A method for establishing a feline osteoarthritis pain model

Abstract

This invention discloses a method for establishing a feline osteoarthritis pain model. Through experimental studies on various aspects such as immunogen, drug concentration, number of immunizations, route of administration, and injection dosage, a mixed immunogen of collagen and 1% carrageenan was used. Cats were immunized by subcutaneous injection at two points near the scapula and on the sole of the paw. The immunization program consisted of two immunizations on day 0 and day 7. A simple and efficient feline osteoarthritis pain model was successfully constructed. The model showed significant swelling of the joints and paws, a decreased claw retraction threshold, and a significant increase in TNF-α factor. It can be used to study the pathogenesis, pathological changes, pain mechanisms, and treatment methods of feline osteoarthritis (OA), and can also be used to evaluate the clinical efficacy of novel feline osteoarthritis analgesics.

Description

Technical Field

[0001] This invention relates to the field of medicine, and more particularly to a method for establishing a feline osteoarthritis pain model. Background Technology

[0002] Osteoarthritis (OA), also known as osteoarthropathy, degenerative arthritis, proliferative arthritis, or senile arthritis, is a chronic joint disease with an unclear etiology, characterized by degenerative changes in articular cartilage and secondary bone hyperplasia.

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

[0004] Osteoarthritis (OA) in animals develops gradually. Examples include Dunkin'-Hartley guinea pigs, STR / ort mice, dogs, and horses. However, the development of OA in animals is difficult to predict, and effective control measures are lacking. Furthermore, different OA models reflect the differences in pain experience among individual OA patients. Currently, there is a lack of research on methods for establishing feline osteoarthritis models.

[0005] Osteoarthritis in cats is a very common chronic joint disease in older cats, characterized by degeneration and wear of the articular cartilage, leading to inflammation, discomfort, and persistent damage within the joint. This disease is widespread and highly prevalent in older cats, with an incidence rate that increases significantly with age, severely impacting their quality of life. Multiple studies have shown that 61% of cats over 6 years of age have at least one instance of osteoarthritis, and this proportion rises to as high as 90% when cats reach 12 years of age.

[0006] As common companion animals, disease and pain management in cats is crucial to ensuring their quality of life. However, because cats have a naturally high pain tolerance and their behavioral changes are often subtle, pain signs are usually difficult to identify. The actual diagnosis rate is far lower than the prevalence rate, at less than 10%, which undoubtedly increases the difficulty of preventing and treating this disease.

[0007] Therefore, in order to conduct in-depth research on the pathogenesis, pathological changes, pain mechanisms, treatment methods, and novel analgesics of feline osteoarthritis, improve the quality of life of cats, and establish a suitable feline osteoarthritis pain model to provide researchers with an important research platform, it is of great significance. Summary of the Invention

[0008] The purpose of this invention is to propose a method for establishing a feline osteoarthritis pain model, providing researchers with a research platform for studying the pathogenesis, pathological changes, pain mechanisms, treatment methods, and analgesic drugs of feline osteoarthritis.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] This invention proposes a method for establishing a feline osteoarthritis pain model. The method uses a collagen immunogen and a 1% carrageenan immunogen as a mixed immunogen. Cats are immunized via subcutaneous injection at two points: near the scapula and on the sole of the paw. The immunization schedule involves two immunizations on days 0 and 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. Half of the dose is injected subcutaneously near the scapula and on the sole of the paw.

[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. After equilibration at 4°C overnight, it is 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 an equal volume of collagen solution with 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 an equal volume of collagen solution with 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 injection points are located subcutaneously on the posterior right metatarsal and subcutaneously near the right scapula.

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

[0016] The method for establishing a feline osteoarthritis pain model proposed in this invention involves experimental studies on various aspects such as immunogens, drug concentrations, immunization frequency, administration routes, and injection doses. A mixed immunogen of collagen and 1% carrageenan was used, and cats were immunized via subcutaneous injection at two points: near the scapula and on the paw. The immunization schedule consisted of two immunizations on days 0 and 7. This successfully constructed a simple and efficient feline osteoarthritis pain model. The model exhibited significant joint and paw swelling, a decreased claw retraction threshold, and a significant increase in TNF-α. It can be used to study the pathogenesis, pathological changes, pain mechanisms, and treatment methods of feline OA, and can also be used to evaluate the clinical efficacy of novel feline osteoarthritis analgesics. Attached Figure Description

[0017] Figure 1 The weight result provided in Embodiment 1 of the present invention.

[0018] Figure 2 The results of foot swelling provided in Embodiment 1 of the present invention.

[0019] Figure 3 The results of TNF-α factor detection provided in Example 1 of this invention.

[0020] Figure 4 The weight result provided in Embodiment 2 of the present invention.

[0021] Figure 5 The results of foot swelling provided in Embodiment 2 of the present invention.

[0022] Figure 6 The result of the cat paw retraction threshold provided in Embodiment 2 of the present invention.

[0023] Figure 7 The results of TNF-α factor detection provided in Example 2 of this invention.

[0024] Figure 8 The weight result provided in Embodiment 3 of the present invention.

[0025] Figure 9 The results of foot swelling provided in Embodiment 3 of the present invention.

[0026] Figure 10 The result of the cat paw retraction threshold provided in Embodiment 3 of the present invention.

[0027] Figure 11 The results of TNF-α factor detection provided in Example 3 of this invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

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

[0031] Example 1

[0032] I. Experimental Methods

[0033] 1. Immunogen preparation

[0034] Adjuvant: Invert Freund's complete adjuvant (20 mg / ml) and mix thoroughly to ensure complete dissolution and homogeneity.

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

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

[0037] 3. In the adjuvant immunization group, Freund's complete adjuvant was injected subcutaneously into the scapula of the cat, with an injection volume calculated at 0.14 ml / kg body weight. In the collagen immunization group, collagen emulsion was injected subcutaneously into the scapula of the cat, at a dose of 0.57 ml / kg, with a booster immunization on day 14. The control group received saline injection at the same location, with an immunization dose of 1 ml / cat.

[0038] 4. Observe weight every 3-4 days after immunization; measure the thickness of the toe pads (footpad area) with calipers; observe changes in diet and activity (especially joint pain and flexibility).

[0039] 5. Blood was collected every week before and after immunization to separate serum. The serum was tested for TNF-α factor using the ELISA method.

[0040] II. Experimental Results

[0041] 1. Clinical observation results

[0042] The weight of the cats after collagen and adjuvant injections did not change significantly compared to the control group. For details on weight changes in each group, please refer to [link to relevant documentation]. Figure 1 .

[0043] Based on measurements of the cat's paw thickness, no significant paw swelling was observed in either the collagen group or the adjuvant group, consistent with the control group. See details for further information. Figure 2 .

[0044] 2. Cytokine detection

[0045] According to the results of the TNF-α factor ELISA assay kit for cat serum, the concentration of TNF-α factor in cat serum did not change significantly after collagen and adjuvant injections, and there was no upward trend. See details below. Figure 3 .

[0046] III. Experiment Summary

[0047] The above data indicate that the feline arthritis model was not successfully established, meaning that the traditional method used to establish a mouse arthritis model is not applicable to cats, and there are differences in immunity and pain experience between different species. This invention aims to optimize the method in terms of immunogen, injection dosage, drug concentration, route of administration, and number of immunizations.

[0048] Example 2

[0049] I. Experimental Methods

[0050] 1. Immunogen preparation

[0051] Adjuvant: Invert Freund's complete adjuvant (20 mg / ml) and mix thoroughly to ensure complete dissolution and homogeneity.

[0052] Collagen: Bovine II collagen lyophilized powder was dissolved in 0.05 mol / L acetic acid to prepare a 4 mg / mL solution, which was then equilibrated overnight at 4°C to obtain a collagen solution. The collagen solution was mixed with an equal volume of Freund's complete adjuvant (Freund's incomplete adjuvant was used for booster immunization) and fully emulsified in an ice bath to obtain a collagen emulsion with a final concentration of 2 mg / mL.

[0053] 2. Grouping

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

[0055] 3. Immunity

[0056] The injections were administered subcutaneously near the scapula and on the plantar surface of the paw. Half of the dose was injected subcutaneously into the right plantar surface, and the remaining half was injected subcutaneously near the right scapula. The adjuvant immunization group received Freund's complete adjuvant at a dose calculated at 0.28 ml / kg body weight, with a booster immunization on day 14. The collagen immunization group received collagen emulsion at 0.57 ml / kg, with booster immunizations on days 7 and 14. The control group received saline injections at the same sites, with an immunization dose of 2 ml per animal.

[0057] 4. Observation

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

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

[0060] II. Experimental Results

[0061] 1. Clinical observation results

[0062] Compared with the control group, cats in the collagen and adjuvant groups showed no significant change in body weight, but slightly reduced food intake and other activities. See details below. Figure 4 .

[0063] In the control group, cats showed no redness or swelling in their joints. Cats injected with collagen and adjuvant showed varying degrees of osteoarthritis between 7 and 14 days after initial immunization, with significant joint swelling occurring between 18 and 25 days later. Measurements of paw swelling revealed that on day 7 after initial immunization, both the collagen and adjuvant groups exhibited significant redness and swelling in their paws. See details for further information. Figure 5 .

[0064] Cats' paw retraction thresholds were measured using a pain meter on days 21 and 25 after the initial immunization. See details for the results. Figure 6 The results showed that on days 21 and 25 after the initial immunization, the collagen group and the adjuvant group differed significantly from the control group. The claw retraction threshold of the cats was lower, meaning that the cats' claws could not withstand as much pain, which indirectly reflected the increase in the degree of joint pain at the paw site.

[0065] 2. Cytokine detection

[0066] Results from the TNF-α factor ELISA assay kit showed that serum TNF-α concentrations were significantly elevated in both the collagen and adjuvant groups 21 days after initial immunization. (See attached results for details.) Figure 7The results show a significant increase in TNF-α, indicating that the body is currently in an inflammatory state.

[0067] III. Experiment Summary

[0068] The data above show that, after comprehensive optimization and improvement of the immunogen, drug concentration, injection dosage, and route of administration, both collagen and adjuvant can successfully establish feline arthritis models. However, considering the drawbacks of multiple drug injections (collagen group) and excessively high drug injection dosages (adjuvant and collagen groups), this invention attempts to further adjust the immunogen, drug concentration, injection dosage, and number of injections to make the model establishment method simpler and more convenient.

[0069] Example 3

[0070] I. Experimental Methods

[0071] 1. Immunogen preparation

[0072] Collagen immunogen: Bovine II collagen lyophilized powder was dissolved in 0.05 mol / L acetic acid to prepare a 4 mg / ml solution, which was then equilibrated overnight at 4°C to obtain a collagen solution. An equal volume of the collagen solution was mixed with Freund's complete adjuvant (Freund's incomplete adjuvant was used for booster immunization) and fully emulsified in an ice bath to obtain a collagen emulsion with a final concentration of 2 mg / ml.

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

[0074] The preparation method of 1% carrageenan immunogen is as follows: Dissolve 0.1g of carrageenan in 10ml of physiological saline, mix well, prepare a 1% carrageenan physiological saline solution, and filter to remove bacteria.

[0075] 2. Grouping

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

[0077] 3. Immunity

[0078] The injections were administered subcutaneously near the scapula and on the plantar surface of the paw. Half of the dose was injected subcutaneously into the right plantar surface, and the remaining half was injected subcutaneously near the right scapula. The mixed immunization group received both collagen immunogen and 1% carrageenan, with the injection volume calculated based on a dose of 0.28 ml / kg body weight for collagen immunogen and 0.1 ml / kg body weight for 1% carrageenan. A booster immunization was administered on day 7. The collagen immunization group received 0.28 ml / kg of collagen emulsion, with a booster immunization on day 7. The control group received saline injections at the same sites, with an immunization dose of 2 ml per animal.

[0079] 4. Observation

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

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

[0082] II. Experimental Results

[0083] 1. Clinical observation results

[0084] Compared with the control group, cats in the mixed immunization group and the collagen immunization group showed no significant change in body weight, but slightly reduced food intake and other activities. See details below. Figure 8 .

[0085] In the control group, cats showed no redness or swelling in their joints. In the mixed-immunization group, varying degrees of osteoarthritis began to appear around day 14 after the initial immunization, with significant joint swelling occurring between days 18 and 30. The results showed a significant difference between the mixed-immunization group and the collagen group; the mixed-immunization group exhibited more pronounced joint swelling. See details below. Figure 9 .

[0086] Cats' paw retraction threshold was measured using a pain meter between days 14 and 30 after the initial immunization. See details for the results. Figure 10 The results showed that, from day 21 to 30 after the initial immunization, the mixed group was significantly different from both the collagen group and the control group. The paw retraction threshold was lower, meaning that the paw could not bear the pain, which indirectly reflected the increased pain in the paw joints.

[0087] 2. Cytokine detection

[0088] Results from a TNF-α ELISA assay of cat serum showed that, 14 days after the initial immunization, the serum TNF-α concentration in both the mixed group and the immunized group was significantly higher than that in the control group. This indicates a significant increase in TNF-α, suggesting the body is currently in an inflammatory response. However, the increase in TNF-α concentration was more significant in the mixed group compared to the collagen group, indicating a more pronounced inflammatory response. (See detailed results below.) Figure 11 .

[0089] III. Experiment Summary

[0090] The data above demonstrate that by adjusting the immunogen, drug concentration, injection dosage, and number of injections, a superior feline arthritis model was successfully established. The mixed immunization model showed even better results and, compared to the method in Example 2, required fewer injections, had a simpler procedure, and required a smaller injection dose.

[0091] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, 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 foregoing 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 uses a mixture of collagen immunogen and 1% carrageenan immunogen as immunogens. Cats are immunized via subcutaneous injection near the scapula and subcutaneously on the paw. The immunization schedule consists of two immunizations on days 0 and 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. Half of the dose is injected subcutaneously near the scapula and subcutaneously on the paw. The collagen immunogen is prepared by dissolving lyophilized bovine II collagen powder in 0.05 mol / L acetic acid to prepare a 4 mg / ml collagen solution. After equilibration at 4°C overnight, an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant is mixed and fully emulsified in an ice bath to obtain a final concentration of 2 mg / mL. For the initial immunization, the collagen immunogen was emulsified by mixing an equal volume of collagen solution with Freund's complete adjuvant to obtain a final concentration of 2 mg / mL collagen emulsion. For the booster immunization, the collagen immunogen was emulsified by mixing an equal volume of collagen solution with Freund's incomplete adjuvant to obtain a final concentration of 2 mg / mL collagen emulsion. The concentration of Freund's complete adjuvant was 20 mg / mL. The two injection sites were the subcutaneous tissue on the right plantar hindfoot and the subcutaneous tissue near the right scapula.

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