The rats were anesthetized using isoflurane and were shaved around the base of the tail. Rats were immunized, using a protocol modified from Nielsen et al. [32 (link)], with 100 μl of 2 mg/ml porcine type II collagen (Chondrex) dissolved in 0.05 M acetic acid and emulsified 1:1 in incomplete Freund’s adjuvant injected intradermally around the base of the tail. One week later, another 100 μl of the same emulsion was injected. Rats that were not immunized were injected with saline.
Porcine type 2 collagen
Manufactured by Chondrex
Sourced in United States
Porcine type II collagen is a natural, soluble protein extracted from porcine cartilage. It serves as a key component in the development and maintenance of articular cartilage.
Automatically generated - may contain errors
Lab products found in correlation
Incomplete freund s adjuvant ifa, by Merck Group (3 mentions)
Incomplete freund s adjuvant, by Merck Group (2 mentions)
Incomplete freund s adjuvant, by Chondrex (1 mentions)
R mct2, by Rigaku (1 mentions)
Pristane, by MP Biomedicals (1 mentions)
Female lewis rats, by Charles River Laboratories (1 mentions)
Labgen 125, by Cole-Parmer (1 mentions)
Pentoxifylline, by Merck Group (1 mentions)
Milrinone, by Cayman Chemical (1 mentions)
Polyethylene glycol peg 400, by Merck Group (1 mentions)
Concanavalin a (cona), by Santa Cruz Biotechnology (1 mentions)
Vinpocetine, by Cayman Chemical (1 mentions)
Papaverine, by Cayman Chemical (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Tofacitinib citrate salt, by LC Laboratories (1 mentions)
10 protocols using porcine type 2 collagen
1
Collagen-Induced Arthritis Protocol
2
Collagen-Induced Arthritis Model in Rats
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CIA was induced in six 7-week-old female Lewis rats by immunizing 200 ml solution of 1 mg/ml porcine type II collagen (Chondrex) dissolved in 0.05 M acetic acid and emulsified in incomplete Freund's adjuvant (Chondrex) at the base of the tail [40 (link)]. Seven days later, the rats received a booster immunization with the collagen. On day 15, the rats received an intra-articular injection, into both right and left ankles, of a 50 μl solution containing 10 μM siRNA–atelocollagen (Koken) complexes targeting p97 (Qiagen) or control. Silencing efficiency of p97 siRNAs was evaluated by Western blotting in in CIA tissues obtained from knee and ankle joints three days after the injection of siRNAs into ankle joints. On days 1, 8, 15, 18, 22, 26 and 29, arthritis was scored according to paw thickness and ankle diameter from 0 (neither erythema nor swelling) to 4 (erythema and severe swelling that encompassed the ankle, foot, and digits or ankylosis of the limb) [41 ]. Bone erosion of each ankle joint was scored in a blinded manner on day 29 by micro–computed tomography (micro-CT) (R_mCT2, Rigaku) from 0 (normal joint) to 3 (severe cartilage and bone erosions) [42 (link)]. The experimental protocol was approved by the Animal Ethics Committee at Hokkaido University.
3
Rodent Models of Arthritis Induction
4
Rat CIA Arthritis Characterization Protocol
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The rat CIA model was used to characterize the effects of RCI therapy on established arthritis [25 (link)–29 ]. Female dark Agouti rats (weight: 120–170 g) were obtained from Harlan Laboratories, Inc. (Indianapolis, IN) and acclimated for approximately 8 days before initiation of CIA. All animal use was in accordance with the guidelines cited in the Guide for the Care and Use of Laboratory Animals [30 ] (Institutional Animal Care Use Committee Protocol BBP12–002). Rats were anesthetized with isoflurane, which has been shown to have a minimal effect on inflammation, and injected with 400 μL of Freund’s incomplete adjuvant (Difco, Detroit, MI) emulsified with 2 mg/mL porcine type II collagen (Chondrex, Inc., Redmond, WA) at 2 sites on the back (200 μL per site) on day 0, and then 100 μL at 1 site on day 7. Arthritis disease was represented by an increase in ankle diameter observed at day 12 and reaching a plateau at days 18 through 20.
5
Collagen-Induced Arthritis Model in Rats
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Female Lewis rats, weighing between 175 and 200 g (Charles River Laboratories; Hollister, CA, United States), were used in these studies. Subjects were randomized into study groups, such that all groups had similar average baseline body weights and activity profiles prior to induction. We employed the CIA model, a well-established and validated rodent rheumatology model (Bendele, 2001 (link); Bevaart et al., 2010 (link); Bolon et al., 2011 (link)). Briefly, to induce arthritis, rats were anesthetized under isoflurane and administered intradermal injections of 2 mg/mL of porcine type II collagen (Chondrex; Redmond, WA, United States) in Incomplete Freund’s Adjuvant (IFA) (Sigma; St. Louis, MO, United States) at two sites on the back of the animal. A booster was given 7 days post-induction. Control animals were injected with IFA only (Figure 1A ).
6
Collagen-Induced Arthritis in Rats
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CIA was induced as described previously (54 , 61 (link)). Briefly, female Lewis rats received a subcutaneous injection of 200 μL of a 1:1 emulsion of 2 mg/mL porcine type II collagen (20031, Chondrex, Redmond, WA) with incomplete Freund’s adjuvant at the base of the tail. After 7 d, rats were given a booster of 100 μL of collagen and adjuvant emulsion. Disease onset was defined as the development of at least one swollen or red paw joint. Clinical scores were determined daily by assigning one point for each swollen or red toe joint, two points for mildly swollen wrist or ankle joints, and five points for each severely swollen wrist or ankle, giving each rat a maximum possible score of 60. Upon disease onset, rats were treated every other day by the subcutaneous injection of P6N buffer vehicle or 0.1 mg/kg ShK-235, or the oral gavage of 1 × 109 cfus LrGusA or 1 × 109 cfus LrS235 daily. CIA is more severe in rats with early disease onset. To avoid biasing our results based on disease severity on the day, each rat developed signs of disease and accounting for differences in the time between immunization, and when a rat developed signs of illness, every rat that developed signs of disease on a given day was placed in a different treatment group to fill all groups in parallel.
7
Collagen-Induced Arthritis Model in Rats
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Female Lewis rats were subcutaneously injected at the base of the tail with the emulsion prepared according to the protocol supplied by Chondrex, Inc. (Redmond, WA, USA) at days 0 and 7 of the experiment. To obtain the emulsion, porcine collagen type II (Chondrex, Inc.) (2 mg/mL) in 0.05 M acetic acid was emulsified with incomplete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA) using a homogenizer with a small blade (LabGen 125, Cole-Parmer, Vernon Hills, IL, USA). On day 20, the animals that developed symptoms of arthritis, i.e., paw swelling above 150% of day 0, were randomly divided into two groups: the control group receiving vehicle alone (n = 6) and the study group treated with the investigated compound at a dose of 20 mg·kg−1, IP once daily for 21 days (n = 6). Relative paw edema of arthritic rats was measured using a digital caliper according to the procedure described by Earp et al. [26 (link)] up to 40 days after the first immunization and then the animals were sacrificed. In addition, the body weight of animals was monitored. After randomization on day 20, using the Mann–Whitney U test, it was verified that there is no significant difference in paw size between the groups.
8
Immunomodulatory Agents Protocol
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Pentoxifylline (PTX), dimethylsulfoxide (DMSO), polyethylene glycol (PEG) 400, LPS (E. coli 055:B5 serotype), incomplete Freund’s adjuvant (IFA), and RPMI 1640 medium were purchased from Sigma-Aldrich (Germany). ConA was purchased from Santa Cruz Biotechnology (USA). Porcine collagen type II (2 mg mL−1 in 0.05 M acetic acid) was obtained from Chondrex, Inc. (USA). Vinpocetine, EHNA, milrinone, zaprinast, and papaverine were purchased from Cayman Chemical (USA). (±)-LSF was obtained from the Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University (Krakow, Poland). GRMS-55 and 4-(8-((Furan-2-ylmethyl)amino)-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-7-yl)-N′-(2-hydroxybenzylidene)butanehydrazide were obtained from the Department of Medicinal Chemistry and rolipram from the Department of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College (Krakow, Poland). Temazepam was a gift from Polfa (Poland). Other reagents and solvents were of HPLC or analytical reagent grade and were purchased from Merck (Germany).
9
Porcine Collagen Immunization Protocol
10
Porcine Collagen Inflammation Model
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