtained from the Comparative Medical Center of Yangzhou University. All mice were housed in an air-conditioned (22 ± 1 ℃) room with a 12 h light/dark cycle and freely available water and food. All animal use and experimental protocols were approved by the Institutional Animal Care and Use Committee and the Animal Ethics Committee of Yangzhou University [SYXK (Su) IACUC 2017-0045].
To generate the ICH model, mice were anesthetized with 10% chloral hydrate (0.25 ml/100 g, intraperitoneally, i.p.) and placed in a stereotaxic frame. Bacterial collagenase type IV (0.05 U) (Sigma-Aldrich Co. St. Louis, MO, USA) in 0.2 μL of saline was injected into the right caudate putamen 3.7 mm laterally and 0.2 mm anteriorly to the bregma, at a depth of 3.8 mm, as described previously [27 (link)].
The injections took 5 min, and the needle was left in place for an additional 5 min. Bone wax was used to seal the burr hole and the scalp was sutured. During the entire surgery, the body temperature of the mice was maintained at 36.5–37.5 ℃ with a heating blanket. Sham-operated mice were treated identically, except they were injected with an equal volume of normal saline.
To generate the ICH model, mice were anesthetized with 10% chloral hydrate (0.25 ml/100 g, intraperitoneally, i.p.) and placed in a stereotaxic frame. Bacterial collagenase type IV (0.05 U) (Sigma-Aldrich Co. St. Louis, MO, USA) in 0.2 μL of saline was injected into the right caudate putamen 3.7 mm laterally and 0.2 mm anteriorly to the bregma, at a depth of 3.8 mm, as described previously [27 (link)].
The injections took 5 min, and the needle was left in place for an additional 5 min. Bone wax was used to seal the burr hole and the scalp was sutured. During the entire surgery, the body temperature of the mice was maintained at 36.5–37.5 ℃ with a heating blanket. Sham-operated mice were treated identically, except they were injected with an equal volume of normal saline.
3
Intracerebral Hemorrhage Induction in Rats
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As in previous studies, cerebral hemorrhage was induced in the right caudate nucleus (Wu et al., 2016 (link)). The rats were anesthetized with isoflurane and fixed in a stereotaxic apparatus. A hole was perforated 4.0 mm laterally to the median line of the brain and 0.1 mm anterior to the bregma, and 1.5 μl saline mixed with 0.38U of bacterial collagenase (type IV; Sigma-Aldrich, St. Louis, MO, United States) was injected via a 5.0-mm needle below the plane of the cranial bones.
4
Intracerebral Hemorrhage Mouse Model
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ICH in mice was induced by intra-striatal injection of autologous blood or bacterial collagenase as previously described (4 (link), 43 (link)). Briefly, mice were anesthetized with ketamine/xylazine mixture by intraperitoneal injection and positioned prone in a stereotactic head frame. A ~1-mm-diameter hole was drilled on the right side of the skull (2.3 mm lateral to midline, 0.5 mm anterior to bregma). In the autologous blood model, 30 μl of nonheparinized blood was withdrawn from angular vein and infused via infusion pump to the coordinates; 5 μl was initially injected at a rate of 1 μl/min at a depth of 3.0 mm, and the remaining 25 μl was injected at an identical rate at 3.7 mm depth. The needle was left for 15 min to prevent backflow and then gently withdrawn. In the collagenase model, 0.0375 U of bacterial collagenase (type IV, Sigma-Aldrich, St. Louis, MO) in 0.5 μl of saline was administered at the same coordinates at rate of 0.5 μl/min. An identical surgical procedure with equal volume of sterile saline injection for sham-operated groups. Cranial burr hole was sealed with bone wax, and incision was sutured. Body temperature was maintained at 37.0° ± 0.5°C throughout the procedures. The total mortality rate of mice subjected to ICH was ~4.8%.
5
Murine Intracerebral Hemorrhage and Tibial Fracture
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The mouse ICH model was established as our previously reported [13, (link)20] (link). In brief, mice were xed in stereotaxic frame (David Kopf Instruments, Tujunga, California) after anesthetized with 4% chloral hydrate by intraperitoneal injection. Then carried out craniotomy after exposed the skull. Subsequently, 0.5 μL (0.1 μL/min) saline containing 0.05 units of bacterial collagenase (type IV, Sigma, USA) was injected into the left striatum at coordinates 1.0 mm anterior and 2.0 mm lateral to bregma and 3.5 mm below the cortical surface, using a microinjection system (LongerPump, China). The needle was kept for another 5 min after injection to prevent leakage. Needle was inserted into the striatum after craniotomy but the collagenase was not injected in sham group (Sham). Tibial fracture (TF) surgery was performed according to the literature described previously [11] (link). Brie y, the bilateral tibia with an intramedullary xation was fractured after mice anesthetized. The model of ICH accompanied by fracture (MI) was performed fracture surgery immediately after ICH. After the surgery, all mice were placed next to a heater to maintain their normal body temperature and put back to cages when they recovered from anesthesia. All of the above operations were performed under aseptic conditions.
6
Induction of Intracerebral Hemorrhage in Mice
7
Mouse Model of Intracerebral Hemorrhage
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Intracerebral hemorrhage was induced in adult male CD-1 mouse (8–12 weeks; n = 43), as reported previously (Sukumari-Ramesh et al., 2012a (link),b (link), 2016 (link); Bonsack et al., 2016 (link); Sukumari-Ramesh and Alleyne, 2016 (link); Ahmad et al., 2017 (link); Chen-Roetling et al., 2017 (link)). Briefly, mouse was anesthetized (ketamine and xylazine) and a small incision was made to expose the skull. Using a high-speed drill, a burr hole (0.5 mm) was made on the skull approximately 2.2 mm lateral to bregma. Then the mouse was placed on to a stereotaxic head frame and a 26-G Hamilton Syringe was used to inject 0.04U of bacterial type IV collagenase (Sigma, St. Louis, MO, United States) in 0.5 μL Phosphate Buffer Saline (pH 7.4; PBS) into the left striatum (3.0 mm) under stereotaxic guidance. Upon removal of the needle, bone wax was used to seal the burr hole. Mice were kept at 37 ± 0.5°C using a small animal temperature controller throughout the procedure. The temporal pattern of hematoma after ICH is provided (Supplementary Figure S1 ).
8
Collagenase-Induced Intracerebral Hemorrhage in Mice
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All animal studies were performed according to protocols approved by the Institutional Animal Care and Use Committee in accordance with the NIH and USDA guidelines. As reported previously (Bonsack et al. 2016 (link); Sukumari-Ramesh et al. 2012a (link), b (link)), ICH was induced in adult male CD-1 mice (8–12 weeks, Charles River, USA). Briefly, mice were anesthetized with ketamine and xylazine and positioned prone on a stereotaxic frame (Stoelting, WI, USA.). Throughout the surgical procedure, the body temperature was maintained at 37 ± 0.5 °C employing a temperature controller (David Kopf Instruments, USA). Using a dental drill (Dremel, USA), a burr hole (0.5 mm) was made 2.2 mm lateral to bregma, not damaging the underlying dura. A Hamilton syringe (26-G) containing 0.04 U of bacterial type IV collagenase (Sigma, USA) in 0.5 μL phosphate-buffered saline (PBS, pH 7.4) was inserted stereotaxically into the left striatum to induce spontaneous ICH. After the needle was removed, the burr hole was covered with bone wax and the incision was sutured. Sham mice underwent the same surgical procedure, but only PBS (0.5 μL) was injected.
9
Spontaneous Intracerebral Hemorrhage Model
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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.
10
Intracerebral Hemorrhage Induction in Aged Mice
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All animal studies were performed according to the protocols approved by the Institutional Animal Care and Use Committee, in accordance with the NIH and USDA guidelines. Intracerebral hemorrhage was induced in aged male C57BL/6 mice (18–24 months), (Jackson Laboratories, Bar Harbor, ME, USA), as previously reported by our laboratory [2 (link),30 (link),31 (link),32 (link),33 (link)]. Briefly, mice were anesthetized with isoflurane and positioned prone on a stereotaxic head frame (Stoelting, Wood Dale, IL, USA). Using a high-speed drill (Dremel, Racine, WI, USA), a burr hole (0.5 mm) was made 2.2 mm lateral to the bregma, and a small animal temperature controller (David Kopf Instruments, Los Angeles, CA, USA) was used to keep the body temperature at 37 ± 0.5 °C. Employing a Hamilton syringe (26-G), 0.04 U of bacterial type IV collagenase (Sigma, St. Louis, MO, USA) in 0.5 μL phosphate-buffered saline (phosphate buffered saline; pH 7.4 (PBS) was injected with the stereotaxic guidance 3.0 mm into the left striatum to induce ICH [2 (link)]. After removing the needle, bone wax was used to seal the burr hole and the incision was stapled. Sham mice underwent the same surgical procedure, but only PBS (0.5 μL) was injected, which served as the experimental control.
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