The induction of the murine intracerebral hemorrhage (ICH) model was achieved through the stereotactic-guided delivery of bacterial collagenase VII (C0773, Sigma-Aldrich, St. Louis, MO, USA) into the right basal ganglia [45 (link)]. Initially, mice were anesthetized with an intraperitoneal administration of pentobarbital (40 mg/kg) and subsequently positioned within a stereotaxic frame (RWD Life Science, Shenzhen, China). Following this, the bacterial collagenase VII (0.1 U in 1 μL) was administered at 0.2 mm posterior and 2.2 mm lateral of the bregma, 3.5 mm in depth, at a speed of 0.1 μL/min. The burr hole was subsequently closed with sterilized medical bone wax after the syringe remained in place for 10 min post administration. For the sham mice, an identical procedure was conducted with the replacement of bacterial collagens with saline. Throughout both the experimental and recovery periods, the animals’ body temperatures were maintained at 37.0 ± 0.5 °C.
C0773
Manufactured by Merck Group
Sourced in United States
C0773 is a laboratory centrifuge designed for general purpose use in research and clinical settings. It is capable of separating samples based on their density and molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 7, by Merck Group (2 mentions)
Stereotaxic frame, by RWD Life Science (1 mentions)
Sprague dawley rat, by Charles River Laboratories (1 mentions)
Chloral hydrate, by Merck Group (1 mentions)
Stereotactic instrument, by Stoelting (1 mentions)
Collagenase, by Merck Group (1 mentions)
Sodium borohydride, by Merck Group (1 mentions)
9 protocols using c0773
1
Murine Intracerebral Hemorrhage Model Induction
2
Collagenase-Induced Striatal Injury Model
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Rats were housed in a 12-h dark (7 pm to 7 am) and 12-h light (7 am to 7 pm) cycle. Animals were anesthetized and placed in a stereotaxic frame. Type VII collagenase (0.5 U/μL × 2.0 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. CX807 (3 mg/kg/d × 3 days) or vehicle was administered i.p. from day 0 to day 2. Animals were sacrificed on day 4 for histological and PCR analysis.
3
Intracerebral Hemorrhage in Mice: ASIV Evaluation
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Male C57 BL/6 mice (12 weeks) were obtained from the Hunan Slake Jingda Laboratory Animal Co., Ltd. (Changsha, China) and housed according to the Animals (Scientific Procedures) Act 1986. All protocols were approved by the Medical Ethics Committee of Central South University (CSU-2022-0168).
Mice were cohoused for one week before surgery. They were randomly assigned into five groups: Sham, ICH, low dose (ASIV-L, 25 mg/kg), median dose (ASIV-M, 50 mg/kg), and high dose (ASIV-H, 100 mg/kg). ASIV was purchased from Source Leaf Biological Co., LTD (S31401, purity: 99.8%, Shanghai, China). ICH was induced by collagenase injection (type VII, 0.075 unit in 0.5 μl, C0773, sigma, St. Louis, MO, USA) into the right globus pallidum (coordinate: 0.5 mm posterior, 2.0 mm lateral to the bregma and 4.0 mm ventral to the skull surface). After being operated, the animals were administrated with ASIV (suspended in distilled water) or an equal volume of distilled water by gavage daily for three days.
On the 3rd day after surgery, the mice were sacrificed and perfused with normal saline and 4% paraformaldehyde. Then, the brains were fixed in 4% paraformaldehyde for 24 h and cut into 3 μm coronal paraffin sections.
Mice were cohoused for one week before surgery. They were randomly assigned into five groups: Sham, ICH, low dose (ASIV-L, 25 mg/kg), median dose (ASIV-M, 50 mg/kg), and high dose (ASIV-H, 100 mg/kg). ASIV was purchased from Source Leaf Biological Co., LTD (S31401, purity: 99.8%, Shanghai, China). ICH was induced by collagenase injection (type VII, 0.075 unit in 0.5 μl, C0773, sigma, St. Louis, MO, USA) into the right globus pallidum (coordinate: 0.5 mm posterior, 2.0 mm lateral to the bregma and 4.0 mm ventral to the skull surface). After being operated, the animals were administrated with ASIV (suspended in distilled water) or an equal volume of distilled water by gavage daily for three days.
On the 3rd day after surgery, the mice were sacrificed and perfused with normal saline and 4% paraformaldehyde. Then, the brains were fixed in 4% paraformaldehyde for 24 h and cut into 3 μm coronal paraffin sections.
4
Collagenase-Induced Intracerebral Hemorrhage Model
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One hundred and sixty-five adult Sprague–Dawley rats (240 g–260 g), obtained from Charles River Laboratories (Wilmington, MA, USA) were accommodated in a temperature-controlled reverse light cycle environment (23°C ± 2°C, 12-hour light/dark cycle) without limited access of food and water. All experiment procedures complied with the National Institute Rules for the Care and Use of Laboratory Animals and were approved by the Guangzhou University of Chinese Medicine Animal Committee. As previously described,22 (link),23 ICH was induced as follows: the rats were anesthetized and fixed in a stereotactic frame in a prone position, then a 1 mm burr hole was made and a 26-gauge needle was inserted into the right basal ganglia. The stereotaxic coordinates were 0.2 mm anterior, 3 mm right lateral to the bregma, and 6 mm ventral to the skull. The ICH model was constructed by type VII collagenase (0.5 U in 2 μL saline per rat, C0773, Sigma-Aldrich Co., St Louis, MO, USA) at a flow rate of 0.4 μL/min. The sham group rats were perforated at the same position and infused in an equal volume of saline into the brain.
5
Collagenase-Induced Intracerebral Hemorrhage in Rats
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Sprague Dawley rats were anesthetized with 3.6% chloral hydrate (10 mL/kg, i.p.; Sigma‐Aldrich, St. Louis, MO, USA) following 12‐h fasting and 4‐h water deprivation. ICH was induced by stereotactically infusing collagenase VII (0.4 U in 0.8 μL sterile saline, C0773; Sigma‐Aldrich) into the caudate nucleus. The injection coordinates were 3.0 mm right, 0.5 mm anterior, and 5.5 mm ventral from bregma at the skull surface using a stereotactic instrument (Stoelting, Wood Dale, IL, USA). Collagenase was continuously injected for 4 min at a speed of 200 nL/min. The needle was then held in place for another 10 min to prevent backflow, and the hole was sealed with bone wax. The rats were then housed in a biologically clean room. Neurological abnormalities were assessed according to a modified neurological severity scores (mNSS), and scores were recorded at 0.5, 1.5, 3, 7, 14, and 28 days post‐ICH by two independent investigators blinded to the experimental treatment scheme. Neurological function was graded on a scale of 0–18 (normal score, 0; maximal deficit score, 18) 10 .
6
Murine Model of Collagenase-Induced Osteoarthritis
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Ten-week-old male C57/BL6 mice (n = 48) were subjected to intra-articular injection collagenase to induce CIOA as previously described [29 ]. Briefly, 1 U of collagenase (C0773; Sigma-Aldrich, St. Louis, MO, USA) was injected into the right knee joint twice on alternate days. Only skin of the right knee joint was resected in the sham-operated group. Some mice from the CIOA group were treated with fargesin (5, 10, or 20 mg/kg, 10 μL, n = 36; PHL82537, Sigma, USA), while others were treated with vehicle (20% DMSO dissolved in saline) by intra-articular injection twice a week for 1, 3, or 6 weeks (10 μL, n = 12). Then, 1, 3, or 6 weeks after operation, mice from each group were sacrificed for collection of the right knee joint (Additional file 1 : Figure S1A). Articular cartilage degeneration was quantified using the Osteoarthritis Research Society International (OARSI) scoring system. H&E staining was used to evaluate synovial activation by scoring synovial lining cell thickness (0–3), as previously described [29 ].. Then, the sum of medial and lateral joint compartments was determined (0–6).
7
Collagenase-induced Intracerebral Hemorrhage Rat Model
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The rats were randomly divided into two groups by lottery method: Sham group (n = 6) and ICH group (n = 18). Rats were anesthetized with 4% isoflurane using an anesthetic gas machine and fixed on a brain stereoscopic injection equipment. 2.5% isoflurane and 1 L/min oxygen were used for continuous anesthesia during surgery. ICH model was constructed by unilaterally stereotactic injection of collagenase VII (0.2 U in 1 μL sterile saline, C0773; Sigma–Aldrich) to striatum region according to the Paxinos and Watson brain atlas [21 ]. The injection coordinates related to bregma was M/L = +0.5 mm, A/P = −3.0 mm, D/V = −5.87 mm. Rats of Sham group were injected with 1 μL sterile saline. After injection, the needle was left in place for an additional 10 min to minimize backflow.
8
Collagenase-Induced Osteoarthritis in Mice
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CIOA was induced as previously described.28 (link) Briefly, mice received an intra-articular injection of one unit of collagenase type VII (C0773, Sigma–Aldrich, US) with saline on days 0 and 2 to induce joint instability in the right knee. The knee joints were harvested and processed for histological examination 4 weeks after CIOA.
9
Quantifying Collagen and Cross-Links
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Analysis of collagen and of collagen cross-links was performed as reported previously [15 (link)]. Briefly, both lungs from two animals were pooled and treated with sodium borohydride (Sigma, 25 mg NaBH4/ml in 0.05 M NaH2PO4/0.15 M NaCl pH 7.4, 1 h on ice, 1.5 h at RT) to stabilize reducible acid-labile cross-links, digested for 12 h at 37 oC with high purity bacterial collagenase (C0773; Sigma, 50 U/ml) and hydrolyzed in 6 N HCl at 110 °C for 24 h. The hydrolysates were precleared by solid phase extraction and analyzed on an amino acid analyzer (Biochrome30, Biochrome, Cambridge, UK). Quantification was based on ninhydrin-generated leucine equivalence factors (DHLNL, HLNL: 1.8). The nomenclature used in the manuscript refers to the reduced variants of cross-links (DHLNL, HLNL). For protein analysis, specimens were digested with bacterial collagenase. After centrifugation, the soluble fraction containing collagen was subjected to hydrolysis and amino acid analysis. Collagen content was calculated based on a content of 14 mg hydroxyproline in 100 mg collagen. The residual fraction was extracted with hot alkali (0.1 N NaOH, 95 °C, 45 min). After centrifugation, the supernatant containing non-collagen/non-elastin proteins and the insoluble residue containing elastin were subjected to hydrolysis and amino acid analysis.
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