Collagen-induced inflammations

Collagen-induced arthritis (CIA) mouse model is currently the most widely used and reliable autoimmune model to study rheumatoid arthritis. In this model, we used bovine type II collagen (CII) and complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (IFA) to form emulsifier, and mice were injected intradermal to induce autoimmune arthritis (CIA). In this model, we ground bovine collagen type II (CII) with complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (IFA) to form emulsifiers, and intradermal injection in mice induced autoimmune arthritis (CIA). This article describes the mouse, CFA strains, key emulsification, anesthesia, and injection immune techniques, as well as the incidence, date of onset, score, pathological results of arthritis. The total time for preparation of reagents and immunization of 20 mice was about 2-2.5 hours. In this protocol, we induced a high incidence of CIA with DBA/1J in genetically susceptible mice and assessed the severity and pathology of the disease, at the same time we found that CII also can induced enteritis, including ileitis and colitis. The initial symptoms of arthritis appear in the 24-26 days of the experiment, that is, 3-5 days after the second immunization, the peak period of inflammation was 30-36 days, the arthritis incidence about 90-100%, at the same time, the incidence of enteritis and arthritis were the same, small intestinal inflammation was more severe, but the duration was short; while the colonic inflammation was mild, and the duration was longer than enteritis, we named it collagen induced inflammations (CIIs).

Collagen-induced arthritis (CIA) mouse model is currently the most widely used and 23 reliable autoimmune model to study rheumatoid arthritis. In this model, we used 24 bovine type II collagen (CII) and complete Freund's adjuvant (CFA) or incomplete 25 Freund's adjuvant (IFA) to form emulsifier, and mice were injected intradermal to 26 induce autoimmune arthritis (CIA). In this model, we ground bovine collagen type II 27 (CII) with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) 28 to form emulsifiers, and intradermal injection in mice induced autoimmune arthritis 29 (CIA). This article describes the mouse, CFA strains, key emulsification, anesthesia, 30 and injection immune techniques, as well as the incidence, date of onset, score, 31 pathological results of arthritis. The total time for preparation of reagents and 32 immunization of 20 mice was about 2-2.5 hours. In this protocol, we induced a high 33 incidence of CIA with DBA/1J in genetically susceptible mice and assessed the 34 severity and pathology of the disease, at the same time we found that CII also can 35 induced enteritis, including ileitis and colitis. The initial symptoms of arthritis 36 appear in the 24-26 days of the experiment, that is, 3-5 days after the second 37 immunization, the peak period of inflammation was 30-36 days, the arthritis incidence 38 about 90-100%, at the same time, the incidence of enteritis and arthritis were the same, 39 small intestinal inflammation was more severe, but the duration was short; while the 40 colonic inflammation was mild, and the duration was longer than enteritis, we named 41 it collagen induced inflammations (CIIs).

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Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, which 47 initially manifests as joint swelling, pain, stiffness, etc., and eventually leads to the 48 erosion of cartilage and bone tissue through long-term invasion of vascular fibers [1].

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. Experiments DBA/1J mice conformed to national and local regulations. with IFA in equal volume, keeping their ratios at 1:1 (Fig.1A).

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Emulsification should pay attention to the indoor temperature and humidity. If the 110 6 temperature is too high, the emulsion is easy to be stratified; the humidity should be 111 kept at 50-60%. If the humidity is too high, it will easily cause the mortar inner wall 112 to liquefy and form water droplets, which will cause the emulsion to be diluted, thus 113 leading to immune failure. When the emulsion droplets do not disperse on the water 114 surface, it means emulsify completely and they can be injected with immunization.

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The emulsion should be put on ice and used up within 60 mins. If the time is too long, 116 the CII will decompose or the liquid will be stratified, leading to immune failure.    133 Inhale CII emulsion into a 1 ml syringe, force the emulsion to the end of the syringe, 134 and push the syringe piston forward to expel air, then put on ice prepare to use.  (Fig. 1C), 0.1 ml/mouse, form a white mass (Fig. 1D).The white mass can be  The arthritis is markedly red and swollen, with limited activity, and identification is 159 not difficult. In the later stage of recovery from arthritis, the redness and swollen 160 subside. Sometimes identification is difficult. As described by Nanakumar etc before 161 [10], scoring was done blindly by a scoring system based on inflamed joints in each 162 paw, inflammation was defined by swelling and redness of jionts. In this scoring 163 system, each inflamed toe or finger joint has a maximum of 1 point, 5 points for each 164 paw, 5 points for metacarpal or metatarsal bones, and 5 points for wrists and ankles. 165 Thus, the maximum score per paw was 15, and the maximum score per mouse was 60. 166 We scored the arthritic paws of each mouse and recorded them every 3 days.  168 The paws of mice were fixed with 4% paraformaldehyde (pH 7.0) for 24 h, Chicago, IL). One-way analysis of variance (ANOVA) following LSD (L) was used 180 for multiple range tests and two-tailed Student's t tests were used for two group 181 comparisons. All data were presented as mean ± SD, p < 0.05 was considered 182 statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

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CIA1 and CIA2 groups have the serious arthritis compared with Con group, we can 187 got the conclusion that all the paws maybe have the arthritis, including the forepaws 188 ( Fig. 2A). Arthritis of CIA1 and CIA2 occurred on the 25th and 26th days of the 189 experiment, respectively, that is, the 4th and 5th days after secondary 190 immunization.The incidence of arthritis in both groups was 100% and 90% after 31 191 and 30 days, respectively (Fig. 2B). Arthritis began to appear on the 24th day of the 192 experiment, and the peak appeared on the 33rd day (Fig. 2C). There was no 193 significant difference between CIA1 and CIA2 groups.  The HE staining pictures of hind paw of mice at the peak of arthritis, there were many 196 inflammatory cell infiltration in the joint cavity of CIA1 and CIA2 groups (Fig. 3A).

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The HE staining pictures of hind paw of mice at the convalescence of arthritis, In 198 10 CIA1 and CIA2 groups, arthritis cells decreased, but bone and cartilage were 199 destroyed (Fig. 3B). Compared with Con group, the HE score of CIA1 and CIA2 was 200 significantly different, but there was no significant difference between the two groups 201 (Fig. 3C-D). 3.3 The bone and joints distruction at convalescence of arthiritis. 203 We used micro-CT scans of the hind paws of convalescent mice to investigate bone 204 and joints distruction. The results showed that the cortical bone and joint injuries in 205 the paws of the CIA1 and CIA2 groups were more severe than Con group (Fig. 4A).

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Bone morphometric parameters of BMC in CIA1 and CIA2 groups were lower than 207 Con group, but there was no significant difference between CIA1 and CIA2 group 208 (Fig. 4B).    The authors declare that they have no known competing financial interests or personal 253 relationships that could have appeared to influence the work in this paper.  Arthritis score after first immunization (n = 10). CIA: collagen induced arthritis, Con:

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Control group. Data are presented as the mean ± SD in (B, C).