Thermal Stimulation Pain Testing Device and Testing Method

Abstract

The present invention relates to the technical field of neuropathic pain of non-human primate experimental animals, and specifically discloses a thermal stimulation pain test device and a test method. It includes a temperature-controlled heat application device and a continuous walking induction device; the temperature-controlled heat application device includes a constant temperature and timing heating module and a fixing belt; the continuous walking induction device includes a transparent limiting box, and the transparent limiting box is composed of a front side plate, a left side plate, a right side plate, a top plate and a rear side plate; a transparent feeder is arranged on the front side of the front side plate; the front side plate is provided with a feeding port, and non-human primate experimental animals walking in the transparent limiting box are fed through the feeding port; the transparent limiting box is fixed directly above the walking track of the treadmill. Using the present invention has the advantages of being able to achieve scientific, effective, accurate and controllable and objective thermal stimulation pain tests, and being able to meet the efficacy detection of new drugs for neuropathic pain, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of neuropathic pain of non-human primate experimental animals, and particularly relates to a thermal stimulation pain test device and a test method. Background Art

[0002] Pain is the most common symptom of various diseases and one of the most serious problems plaguing human health today. Currently, the types of analgesics used clinically are limited, and most of them have obvious side effects such as weak analgesic effects and / or addiction and tolerance, resulting in a large number of pain patients not receiving effective treatment. Therefore, at present, each pharmaceutical factory or drug research and development institution is developing new non-addictive and low-side-effect analgesics. However, since it is difficult to directly conduct pain and drug efficacy tests on humans after new drugs are developed, pain animal models are often established to screen and identify analgesic candidate drugs or detect the drug efficacy and side effects of new drugs. In particular, pain disease modeling and new drug testing using non-human primate experimental animals are the most important. First, the first step is pain disease modeling, and then the drug efficacy of new drugs is tested. Currently, there is still little research on pain disease modeling, especially for pain disease modeling of non-human primate experimental animals, there is no in-depth research and feasible plan, and there is no effective animal disease model, so the testing of new drugs is out of the question. At the same time, during the pain test process, because non-human primate experimental animals cannot communicate with humans, how to objectively and intuitively evaluate indicators such as pain indexes during the relevant test process is also a major technical problem faced in the pain test of non-human primate experimental animals. Therefore, there is still a lack in current pain testing and methods. The above technical problems cannot be effectively solved, directly resulting in the inability to effectively conduct tests on non-human primate experimental animals for relevant pain new drugs, seriously hindering the evaluation and testing of pain new drugs. Summary of the Invention

[0003] The present invention provides a thermal stimulation pain test device and a test method with good reliability for the above technical problems.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A thermal stimulation pain test device includes a temperature-controlled heat application device and a continuous walking induction device; the temperature-controlled heat application device includes a constant temperature and timing heating module and a fixing belt; the continuous walking induction device includes a transparent limiting box, and the transparent limiting box is composed of a front side plate, a left side plate, a right side plate, a top plate and a rear side plate; a transparent feeder is arranged on the front side of the front side plate; the front side plate is provided with a feeding port, and non-human primate experimental animals walking in the transparent limiting box are fed through the feeding port; the transparent limiting box is fixed directly above the walking track of a treadmill.

[0006] Preferably, a plurality of ventilation holes are further provided in the upper front part of the transparent defining box.

[0007] Preferably, a fan is further provided in the upper front part of the transparent defining box, and the fan is arranged outside the ventilation holes.

[0008] Preferably, the transparent feeder includes a transparent food storage chamber, a food falling opening is provided at the lower part of the transparent food storage chamber, and a valve is provided at the food falling opening; a drag-type feeding box is provided at the lower end of the food falling opening; the drag-type feeding box is movably arranged at the feeding opening.

[0009] Preferably, the transparent feeder further includes a feeding box support base, the feeding box support base is arranged below the drag-type feeding box, and a locking device is correspondingly arranged between the feeding box support base and the drag-type feeding box.

[0010] A test method using the heat-stimulated pain test device as described above includes the following steps:

[0011] (1) Disease modeling

[0012] S1: Injecting capsaicin: Intradermally injecting 100 μl / 50 μg of capsaicin into the inner thigh of a non-human primate experimental animal.

[0013] S2: Heating induction: Using the temperature-controlled heat application device to apply a temperature above 38°C and maintain it for more than 100 s to the injection area, so as to prompt the non-human primate experimental animal to exhibit neuropathic pain symptoms.

[0014] (2) Using the heat-stimulated pain test device to detect symptom manifestations

[0015] Step 1: At the latest 100 s before starting to apply heat to the non-human primate experimental animal using the temperature-controlled heat application device, ensure that the non-human primate experimental animal walks normally on the walking belt of the treadmill in the heat-stimulated pain test device, start timing after starting the heat application, and at the same time record the non-human primate experimental animal.

[0016] Step 2: Stop timing when pain manifestations appear in the legs of the non-human primate experimental animal. Preferably, for the heating induction, use the temperature-controlled heat application device to apply 43°C to the injection area and maintain it for more than 100 s.

[0017] Preferably, for the heating induction, a dual-control heating method is adopted, that is, first use the temperature-controlled heat application device to apply 38°C to the injection area and maintain it for more than 15 s, and then apply 43°C and maintain it for more than 120 s.

[0018] Preferably, when testing the effect of a new drug to be tested, the administration of the new drug to be tested is completed before injecting capsaicin into the non-human primate experimental animal.

[0019] Preferably, 30 minutes after the administration of the new drug to be tested and / or 60 minutes after the administration of the new drug to be tested and / or 120 minutes after the administration of the new drug to be tested, the disease modeling and the symptom characterization detection are carried out respectively.

[0020] The beneficial effects of the present invention compared with the prior art:

[0021] 1. The thermal stimulation pain test device of the present invention induces pain manifestations in the disease model of non-human primate experimental animals through the continuous walking induction device, thereby ensuring the objectivity of pain test evaluation and solving the problem that effective and objective evaluation of pain tests for non-human primate experimental animals has not been achieved. The present invention also uses a transparent feeder to stimulate non-human primate experimental animals to continuously cooperate in walking, ensuring that pain manifestations can be well reflected.

[0022] 2. The present invention uses the scheme of capsaicin + temperature-controlled heat application device + continuous walking induction device for disease model induction and detection, which can achieve scientific, effective, accurate, controllable and objective thermal stimulation pain tests, and can meet the efficacy detection of new drugs for neuropathic pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic structural diagram of the transparent feeder of the continuous walking induction device of the present invention in a closed state;

[0024] Figure 2 is a schematic structural diagram of the transparent feeder of the continuous walking induction device of the present invention in a feeding state;

[0025] Figure 3 is a schematic structural diagram of the valve area of the transparent feeder of the present invention;

[0026] Figure 4 is a cross-sectional view of the transparent feeder of the present invention;

[0027] Figure 5 is a cross-sectional view of the temperature-controlled heat application device of the present invention;

[0028] Figure 6 is the latency of capsaicin-induced thermal stimulation pain response in cynomolgus monkey 1 after oral administration of gabapentin in Table 2;

[0029] Figure 7 is the latency of capsaicin-induced thermal stimulation pain response in cynomolgus monkey 2 after oral administration of gabapentin in Table 2;

[0030] Figure 8 It is the latency of capsaicin-induced thermal stimulation pain response in cynomolgus monkey 3 after oral administration of gabapentin in Table 2;

[0031] Figure 9 It is the latency of capsaicin-induced thermal stimulation pain response in cynomolgus monkey 4 after oral administration of gabapentin in Table 2. Detailed implementation manners

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "inner", "front", "rear", "left", "right", etc. is based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship in which the product of the invention is usually placed during use. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0034] In the description of the present invention, it should also be noted that unless otherwise clearly specified and limited, the terms "set" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0035] As Figures 1 to 5 shown, the thermal stimulation pain test device includes a temperature control heat application device and a continuous walking induction device; the temperature control heat application device includes a constant temperature and timing heating module 23 and a fixing belt 26; in this embodiment, the constant temperature and timing heating module 23 includes a heating metal plate 25 and a control circuit 24, and can also be integrated with a power supply and a start switch, etc. When in use, the heating mode and constant temperature time are stored in the chip, and the heating stops after reaching the constant temperature time to meet the test requirements and not harm the animals.

[0036] The continuous walking induction device includes a transparent confinement box, which is composed of a front side plate 10, a left side plate, a right side plate 13, a top plate 12 and a rear side plate 14; a transparent feeder 7 is arranged on the front side of the front side plate 10; the front side plate 10 is provided with a feeding port 10-1, and non-human primate experimental animals walking in the transparent confinement box are fed through the feeding port 10-1; the transparent confinement box is fixed directly above the walking track 1 of the treadmill. During use, by using food as a reward, non-human primate experimental animals are induced to continuously cooperate to walk on the walking track 1 of the treadmill, that is: first, the transparent confinement box is clamped to the body on both sides of the walking track 1 of the treadmill through the fixing block 2 at the bottom by a U-shaped clamp, then the non-human primate experimental animal is placed in the transparent confinement box, and then the food is placed in the transparent confinement box. Through long-term training of the non-human primate experimental animal before the test, that is, to prompt the non-human primate experimental animal to be willing to cooperate and walk continuously for a certain period of time, then the treadmill is stopped and reward food is immediately given from the feeding port 10-1. If it does not cooperate to walk or the continuous walking does not meet the requirements, no reward food is given; after long-term training, it can induce non-human primate experimental animals to produce conditioned reflexes, and then relevant tests can be carried out. In the present invention, by placing the reward food in the transparent feeder 7, non-human primate experimental animals can see the reward food in time, thereby improving the cooperation degree of non-human primate experimental animals and effectively ensuring that non-human primate experimental animals cooperate to complete the whole process of pain testing.

[0037] Preferably, a plurality of ventilation holes 11 are further arranged at the upper front part of the transparent confinement box, and a fan 8 is arranged outside the ventilation hole 9 of the front side plate 10. Of course, a fan can also be installed outside the ventilation hole 11. During use, by blowing air with the fan 8, the air flow can be promoted to blow in from the upper front part and exit from the lower part of the transparent confinement box (there is a gap between the bottom of the transparent confinement box and the walking track 1), which can reduce the stuffiness and reduce the peculiar smell in the transparent confinement box for non-human primate experimental animals, improve the comfort of non-human primate experimental animals, and thus improve the cooperation degree of non-human primate experimental animals. In this embodiment, the rear side plate 14 is connected to the left side plate and the right side plate 13 in a way of being pulled up and down. The purpose of this design is that: the rear side plate 14 serves as a sliding door, which is convenient for non-human primate experimental animals to enter and exit.

[0038] Preferably, the transparent feeder 7 includes a transparent food storage chamber, a food dropping port 16 is arranged at the lower part of the transparent food storage chamber, and a valve 6 is arranged at the food dropping port 16; a drag-type feeding box 5 is arranged at the lower end of the food dropping port 16; the drag-type feeding box 5 is movably arranged at the feeding port 10-1. In this embodiment, as Figure 3As shown, the bottom surface of the transparent food storage is inclined, and the valve 6 is of the pull-out type. An opening 18 is provided in the middle thereof. Then, a valve socket 17 corresponding to the pull-out valve 6 is provided at the food falling port 16. During use, by pushing and pulling the valve 6, the food is caused to fall from the opening 18, realizing the controllability of the food falling.

[0039] Preferably, the transparent feeder further includes a feeder box support base 3. The feeder box support base 3 is arranged below the pull-type feeder box 5, and a locking device is correspondingly arranged between the feeder box support base 3 and the pull-type feeder box 5. The locking device in this embodiment can be a spring plunger 4. Then, a plunger locking hole 21 is correspondingly arranged on the bottom surface 20 of the pull-type feeder box 5, and its main function is to realize the self-locking function of the pull-type feeder box 5. During use, first let the food in the transparent food storage fall into Figure 1 the feeding trough 22 of the pull-type feeder box 5 in the state. Then, when it is necessary to give reward food to non-human primate experimental animals, the handle 19 of the pull-type feeder box 5 is held by hand and pushed forcefully inward, so that the plunger locking hole 21 is disengaged from the spring plunger 4, and then the feeding trough 22 is pushed into Figure 2 the state. At this time, non-human primate experimental animals can grab the reward food, establish a conditioned reflex, and improve the continuous cooperation degree of non-human primate experimental animals.

[0040] The test method using the thermal stimulation pain test device as described above includes the following steps:

[0041] (1) Disease modeling

[0042] S1: Inject capsaicin: Intradermally inject 100 μl / 50 μg of capsaicin into the inner thigh of non-human primate experimental animals;

[0043] S2: Temperature increase induction: Apply a temperature of more than 38°C and maintain it for more than 100 s to the injection area using the temperature control heating device, so as to cause non-human primate experimental animals to exhibit neuropathic pain symptoms;

[0044] (2) Use the thermal stimulation pain test device to detect symptom manifestations

[0045] Step 1: At the latest 100 s before starting to apply heat to non-human primate experimental animals using the temperature control heating device, ensure that the non-human primate experimental animals walk normally on the walking belt of the treadmill in the thermal stimulation pain test device, and start timing after starting the heat application and record the non-human primate experimental animals at the same time;

[0046] Step 2: Stop timing when pain symptoms appear in the legs of the non-human primate experimental animals. Preferably, for the heating induction, the temperature-controlled heat application device is used to apply 43°C to the injection area and maintain it for more than 100 s.

[0047] Preferably, for the heating induction, a dual-control heating method is adopted, that is, the temperature-controlled heat application device is first used to apply 38°C to the injection area and maintain it for more than 15 s, and then 43°C is applied and maintained for more than 120 s.

[0048] Preferably, when testing the effect of the new drug to be tested, the administration of the new drug to be tested is completed before injecting capsaicin into the non-human primate experimental animals.

[0049] Preferably, 30 min after the administration of the new drug to be tested and / or 60 min after the administration of the new drug to be tested and / or 120 min after the administration of the new drug to be tested, the disease modeling and the symptom characterization detection are carried out respectively.

[0050] The specific test process is as follows:

[0051] (1) Study on the latency of thermal stimulation pain response

[0052] A pain model of cynomolgus monkeys is induced by capsaicin, and the latency of the pain response of cynomolgus monkeys is detected by thermal stimulation.

[0053] 1. Establish a baseline

[0054] That is, when no capsaicin is injected, the sensitivity test of non-human primate experimental animals to thermal stimulation is carried out. As shown in Table 1, the average response latency is 117.1 s.

[0055] 2. Study on the latency of thermal stimulation pain response

[0056] After injecting capsaicin, the sensitivity of non-human primate experimental animals to thermal stimulation is studied. As shown in Table 1, the shortest average response latency is 14.9 s at 90 minutes; then the latency gradually extends, and the average response latency is 105.6 s at 8 hours.

[0057] Table 1 Latency of capsaicin-induced thermal stimulation pain response in cynomolgus monkeys

[0058]

[0059] Through the above tests, it is concluded that: from 30 minutes to 4 hours after injecting capsaicin, cynomolgus monkeys are most sensitive to thermal pain, and then 30 minutes, 60 minutes, and 120 minutes after drug administration are preferably used as the time for standard tests.

[0060] (2) Simulated drug treatment test

[0061] Gabapentin was first developed by Warner-Lambert Company in the United States and is currently recognized as a therapeutic drug that shows significant effects in animal models of spasm, analgesia, and amyotrophic lateral sclerosis. Therefore, in this experiment, a capsaicin-induced pain model of cynomolgus monkeys with better reaction stability was screened out. Then, before and after oral administration of gabapentin (at a dosage of 30 mg / KG), the pain reaction latency of cynomolgus monkeys was detected by thermal stimulation. As shown in Table 2, before injecting capsaicin, the average pain reaction latency of cynomolgus monkeys to thermal stimulation was 43 s. Before drug administration (after injecting capsaicin), the average pain reaction latency of cynomolgus monkeys to thermal stimulation was 12 s. At 30 minutes, 60 minutes, and 120 minutes after drug administration, the average reaction latencies were 83 s, 37 s, and 77 s, respectively.

[0062] Table 2 Latency of capsaicin-induced thermal stimulation pain reaction in cynomolgus monkeys after oral administration of gabapentin

[0063]

Claims

1. Thermal stimulation pain testing device, characterized in that: It includes a temperature-controlled heat application device and a continuous walking induction device; the temperature-controlled heat application device includes a constant temperature and timing heating module and a fixing belt; the continuous walking induction device includes a transparent limiting box, which is composed of a front side plate, a left side plate, a right side plate, a top plate and a rear side plate; a transparent feeder is arranged on the front side of the front side plate; the front side plate is provided with a feeding port, and non-human primate experimental animals walking in the transparent limiting box are fed through the feeding port; the transparent limiting box is fixed directly above the walking track of the treadmill. The transparent feeder includes a transparent food storage chamber, a food dropping port is arranged at the lower part of the transparent food storage chamber, and a valve is arranged at the food dropping port; a drag-type feeding box is arranged at the lower end of the food dropping port; the drag-type feeding box is movably arranged at the feeding port; the transparent feeder further includes a feeding box support base, the feeding box support base is arranged below the drag-type feeding box, and a locking device is correspondingly arranged between the feeding box support base and the drag-type feeding box; the bottom surface of the transparent food storage chamber is designed to be inclined, the valve is of a pull-out type, an opening is arranged in the middle of it, and a valve socket is correspondingly arranged at the food dropping port for the pull-out type valve.

2. The thermal stimulation pain testing device according to claim 1, wherein: A plurality of ventilation holes are further arranged at the upper front part of the transparent limiting box.

3. The thermal stimulation pain testing device according to claim 2, wherein: A fan is further arranged at the upper front part of the transparent limiting box, and the fan is arranged outside the ventilation holes.

4. A testing method using the thermal stimulation pain testing device according to any one of claims 1 to 3, characterized in that, It includes the following steps: (1) Disease modeling S1: Inject capsaicin: Intradermally inject 100 μl / 50 μg of capsaicin into the inner thigh of non-human primate experimental animals. S2: Temperature increase induction: Use the temperature-controlled heat application device to apply a temperature above 38°C and maintain it for more than 100 s to the injection area, so as to prompt non-human primate experimental animals to exhibit neuropathic pain symptoms. (2) Use the thermal stimulation pain test device to detect symptom manifestations Step 1: At the latest 100 s before starting to apply heat to non-human primate experimental animals using the temperature-controlled heat application device, ensure that the non-human primate experimental animals walk normally on the walking track of the treadmill in the thermal stimulation pain test device, and start timing after starting the heat application, and at the same time record the non-human primate experimental animals. Step 2: Stop timing when non-human primate experimental animals show pain manifestations.

5. The test method according to claim 4, characterized in that: For the temperature increase induction, use the temperature-controlled heat application device to apply 43°C to the injection area and maintain it for more than 100 s.

6. The test method according to claim 4, characterized in that: For the temperature increase induction, a dual-control temperature increase method is adopted, that is, first use the temperature-controlled heat application device to apply 38°C to the injection area and maintain it for more than 15 s, and then apply 43°C and maintain it for more than 120 s.

7. The test method according to claim 4, characterized in that: When testing the effect of the new drug to be tested, administer the new drug to be tested before injecting capsaicin into non-human primate experimental animals.

8. The test method according to claim 7, wherein: After 30 min and / or 60 min and / or 120 min after completing the administration of the new drug to be tested, then carry out the disease modeling and the symptom manifestation detection respectively.

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