Method for establishing cat osteoarthritis pain model

Abstract

The invention discloses a method for establishing a cat osteoarthritis pain model, which comprises the following steps of: performing experimental research on immunogen, drug concentration, immunization frequency, administration route, injection dosage and the like, and adopting collagen immunogen and 1% carrageenan immunogen as mixed immunogen; a cat is immunized by adopting a two-point injection mode of subcutaneous injection near a shoulder blade and plantar subcutaneous injection, the immunization procedure adopts twice immunization of 0 day and 7 days, a simple and efficient cat osteoarthritis pain model is successfully constructed, the model has obvious joint and claw swelling, a cat claw retraction threshold value is reduced, a TNF-a factor is obviously increased, and the model can be used for researching the pain of the cat osteoarthritis, the pain of the cat osteoarthritis and the pain of the cat osteoarthritis. The method can be used for researching the pathogenesis, pathological change, pain mechanism and OA treatment method of the cat osteoarthritis, and can also be used for evaluating the clinical effect of a novel analgesic drug for the cat osteoarthritis.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a method for establishing a cat osteoarthritis pain model. Background Art

[0002] Osteoarthritis (OA), also known as osteoarthritis, degenerative arthritis, proliferative arthritis, and senile arthritis, is a chronic joint disease with unknown etiology, characterized by degeneration of articular cartilage and secondary bone hyperplasia.

[0003] Animal models not only help scientists to study the pain mechanism of osteoarthritis in depth, but also provide a basis for finding new treatments. However, previous OA models were mainly used to reflect structural pathological changes. Until recently, the effectiveness of OA models for studying human OA pain has not been elucidated in detail. Animal OA models include two major categories: (1) Surgical models: transection of the meniscus and / or cruciate ligament, or joint instability, etc. (2) Chemical models: intra-articular injection of sodium iodoacetate, etc. The above models have been used to study OA pathology and the mechanism of OA pain.

[0004] Animal OA develops gradually. For example, Dunkin-Hartley guinea pigs, STR / ort mice, dogs and horses. However, the development of animal OA is difficult to predict and lacks effective control measures. At the same time, the difference in OA models also reflects the difference in pain experience between individual OA patients. At present, there is a lack of research on the method of establishing a feline osteoarthritis model.

[0005] Feline osteoarthritis is a very common chronic joint disease in older cats, characterized by the degeneration and wear of articular cartilage, leading to inflammation, discomfort and persistent damage within the joints. This disease is widespread and highly prevalent in older cats, with the incidence rate increasing significantly with age, which has a serious impact on the quality of life of cats. According to multiple studies, 61% of cats over the age of 6 suffer from at least one osteoarthritis, and when cats reach the age of 12, this proportion is as high as 90%.

[0006] Cats are common companion animals, and their disease and pain management is crucial to ensuring their quality of life. However, because cats are born with a strong ability to tolerate pain, and their behavioral changes are relatively hidden, the signs of pain are usually subtle and difficult to identify. The actual diagnosis rate is much lower than the prevalence rate, only less than 10%, which undoubtedly increases the difficulty of prevention, control and treatment of the disease.

[0007] Therefore, in order to deeply study the pathogenesis, pathological changes, pain mechanism, treatment methods and new analgesics of feline osteoarthritis and improve the quality of life of cats, it is of great significance to establish a suitable feline osteoarthritis pain model to provide an important research platform for scientific researchers. Summary of the invention

[0008] The purpose of the present invention is to propose a method for establishing a feline osteoarthritis pain model, and to provide a research platform for researchers on the pathogenesis, pathological changes, pain mechanisms, treatment methods and analgesics of feline osteoarthritis.

[0009] In order to achieve the above object, the technical solution of the present invention is as follows:

[0010] The invention provides a method for establishing a feline osteoarthritis pain model. The method adopts a collagen immunogen and a 1% carrageenan immunogen as a mixed immunogen, and adopts a two-point injection mode of subcutaneous injection near the shoulder blade and subcutaneous injection at the sole of the foot to immunize the cat, and the immunization procedure adopts two immunizations at 0 days and 7 days; the final concentration of bovine II collagen in the collagen immunogen is 2 mg / mL, and the final concentration of carrageenan in the 1% carrageenan immunogen is 10 mg / mL; the immunization dose of the collagen immunogen is 0.28 ml / kg, and the immunization dose of the 1% carrageenan immunogen is 0.1 ml / kg, and one half of the dose is respectively injected subcutaneously near the shoulder blade and subcutaneously at the sole of the foot.

[0011] Furthermore, the preparation method of the collagen immunogen is as follows: bovine II collagen lyophilized powder is dissolved in 0.05 mol / L acetic acid solvent to prepare a 4 mg / ml collagen solution, and then equilibrated at 4°C overnight, and then mixed with an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant, and fully emulsified in an ice bath to obtain a collagen emulsion with a final concentration of 2 mg / mL.

[0012] Furthermore, during the initial immunization, the collagen immunogen was emulsified by mixing equal volumes of collagen solution and Freund's complete adjuvant to obtain a collagen emulsion with a final concentration of 2 mg / mL; during the booster immunization, the collagen immunogen was emulsified by mixing equal volumes of collagen solution and Freund's incomplete adjuvant to obtain a collagen emulsion with a final concentration of 2 mg / mL.

[0013] Furthermore, the concentration of the Freund's complete adjuvant is 20 mg / ml.

[0014] Furthermore, the two-point injection locations are subcutaneous on the right plantar and subcutaneous near the right scapula.

[0015] Compared with the prior art, the technical effects of the present invention are:

[0016] The method for establishing a feline osteoarthritis pain model proposed in the present invention uses collagen immunogen and 1% carrageenan immunogen as mixed immunogens through experimental studies on immunogens, drug concentrations, immunization times, administration routes, injection doses, etc., and immunizes cats by two-point injections near the scapula and subcutaneous injections at the sole of the foot. The immunization procedure uses two immunizations on day 0 and day 7, and successfully constructs a simple and efficient feline osteoarthritis pain model. The model has obvious swelling of joints and paws, a reduced cat paw retraction threshold, and a significantly increased TNF-a factor. The model can be used to study the pathogenesis, pathological changes, pain mechanism and OA treatment methods of feline OA, and can also be used to evaluate the clinical effects of new feline osteoarthritis analgesics. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The weight results provided in Example 1 of the present invention.

[0018] Figure 2 This is the paw swelling result provided in Example 1 of the present invention.

[0019] Figure 3 This is the TNF-a factor detection result provided in Example 1 of the present invention.

[0020] Figure 4 The weight results provided for Example 2 of the present invention.

[0021] Figure 5 This is the paw swelling result provided in Example 2 of the present invention.

[0022] Figure 6 This is the cat paw retraction threshold result provided in Example 2 of the present invention.

[0023] Figure 7 This is the TNF-a factor detection result provided in Example 2 of the present invention.

[0024] Figure 8 The weight results provided in Example 3 of the present invention.

[0025] Fig. 9 This is the paw swelling result provided in Example 3 of the present invention.

[0026] Fig.10 This is the cat paw retraction threshold result provided in Example 3 of the present invention.

[0027] Fig.11 This is the TNF-a factor detection result provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention.

[0029] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0030] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0031] Example 1

[0032] 1. Experimental Methods

[0033] 1. Immunogen Preparation

[0034] Adjuvant: Mix Freund's complete adjuvant (20 mg / ml) by inversion to ensure complete dissolution and homogeneity.

[0035] Collagen: Mix equal volumes of bovine II collagen solution (concentration 2 mg / mL) and Freund's complete adjuvant (Freund's incomplete adjuvant for booster immunization), fully emulsify in an ice bath to obtain a collagen emulsion with a final concentration of 1 mg / mL.

[0036] 2. The cats (1-3 years old American Shorthair) were weighed in groups (weight range was 2.5-5.5kg); cats with good mental state and normal eating were selected for the experiment; the cats were randomly divided into 3 groups, including collagen immunization group (referred to as collagen group), adjuvant immunization group (referred to as adjuvant group), and control group, with 5 cats in each group.

[0037] 3. Immunity adjuvants: Freund's complete adjuvant was injected subcutaneously at the scapula of cats in the immunization group, and the injection volume was calculated based on 0.14 ml / kg body weight; collagen emulsion was injected subcutaneously at the scapula of cats in the collagen immunization group, 0.57 ml / kg, and booster immunization was performed on D14. Normal saline was injected at the same location in the control group, and the immunization dose was 1 ml / cat.

[0038] 4. Observe and measure body weight every 3 to 4 days after immunization; measure the thickness of the paw (foot pad) with a vernier caliper; observe changes in diet and activity (especially pain and flexibility in the joints).

[0039] 5. Blood was collected before immunization and every week after immunization to separate serum, and the TNF-α factor was detected by ELISA method.

[0040] 2. Experimental Results

[0041] 1. Clinical observation results

[0042] The weight of cats after collagen and adjuvant injection did not change significantly compared with the control group. The weight changes of cats in each group are detailed in Figure 1 .

[0043] According to the thickness of cat paws, there was no obvious swelling in the collagen group and adjuvant group, which was consistent with the results of the control group. Figure 2 .

[0044] 2. Cytokine Detection

[0045] According to the results of the TNF-a factor ELISA test kit for cat serum, the concentration of TNF-a factor in cat serum did not change significantly and did not show an upward trend after collagen and adjuvant injection. Figure 3 .

[0046] 3. Experimental Summary

[0047] The above data show that the establishment of a feline arthritis model was unsuccessful, that is, the traditional mouse arthritis model establishment method is not suitable for animal cats, and there are differences in immunity and pain experience between different species. The present invention considers optimizing the method from the aspects of immunogen, injection dose, drug concentration, administration route, and number of immunizations.

[0048] Example 2

[0049] 1. Experimental Methods

[0050] 1. Immunogen Preparation

[0051] Adjuvant: Mix Freund's complete adjuvant (20 mg / ml) by inversion to ensure complete dissolution and homogeneity.

[0052] Collagen: Dissolve bovine II collagen lyophilized powder in 0.05 mol / L acetic acid solvent to 4 mg / ml, and equilibrate at 4°C overnight to obtain collagen solution. Mix the collagen solution with Freund's complete adjuvant (Freund's incomplete adjuvant for booster immunization) in equal volumes, fully emulsify in an ice bath, and obtain a collagen emulsion with a final concentration of 2 mg / mL.

[0053] 2. Grouping

[0054] The body weight of cats (the same as in Example 1) was measured (the body weight range was 2.5 to 5.5 kg); cats with good mental state and normal eating were selected for the experiment, and the cats in good condition were randomly divided into 3 groups, a collagen immunization group (referred to as the collagen group), an adjuvant immunization group (referred to as the adjuvant group) and a control group, with injection numbers, and all cats were raised in a free-range form, with 5 cats in each group.

[0055] 3. Immunization

[0056] The injection was performed subcutaneously near the scapula and the sole of the foot. Half of the drug was injected subcutaneously at the right sole of the foot after half of the drug, and half of the drug was injected subcutaneously near the right scapula. The adjuvant immunization group was injected with Freund's complete adjuvant, and the injection volume was calculated according to the dose of 0.28ml / kg body weight. The booster immunization was performed on D14; the collagen immunization group was injected with collagen emulsion, 0.57ml / kg, and booster immunization was performed on D7 and D14. The control group was injected with normal saline at the same location, and the immunization dose was 2ml / mouse.

[0057] 4. Observation

[0058] Measure body weight every 2 to 4 days after immunization; measure the thickness of the cat's paw with a vernier caliper; observe changes in diet and activity (especially pain and flexibility in the joints); use a handheld force sensor to determine the cat's paw retraction threshold, expressed in grams-force (gf).

[0059] 5. Blood was collected before and after immunization to separate serum, and the serum was tested for TNF-α factor using ELISA method.

[0060] 2. Experimental Results

[0061] 1. Clinical observation results

[0062] Compared with the cats in the control group, the cats in the collagen group and adjuvant group showed no significant changes in body weight, and their food intake and other activities were slightly reduced. The results are detailed in Figure 4 .

[0063] Cats in the control group showed no redness or swelling in their joints. Cats injected with collagen and adjuvant showed varying degrees of osteoarthritis from 7 to 14 days after the initial immunization. Around 18 to 25 days, cats' joints became swollen. According to the measurement of cat paw swelling, cats in both the collagen and adjuvant groups showed obvious redness and swelling of their paws on the 7th day after the initial immunization. For details, see Figure 5 .

[0064] The cat claw withdrawal threshold was measured using a pain meter on days 21 and 25 after the initial immunization. The results are detailed in Figure 6 From the results, it can be seen that there are significant differences between the collagen group and the adjuvant group and the control group on the 21st and 25th days after the initial immunization. The cat's claw retraction threshold is reduced, that is, the degree of pain the cat's claw endures is reduced, which indirectly reflects the increase in the degree of joint pain at the cat's claw.

[0065] 2. Cytokine Detection

[0066] According to the results of cat serum detection by TNF-a factor ELISA detection kit, the concentration of TNF-a factor in serum of collagen group and adjuvant group increased significantly 21 days after the first immunization. For detailed results, see Figure 7From the results, we can see that TNF-a factor increased significantly, proving that the body is currently in an inflammatory response state.

[0067] 3. Experimental Summary

[0068] The above data show that after comprehensive optimization and improvement of immunogen, drug concentration, injection dose, administration route, etc., collagen and adjuvant can successfully establish a feline arthritis model. However, considering the shortcomings of multiple drug injection times (collagen group) and large drug injection dose (adjuvant group and collagen group), the present invention attempts to further adjust the immunogen, drug concentration, injection dose and injection times to make the model establishment method simpler and more convenient.

[0069] Example 3

[0070] 1. Experimental Methods

[0071] 1. Immunogen Preparation

[0072] Collagen immunogen: Dissolve bovine II collagen lyophilized powder in 0.05 mol / L acetic acid solvent to 4 mg / ml, and equilibrate at 4°C overnight to obtain collagen solution. Mix the collagen solution with Freund's complete adjuvant (Freund's incomplete adjuvant for booster immunization) in equal volumes, fully emulsify in an ice bath, and obtain a collagen emulsion with a final concentration of 2 mg / ml.

[0073] Mixed immunogens: Collagen immunogen and 1% carrageenan were mixed for immunization. The injection volume was calculated based on the collagen immunogen dose of 0.28 ml / kg body weight and the 1% carrageenan dose of 0.1 ml / kg body weight.

[0074] The preparation method of 1% carrageenan immunogen is as follows: 0.1 g of carrageenan is dissolved in 10 ml of normal saline, mixed to prepare a 1% carrageenan normal saline solution, and filtered to sterilize.

[0075] 2. Grouping

[0076] The body weight of cats (the same as in Example 1) was measured (the body weight range was 2.5 to 5.5 kg); cats with good mental state and normal eating were selected for the experiment, and their mental state was evaluated. The cats in good condition were randomly divided into 3 groups, a mixed immunization group (referred to as the mixed group), a collagen immunization group (referred to as the collagen group) and a control group, with 5 cats in each group, with injection numbers, and all cats were raised in a free-range manner.

[0077] 3. Immunization

[0078] The injection was performed subcutaneously near the scapula and the sole of the foot. Half of the drug was injected subcutaneously at the right sole of the foot after half of the drug, and half of the drug was injected subcutaneously near the right scapula. The mixed immunization group was injected with collagen immunogen and 1% carrageenan. The injection volume was calculated according to the collagen immunogen 0.28ml / kg body weight and the 1% carrageenan 0.1ml / kg body weight. The booster immunization was performed on D7. The collagen immunization group was injected with collagen emulsion, 0.28ml / kg, and the booster immunization was performed on D7. The control group was injected with normal saline at the same location, and the immunization dose was 2ml / mouse.

[0079] 4. Observation

[0080] Measure body weight every 2 to 4 days after immunization; measure the thickness of the cat's paw with a vernier caliper; observe changes in diet and activity (especially pain and flexibility in the joints); use a handheld force sensor to determine the cat's paw retraction threshold.

[0081] 5. Blood was collected before and after immunization to separate serum, and the serum was tested for TNF-α factor using ELISA method.

[0082] 2. Experimental Results

[0083] 1. Clinical observation results

[0084] Compared with the cats in the control group, the cats in the mixed immunization group and the collagen immunization group showed no significant changes in body weight, and their food intake and other activities were slightly reduced. The results are detailed in Figure 8 .

[0085] Cats in the control group showed no redness or swelling in their joints. Cats in the mixed immunization group developed varying degrees of osteoarthritis around the 14th day after the initial immunization. Around the 18th to 30th day, cats' joints became swollen significantly. From the results, there was a significant difference between the mixed immunization group and the collagen group. The joint swelling in the mixed immunization group was more obvious. For details, see Fig. 9 .

[0086] The cat claw withdrawal threshold was measured using a pain meter 14 to 30 days after the initial immunization. The results are detailed in Fig.10 From the results, it can be seen that on the 21st to 30th day after the initial immunization, there were significant differences in the mixed group compared with the collagen group and the control group, and the cat's claw retraction threshold was reduced, that is, the degree of pain the cat's claw endured was reduced, which indirectly reflected the increase in the degree of joint pain at the cat's claw.

[0087] 2. Cytokine Detection

[0088] According to the results of the TNF-a factor ELISA test kit for cat serum, the TNF-a factor concentration in the serum of the mixed group and the immune group 14 days after the initial immunization was significantly higher than that of the control group. From the results, it can be seen that the TNF-a factor increased significantly, proving that the body is currently in an inflammatory response state. However, compared with the collagen group, the TNF-a factor concentration in the mixed group increased more significantly, and the inflammatory response state was more obvious. For detailed results, see Fig.11 .

[0089] 3. Experimental Summary

[0090] The above data show that by adjusting the immunogen, drug concentration, injection dose and injection times, a better feline arthritis model was successfully established. The mixed immunization group model has a better effect, and compared with the method of Example 2, the injection times are fewer, the procedure is simpler, and the injection dose is less.

[0091] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for establishing a feline osteoarthritis pain model, characterized in that: The method adopts collagen immunogen and 1% carrageenan immunogen as mixed immunogens, and adopts two-point injection mode of subcutaneous injection near shoulder blade and subcutaneous injection at the sole of foot to immunize cat, and the immunization procedure adopts two immunizations on day 0 and day 7; the final concentration of bovine II collagen in the collagen immunogen is 2 mg / mL, and the final concentration of carrageenan in the 1% carrageenan immunogen is 10 mg / mL; the immunization dose of the collagen immunogen is 0.28 ml / kg, and the immunization dose of the 1% carrageenan immunogen is 0.1 ml / kg, and half of the dose is respectively injected subcutaneously near shoulder blade and subcutaneously at the sole of foot.

2. The method for establishing a feline osteoarthritis pain model according to claim 1, characterized in that: The preparation method of the collagen immunogen is as follows: bovine II collagen lyophilized powder is dissolved in 0.05 mol / L acetic acid solvent to prepare a 4 mg / ml collagen solution, and then the solution is balanced at 4°C overnight, and then mixed with an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant, and fully emulsified in an ice bath to obtain a collagen emulsion with a final concentration of 2 mg / mL.

3. The method for establishing a feline osteoarthritis pain model according to claim 2, characterized in that: During the initial immunization, the collagen immunogen was emulsified by mixing equal volumes of collagen solution and Freund's complete adjuvant to obtain a collagen emulsion with a final concentration of 2 mg / mL; during the booster immunization, the collagen immunogen was emulsified by mixing equal volumes of collagen solution and Freund's incomplete adjuvant to obtain a collagen emulsion with a final concentration of 2 mg / mL.

4. The method for establishing a feline osteoarthritis pain model according to claim 2, characterized in that: The concentration of the Freund's complete adjuvant was 20 mg / ml.

5. The method for establishing a feline osteoarthritis pain model according to claim 1, characterized in that: The two-point injection sites are subcutaneous on the right plantar and subcutaneous near the right scapula.

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