Mouse model for detecting endogenous pain regulation function and construction method and application thereof

Abstract

The invention discloses a mouse model for detecting an endogenous pain regulation function as well as a construction method and application of the mouse model, and relates to the technical field of experimental animal models. The construction method of the mouse model comprises the following steps: screening out mice with normal mechanical pain thresholds for model construction by measuring the mechanical pain thresholds of the mice; injecting a complete Freund's adjuvant into the front limbs of the screened mice or clamping the front limbs by using a clip; capsaicin is injected into the hind limbs of the mouse, and the mouse model for detecting the endogenous pain regulation function is obtained through pain behavior verification. The construction method of the mouse model is simple, the success rate of model construction is high, and the mouse model has important value and significance for pathological research of chronic pain and screening and evaluation of drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of experimental animal models, and in particular to a mouse model for detecting endogenous pain regulation function, and a construction method and application thereof. Background Art

[0002] Endogenous pain modulation refers to a series of behaviors of the central nervous system that can reduce or enhance pain. These behaviors are usually initiated in different brain regions, output in the form of a spectrum, converge in the brainstem pain center, and then send descending information to the spinal cord, which can be inhibitory or facilitatory, respectively reducing or enhancing the nociceptive signals from the periphery. Descending pain control pathways can be activated by a "top-down" mode, that is, from the brain to the brainstem, such as through psychological intervention. In addition, they can also be induced by a "bottom-up" mode, that is, from the periphery and spinal cord to the brainstem, such as noxious stimulation. The latter is often called "counterstimulation" or "pain suppression by pain". Since this century, there has been a large amount of human research literature that unanimously recommends the use of the term "conditioned pain modulation" (CPM).

[0003] Compared with normal controls, patients with idiopathic pain syndromes are characterized by inefficient CPM, including fibromyalgia, irritable bowel syndrome, migraine, tension headache, temporomandibular joint disorder, osteoarthritis, myalgia, and whiplash. Inefficient CPM is common among patients with various pain syndromes. One possibility is that inefficient pain inhibition is secondary to the presence of pain, while another possibility is that inefficient pain inhibition is the main cause of pain in clinical practice and serves as a risk factor for the development of pain. Patients who had no pain before thoracotomy were tested for pain modulation and followed up for the occurrence of chronic pain after surgery. CPM efficiency was found to predict chronic pain after thoracotomy; patients with inefficient CPM had a higher risk of developing chronic pain.

[0004] A comprehensive understanding of the function of an individual's endogenous pain regulation system can help accelerate the progress of pain mechanism research, help accurately screen drug targets, and promote precision medicine for pain drugs. In addition, the efficiency of endogenous pain regulation in the body can also predict the risk of chronic pain. By detecting the endogenous regulation function of pain, it is beneficial to effectively intervene in chronic pain at an early stage, thereby reducing the pain caused by chronic pain and potentially effectively reducing social medical costs. However, the function of endogenous pain modulation in the body is relatively complex. Therefore, it is necessary to establish a method or model for evaluating an individual's pain regulation ability in order to conduct in-depth mechanism research and provide theoretical support and ideas for corresponding clinical applications.

[0005] Chinese patent CN116584436A discloses a method for constructing a chronic primary pain mouse model and its application, the method comprising the following steps: (1) screening mice with normal pain threshold by measuring the baseline of mechanical pain threshold of mice for model construction; (2) using low-frequency transcutaneous electrical stimulation to obtain a chronic primary pain mouse model by verifying pain behavior of the mice screened in step (1). The mouse model can be used to study the pathology of chronic primary pain and evaluate treatment methods such as drug therapy, but cannot be used to detect endogenous pain regulation function.

[0006] In view of this, in order to address the deficiencies of the prior art, the present invention provides a mouse model for detecting endogenous pain regulation function, and a construction method and application thereof. Summary of the invention

[0007] The purpose of the present invention is to provide a mouse model for detecting endogenous pain regulation function, a construction method and application thereof, to construct a model in which a pain stimulus (conditioned stimulus) inhibits the pain perception caused by a pain stimulus (experimental stimulus) applied to another part of the body, to realize the detection of the endogenous pain regulation function of mice, and to provide a method for detecting the degree of pain inhibition.

[0008] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a method for constructing a mouse model for detecting endogenous pain regulation function, comprising the following steps:

[0010] (1) By measuring the mechanical pain threshold of mice, mice with normal mechanical pain threshold were selected for model construction;

[0011] (2) Injection of complete Freund's adjuvant or clamping of the forelimbs of the selected mice;

[0012] (3) Capsaicin was injected into the hind limbs of mice, and the pain behavior was verified to obtain a mouse model for detecting endogenous pain regulation function.

[0013] Preferably, in step (1), the mouse is an adult C57BL / 6 mouse.

[0014] Preferably, in step (1), the normal mechanical pain threshold means that the mechanical pain threshold is ≥ 0.3g.

[0015] Preferably, in step (2), the volume of the injected complete Freund's adjuvant is 5-20 μl.

[0016] Further preferably, in step (2), the concentration of the injected complete Freund's adjuvant is 0.5-2 mg / mL.

[0017] Preferably, step (2) also includes a mechanical pain threshold measurement process, which is as follows: the screened mice are anesthetized, complete Freund's adjuvant is injected into the forelimbs or clamped using a clamp, and the mechanical pain threshold of the corresponding part is measured after the mice wake up.

[0018] Preferably, in step (3), the volume of the injected capsaicin is 5-20 μl.

[0019] Further preferably, in step (3), the concentration of the injected capsaicin is 0.5-2 mg / mL.

[0020] Preferably, in step (3), the pain behavior verification comprises: measuring the mechanical pain threshold and spontaneous pain score after injecting capsaicin into the hind limbs of mice.

[0021] In another aspect, the present invention provides a mouse model for detecting endogenous pain regulation function, wherein the mouse model is constructed by the above-mentioned construction method.

[0022] Finally, the present invention provides the application of the above mouse model in exploring the pathogenesis of chronic pain, screening and evaluating drugs for treating chronic pain.

[0023] The beneficial effects of the present invention are:

[0024] The invention discloses a mouse model for detecting endogenous pain regulation function. The mouse model has a simple construction method and a high success rate of model construction, and has important value and significance for pathological research on chronic pain and screening and evaluation of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The schematic diagram of pain in the forelimb of mice and the changes in pain are constructed for Example 1, wherein a is the pain caused by injection of CFA into the forelimb of mice; b is the pain caused by stimulation of the forelimb of mice with crocodile clips.

[0026] Figure 2 This is a schematic diagram of how pain caused by CFA injection into the forelimbs of mice inhibits pain in the hindlimbs and pain changes in Example 1, wherein a is a schematic diagram of how pain caused by CFA injection into the forelimbs of mice inhibits pain in the hindlimbs; b is the pain changes in mice.

[0027] Figure 3 This is a schematic diagram of how the pain induced by applying crocodile clips to the forelimbs of mice suppresses the pain in the hindlimbs and the pain changes in Example 1, wherein a is a schematic diagram of how the pain induced by applying crocodile clips to the forelimbs of mice suppresses the pain in the hindlimbs; b is the pain changes in mice.

[0028] Figure 4This is a schematic diagram of the pain suppression and pain changes in the hind limbs caused by injection of different doses of CFA into the forelimbs of mice in Example 2, wherein a and b are schematic diagrams of the pain suppression and pain changes in the hind limbs caused by injection of 5uL CFA into the forelimbs of mice; c and d are schematic diagrams of the pain suppression and pain changes in the hind limbs caused by injection of 10uL CFA into the forelimbs of mice.

[0029] Figure 5 This is a schematic diagram of detecting and evaluating the CPM efficiency and pain changes of SNI mice in Example 3, wherein a is a schematic diagram of detecting hind limb pain and pain changes under the CPM model of SNI mice, and b is a schematic diagram of administering duloxetine to SNI mice to enhance the CPM efficiency of mice.

[0030] Figure 6 This is a schematic diagram of the inhibition of acute pain in the hind limbs by carrageenan in the forelimbs of mice and the pain changes in comparative example 1, wherein a is a schematic diagram of the inhibition of hind limb pain by pain caused by injection of CFA in the forelimbs of mice; b is the pain changes in mice; c is a schematic diagram of the inhibition of hind limb pain by pain caused by crocodile clips in the forelimbs of mice; d is the pain changes in mice.

[0031] Figure 7 This is a schematic diagram of the inhibition of hindlimb pain caused by injection of formalin in the forelimbs of mice and the pain changes in comparative example 2, wherein a is a schematic diagram of the inhibition of hindlimb pain caused by injection of formalin in the forelimbs of mice; b is the pain changes in mice.

[0032] Figure 8 This is a schematic diagram of comparative example 3 for detecting and evaluating the CPM efficiency and pain changes of SNI mice using CFA and 5 μl of capsaicin, wherein a is a schematic diagram for detecting hind limb pain under the CPM model of SNI mice, and b is the pain changes after duloxetine was applied to SNI mice.

[0033] Figure 1-8 In the table, *, **, and *** represent significant differences between groups, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. DETAILED DESCRIPTION

[0034] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and they should also fall within the scope of the present invention.

[0035] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0036] Example 1

[0037] A method for constructing a mouse model for detecting endogenous pain regulation function, specifically comprising the following steps:

[0038] (1) A group of adult C57BL / 6 mice (male and female) were selected, and the mechanical pain threshold of all mice was measured at the same time every day from day 5 to 7. Mice with abnormal mechanical pain threshold of hind limbs (less than 0.3 g) were eliminated, and the remaining mice were used to construct the model, ensuring that the number of mice used for behavioral statistics in each group was ≥ 4 mice / group;

[0039] (2) Mice were anesthetized by steady inhalation of 2%-3% isoflurane. The forelimbs of the mice in the experimental group were injected with 20 μL of 1 mg / mL complete Freund's adjuvant (CFA) or clamped with crocodile clips. The mechanical pain threshold of the corresponding part was measured 60 minutes after the mice woke up. The forelimbs of the mice in the control group were injected with 20 μL of normal saline or the crocodile clips were attached to the forelimbs of the mice with tape. It was found that the forelimbs of the mice showed obvious pain sensitivity after injection of CFA and clamping with crocodile clips (such as Figure 1 );

[0040] (3) 10 μL of 1 mg / mL capsaicin (CAP) was injected into the hind limbs of mice in the experimental and control groups, and the mechanical pain threshold and spontaneous pain score of the hind limbs of the mice were tested. It was found that the pain threshold of the hind limbs of mice with forelimb pain was significantly increased (e.g. Figure 2 and Figure 3 ), indicating that a model has been successfully established in which a painful stimulus (conditioned stimulus) inhibits the pain perception caused by a painful stimulus (experimental stimulus) applied to another part of the body, and suggesting that normal mice have good endogenous pain inhibition function.

[0041] The mechanical pain threshold is measured using the von Frey experiment, and the specific process is: use von Frey fibers (0.04-1.0 grams) to act vertically on the surface of the skin on the sole of the mouse's foot, and apply sufficient force to make it bend. When the mouse retracts its paw or licks its paw, the fiber threshold at this time is recorded, and the average value of the minimum fiber applied five times is calculated as its mechanical pain threshold.

[0042] The specific process of detecting the spontaneous pain score is as follows: the behavioral performance of each mouse is recorded by video for 30 minutes, and then the video clips are played back in slow motion, and the occurrence of licking, retracting, shaking or lifting events involving the hind paws is manually counted, and actions related to movement, body repositioning and grooming are excluded from the count; each lick is scored as 2 points, and each retracting, shaking or lifting is scored as 1 point, and the accumulated score obtained is the spontaneous pain score.

[0043] Statistical analysis: GraphPad Prism 8.0 was used to process and plot statistical results. All data met normality, and the inter-group comparisons were analyzed using T-tests (unpaired two-tailed Student's t-tests). Differences were considered statistically significant when P < 0.05.

[0044] Example 2 Effects of different doses of CFA injected into the forelimbs on hindlimb pain

[0045] This example uses the mouse model constructed in Example 1 for evaluation to detect the inhibitory effect of different intensities of forelimb pain on hindlimb pain. The specific method is as follows:

[0046] (1) Ten SPF-grade adult C57BL / 6 mice (weight 20±2 g, half male and half female) were selected as the research subjects. The experimental animals were purchased from Weitonglihua Biotechnology Co., Ltd., with ethical review approval number: USTCACUC26120123010. The mice were raised in standard conditions (relative humidity 50±10%, 12-hour light-dark cycle, temperature 22±2℃), 5 per cage, fed with ordinary feed, and free access to food and water. They were randomly divided into 2 groups, namely high-dose group (n=5) and low-dose group (n=5);

[0047] (2) Low-dose group: The mice were anesthetized by steady inhalation of 2%-3% isoflurane, and 5 μL of 1 mg / mL CFA was injected into the left forelimb of the mice. The mechanical pain threshold was measured 60 minutes after the mice woke up.

[0048] (3) High-dose group: The mice were anesthetized by steady inhalation of 2%-3% isoflurane, and 10 μL of 1 mg / mL CFA was injected into the left forelimb of the mice. The mechanical pain threshold was measured 60 minutes after the mice woke up.

[0049] (4) 10 μL of 1 mg / mL capsaicin was injected into the left hind limbs of the two groups of mice, and the mechanical pain threshold and spontaneous pain score of the hind limbs of the mice were tested. It was found that the hind limb pain of the mice injected with 5 μL CFA was not significantly relieved, while the hind limb pain of the mice injected with 10 μL CFA was significantly relieved (e.g. Figure 4 ).

[0050] Example 3 Application of mouse model

[0051] Individuals with chronic pain often display inefficient CPMs, so this model can be used to evaluate the efficacy of drugs to treat inefficient CPMs. The specific methods are as follows:

[0052] (1) A group of adult C57BL / 6 mice (male and female) were selected and the right hind limb nerve was ligated to obtain chronic pain-sparing nerve injury (SNI) mice. Control mice were also operated on without nerve ligation.

[0053] (2) Mice were anesthetized by steady inhalation of 2%-3% isoflurane, and 20 μL of 1 mg / mL CFA was injected into the left forelimb of SNI and control mice, and 10 μL of 1 mg / mL capsaicin was injected into the left hindlimb. The mechanical pain threshold and spontaneous pain score of the left hindlimb of the mice were tested. It was found that SNI mice did not show hindlimb pain relief, suggesting that CPM efficiency of chronic pain mice is low (e.g. Figure 5 );

[0054] (3) The model was constructed by intraperitoneal injection of duloxetine (24 mg / kg) into SNI mice, and it was found that the pain in the hind limbs of SNI mice was relieved, indicating that duloxetine can be used to improve the efficiency of CPM and enhance the endogenous pain regulation function of mice (such as Figure 5 ).

[0055] Comparative Example 1

[0056] The capsaicin-induced pain model establishment method in Example 1 was replaced by the carrageenan-induced pain model establishment method, and the remaining steps were the same as in Example 1.

[0057] (1) A group of adult C57BL / 6 mice (male and female) were selected, and the mechanical pain threshold of all mice was measured at the same time every day from day 5 to 7. Mice with abnormal mechanical pain threshold of hind limbs (less than 0.3 g) were eliminated, and the remaining mice were used to construct the model, ensuring that the number of mice used for behavioral statistics in each group was ≥ 4 mice / group;

[0058] (2) Mice were anesthetized by steady inhalation of 2%-3% isoflurane. The forelimbs of mice in the experimental group were injected with 20 μL of 1 mg / mL complete Freund's adjuvant (CFA) or clamped with crocodile clips. The mechanical pain threshold of the corresponding part was measured 60 minutes after the mice woke up. The forelimbs of mice in the control group were injected with 20 μL of normal saline or the crocodile clips were attached to the forelimbs of the mice with tape.

[0059] (3) 15 μL of 5 mg / mL carrageenan was injected into the hind limbs of mice in the experimental and control groups, and the mechanical pain threshold and spontaneous pain score of the hind limbs of the mice were tested. It was found that the pain threshold of the hind limbs of mice with forelimb pain was increased (e.g. Figure 6 ), but the analgesic effect was not as obvious as that of the complete Freund's adjuvant and capsaicin combination model.

[0060] Comparative Example 2

[0061] The complete Freund's adjuvant pain model in Example 1 was replaced by a formalin pain model, and the remaining steps were the same as in Example 1.

[0062] (1) A group of adult C57BL / 6 mice (male and female) were selected, and the mechanical pain threshold of all mice was measured at the same time every day from day 5 to 7. Mice with abnormal mechanical pain threshold of hind limbs (less than 0.3 g) were eliminated, and the remaining mice were used to construct the model, ensuring that the number of mice used for behavioral statistics in each group was ≥ 4 mice / group;

[0063] (2) The mice were anesthetized by steady inhalation of 2%-3% isoflurane. The forelimbs of the mice in the experimental group were injected with 10 μL of 5% formalin solution. The mechanical pain threshold of the corresponding part was measured 30 minutes after the mice woke up. The forelimbs of the mice in the control group were injected with 10 μL of normal saline.

[0064] (3) 10 μL of 1 mg / mL capsaicin was injected into the hind limbs of mice in the experimental and control groups, and the mechanical pain threshold and spontaneous pain score of the hind limbs of the mice were tested. It was found that the pain threshold of the hind limbs of mice injected with formalin in the forelimbs was increased (e.g. Figure 7 ), but the analgesic effect was not as obvious as that of the complete Freund's adjuvant and capsaicin combination model.

[0065] Comparative Example 3

[0066] The 10 μL of 1 mg / mL capsaicin in step (2) of Example 3 was replaced with 5 μL of 1 mg / mL capsaicin, and the remaining steps were the same as in Example 3.

[0067] (1) A group of adult C57BL / 6 mice (male and female) were selected and the right hind limb nerve was ligated to obtain chronic pain-sparing nerve injury (SNI) mice. Control mice were also operated on without nerve ligation.

[0068] (2) Mice were anesthetized by steady inhalation of 2%-3% isoflurane, and 20 μL of 1 mg / mL CFA was injected into the left forelimb of SNI and control mice, and 5 μL of 1 mg / mL capsaicin was injected into the left hindlimb;

[0069] (3) The model was constructed after intraperitoneal injection of duloxetine (24 mg / kg) into SNI mice. It was found that the pain in the hind limbs of SNI mice was relieved, but the pain relief was not as significant as that after 10 μL of capsaicin injection, suggesting that compared with 5 μL of capsaicin, 10 μL of capsaicin can achieve accurate evaluation of the efficacy of treating inefficient CPM (e.g. Figure 8 ).

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a mouse model for detecting endogenous pain regulation function, characterized in that: The following steps are involved: (1) By measuring the mechanical pain threshold of mice, mice with normal mechanical pain threshold were selected for model construction; (2) Injection of complete Freund's adjuvant or clamping of the forelimbs of selected mice; (3) Capsaicin was injected into the hind limbs of mice, and the pain behavior was verified to obtain a mouse model for detecting endogenous pain regulation function.

2. The construction method according to claim 1, characterized in that: In step (1), the mouse is an adult C57BL / 6 mouse.

3. The construction method according to claim 1, characterized in that: In step (1), the normal mechanical pain threshold means that the mechanical pain threshold is ≥ 0.3g.

4. The construction method according to claim 1, characterized in that: In step (2), the volume of the injected complete Freund's adjuvant is 5-20 μl.

5. The construction method according to claim 4, characterized in that: In step (2), the concentration of the injected complete Freund's adjuvant is 0.5-2 mg / mL.

6. The construction method according to claim 1, characterized in that: Step (2) also includes a mechanical pain threshold measurement process, which is as follows: the screened mice are anesthetized, complete Freund's adjuvant is injected into the forelimbs or clamped with a clamp, and the mechanical pain threshold of the corresponding part is measured after the mice wake up.

7. The construction method according to claim 1, characterized in that: In step (3), the volume of the injected capsaicin is 5-20 μL; the concentration of the injected capsaicin is 0.5-2 mg / mL.

8. The construction method according to claim 1, characterized in that: In step (3), the pain behavior verification includes: measuring the mechanical pain threshold and spontaneous pain score after injecting capsaicin into the hind limbs of mice.

9. A mouse model for detecting endogenous pain regulation function, characterized in that: The mouse model is constructed by the construction method described in any one of claims 1-8.

10. Use of the mouse model according to claim 9 in exploring the pathogenesis of chronic pain, and screening and evaluating drugs for treating chronic pain.

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