A method for constructing an animal model of neuropathic pain induced by thermal injury

Abstract

The present invention provides a method for constructing an animal model of neuropathic pain caused by thermal injury, the method comprising: performing radiofrequency thermocoagulation on the sciatic nerve of an adult male SD rat; the radiofrequency thermocoagulation method is a bipolar radiofrequency mode, the position is 0.5 cm and 1 cm from the bifurcation of the rat sciatic nerve to the head end, and the radiofrequency parameters are as follows: temperature 65°C, pulse rate 2Hz, pulse width 20ms, duration 1min, and two radiofrequency thermocoagulation treatments are performed, with an interval of 5 minutes. The present invention constructs an animal model of neuropathic pain caused by thermal injury, the sensitivity to mechanical pain stimulation and the sensitivity to thermal pain stimulation are both increased, the sciatic nerve is damaged by light microscopy, the expression of Nav1.8mRNA is increased in the dorsal root ganglion, and a sequence of changes after radiofrequency thermocoagulation is consistent with the characteristics of neuropathic pain.

Description

Technical Field

[0001] The invention relates to a method for constructing an animal model of neuropathic pain induced by thermal injury, and belongs to the technical field of animal model construction. Background Art

[0002] Neuropathic pain is pain caused by damage or disease of the somatic sensory nervous system. Spontaneous pain and allodynia are its main clinical features. Its pathogenesis is complex and has multiple pathophysiological characteristics.

[0003] In the prior art, physical and chemical factors are used to cause nerve damage, thereby simulating neuropathic pain caused by various clinical reasons. For example, physical factors and inflammatory factors are simulated by chronic compression or ligation of the sciatic nerve to establish an animal model of neuropathic pain caused by peripheral nerve damage. The tightness and strength of the compression can only be judged by human observation, which has certain human errors and is difficult to balance, and the consistency of the degree of damage cannot be fully ensured. The situation where chemical factors cause nerve damage includes the establishment of an animal model of pathological pain of diabetic peripheral neuropathy through nerve damage caused by high blood sugar.

[0004] Due to the different causes of neuropathic pain, although the clinical manifestations are similar, there are obvious differences in their pathogenesis and treatment strategies. At present, with the widespread application of thermal effect clinical treatment methods, the incidence of neuropathic pain after related treatments has also increased. Common thermal effect treatment methods include microwave ablation, radiofrequency ablation, percutaneous laser ablation, and high-intensity focused ultrasound. Their working mechanism is to produce local thermal effects on target tissues through different energy methods, and to achieve the purpose of inactivating cells and tissues by causing tissue coagulation and necrosis through high temperature. Due to the close relationship between the treatment target and the nerves and problems related to surgical operations, the occurrence of pain caused by postoperative nerve damage is difficult to avoid.

[0005] Although neuralgia after thermal injury exists, there are few studies related to the repair and treatment of thermal injury to nerves. The fundamental reason is the lack of clinical models related to thermal injury to nerves. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for constructing an animal model of neuropathic pain induced by thermal injury, so as to achieve the following invention objectives: to construct an animal model of neuropathic pain induced by thermal injury, in which mechanical stimulation neuralgia is formed 5 days after treatment and thermal stimulation neuralgia is formed 7 days after treatment, and the duration of neuralgia is long.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A method for constructing an animal model of neuropathic pain induced by thermal injury, wherein adult male SD rats are subjected to radiofrequency thermocoagulation; the radiofrequency thermocoagulation method is to use a bipolar radiofrequency mode, with a radiofrequency temperature of 65°C, a pulse rate of 2Hz, a pulse width of 20ms, and a duration of 1min, and perform radiofrequency thermocoagulation twice, with an interval of 5 minutes. The radiofrequency position is 0.5cm and 1cm from the bifurcation of the rat sciatic nerve to the head end.

[0009] After the radiofrequency thermocoagulation needle touches the sciatic nerve, a foot twitch occurs, indicating that the preset position has been reached.

[0010] Before radiofrequency thermocoagulation, the position and depth of the sciatic nerve bifurcation at 0.5 cm and 1 cm toward the head of the rat were located by ultrasound and marked on the skin.

[0011] The rats were raised for 5 days before radiofrequency thermocoagulation; the raising method was to select adult male SD rats, control the temperature of the animal room and laboratory at 23-26°C, and the relative humidity at 40-60% to ensure the biological rhythm of the animals, implement a 12-hour light / dark cycle environment setting, and provide food and water freely; animal behavior experiments were all carried out during the day, and the rats were placed in an observation box for 30 minutes every day for 3 days before the experiment to adapt.

[0012] Compared with the prior art, the present invention achieves the following beneficial effects:

[0013] (1) This application creates a stable animal model of pathological neuropathic pain after thermal injury through radiofrequency thermocoagulation technology, which can be applied to the research of nerve repair after thermal injury and subsequent clinical treatment.

[0014] (2) The present invention constructs an animal model of thermal injury-induced neuropathic pain, which increases the sensitivity to mechanical pain stimulation and thermal pain stimulation. The behavioral changes of rats after radiofrequency thermocoagulation are consistent with the characteristics of neuropathic neuralgia. Mechanical stimulation neuralgia is formed 5 days after radiofrequency thermocoagulation, and thermal stimulation neuralgia is formed 7 days after radiofrequency thermocoagulation. The neuralgia lasts until 30 days after radiofrequency thermocoagulation. Light microscopy shows signs of sciatic nerve injury, and the dorsal root ganglion shows increased expression of Nav1.8 (voltage-gated sodium channel 1.8) mRNA BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a light microscopic image of the sciatic nerve of the control rat at 14 days under 20 times magnification;

[0016] Figure 2 This is a light microscopic image at 20 times magnification of the sciatic nerve of a rat 14 days after 65°C radiofrequency thermocoagulation;

[0017] Figure 3The results of RT-PCR of Nav1.8 mRNA in dorsal root ganglion of rats in the control group and 14 days after 65℃ radiofrequency thermocoagulation. DETAILED DESCRIPTION

[0018] Example 1 A method for constructing an animal model of thermal injury-induced neuropathic pain

[0019] (1) Animals and breeding

[0020] Adult male SD rats were selected, and the temperature of the animal room and laboratory was controlled at 23-26℃, and the relative humidity was 40-60% to ensure the biological rhythm of the animals. A 12-hour light / dark cycle environment was implemented, and food and water were provided freely. Animal behavior experiments were all carried out during the day, and the rats were placed in an observation box for 30 minutes every day for 3 days before the experiment to adapt.

[0021] Twenty-four rats were randomly divided into four groups according to the random number table method: 6 rats in each group.

[0022] The control group only placed radiofrequency needles without radiofrequency thermocoagulation. The other three groups were set at different radiofrequency temperatures and underwent bipolar radiofrequency thermocoagulation, namely the 55℃ group (denoted as RF55), the 65℃ group (denoted as RF65) and the 75℃ group (denoted as RF75), and the other radiofrequency parameters were the same.

[0023] (2) Radiofrequency thermocoagulation

[0024] Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). The rats were placed in a prone position with their limbs stretched out and fixed on a fixation frame. The surface hair of the rats was cleaned near the right femur of the rats. Ultrasound scanning was performed behind the thigh to find the bifurcation of the sciatic nerve. Then, the position and depth of 0.5 cm and 1 cm toward the head were located and marked on the skin. The rats were disinfected with iodine three times.

[0025] The radiofrequency thermocoagulation needle (22G, 5 cm in length, 5 mm in exposed end) was inserted, and the foot twitched, indicating that the puncture reached the preset position.

[0026] The control group only punctured the sciatic nerve at the preset position with a radiofrequency thermocoagulation needle, and no radiofrequency thermocoagulation was performed. The other three groups underwent bipolar radiofrequency treatment, namely the 55°C group, the 65°C group, and the 75°C group. The other radiofrequency parameters were: pulse rate 2Hz, pulse width 20ms, and duration 1min. Radiofrequency thermocoagulation was repeated twice with an interval of 5 minutes. After the experiment, the radiofrequency needles of each group were removed.

[0027] Example 2 Spontaneous pain response measurement

[0028] Before the experiment, the rats were placed in the test box to adapt for more than 30 minutes (the purpose was to make the rats familiar with the subsequent equipment and reduce the influence of factors such as tension and panic). On the first and third days after radiofrequency thermocoagulation, the number of spontaneous paw reflexes of the rats was observed every 5 minutes within 20 minutes, and the corresponding records were made to evaluate the degree of spontaneous pain in the rats. Scoring criteria: 0 points if the rat's hind feet can be placed flat on the ground normally; 1 point if the rat's hind feet are placed flat on the ground with the toes together and facing downward; 2 points if only the inner edge of the hind paw of the rat's hind foot touches the ground; 3 points if only the heel of the rat touches the ground and the toes are curled inward; 4 points if the rat's whole foot is raised and away from the ground; 5 points if the rat licks its feet.

[0029] The results are shown in Table 1.

[0030] Table 1 Spontaneous pain scores of rats (n=6)

[0031]

[0032] From the results in Table 1, it can be seen that on the 1st and 3rd day after radiofrequency thermocoagulation, the spontaneous pain of rats was higher than that before treatment and in the control group. The higher the temperature of radiofrequency thermocoagulation, the higher the score of spontaneous pain.

[0033] After waking up from radiofrequency thermocoagulation, rats treated with radiofrequency thermocoagulation had spontaneous pain behavior on the same day, and the number of foot lifting and licking did not increase significantly. However, the number of spontaneous foot licking began to increase one day after radiofrequency thermocoagulation, and the toes of the foot on the radiofrequency thermocoagulation side began to bite. In the resting position, rats treated with radiofrequency thermocoagulation intermittently and suddenly licked the hind paw on the same side of the treatment side, and gently bit or pulled the nails with their mouths. This was common within 20 days after radiofrequency thermocoagulation, but the frequency was lower in 25-40 days. RF65 and RF75 showed autophagy, which was most obvious from 3-7 days after radiofrequency thermocoagulation, often in a state of dripping blood and repeated biting. 20 days after radiofrequency thermocoagulation, the autophagy of rats improved, and the wounds healed with scabs. The feet of rats on the radiofrequency thermocoagulation side were mostly bent, the toes were gathered, and the hind paws were everted.

[0034] Example 3 Determination of mechanical withdrawal threshold (MWT) in rats

[0035] MWT is an important data for determining the mechanical pain threshold of rats. In order to eliminate the influence of psychological factors on the test results, rats were placed in the test box for 20 minutes before the test. The experimental environment was controlled at (23±2)℃, and the laboratory was maintained in a quiet environment. Then, in a transparent container separated from each other by a perforated metal grid at the bottom, von Frey fibers of different masses were selected to vertically stimulate the outer side of the right hind paw of the rat. The unit was set to g, starting with a small intensity stimulation and gradually increasing. Each pressure stimulation was 10 times, with an interval of 3-5s. If more than 5 out of 10 stimulations resulted in foot retraction, shaking and licking, it was the mechanical pain threshold of the rat.

[0036] Each test was performed at a fixed time between 8:00 and 12:00 every day, and was conducted on the 1st, 3rd, 5th, 7th, 10th, 15th, 20th, 25th, 30th, 35th, and 40th day after radiofrequency thermocoagulation treatment.

[0037] The experimental results showed that there was no significant change in MWT during the entire observation period in the control group, and there was only a short-term retraction and avoidance reaction when the stimulation threshold was reached. Rats treated with radiofrequency thermocoagulation showed a longer-term avoidance reaction to the threshold stimulation. After radiofrequency thermocoagulation in the RF55 group, the MWT sensitivity increased from d3 to d10 (p<0.05), but it could be restored to the state before radiofrequency thermocoagulation around d15 after radiofrequency thermocoagulation. In the RF65 group, the MWT increased on the first day after radiofrequency thermocoagulation, and the sensitivity increased from d5 to d30 (p<0.05). On d35, the MWT of rats returned to the level before radiofrequency thermocoagulation. In the RF75 group, the thresholds on d1-d15 increased significantly, and the sensitivity to mechanical pain stimulation decreased significantly (p<0.05). During the entire observation period, the MWT threshold was higher than before radiofrequency thermocoagulation. It can be seen that neuralgia began to form 5 days after radiofrequency thermocoagulation in group RF65, lasted for 25 days, and gradually disappeared 30 days after radiofrequency thermocoagulation. The duration of neuralgia was significantly longer than that in group RF55.

[0038] Table 2 Comparison of mechanical stimulation paw withdrawal thresholds in rats (n=6, unit: g)

[0039]

[0040] Compared with the control group * p<0.05, ** p<0.01, compared with the same group before treatment, #p<0.05, ##p<0.01

[0041] Example 4 Determination of Thermal Withdrawal Latency (HWL) in Rats

[0042] HWL is an important index for measuring the rat's response to thermal pain stimulation. Before the test, the rat was kept in a resting state for 20 minutes, the experimental environment was controlled at (23±2)℃, and the laboratory was maintained in a quiet environment. The rat was placed in a plexiglass cover, and the sole of the rat was irradiated by the thermal pain stimulation system. The parameters were set to 60% intensity and 30 seconds of interruption time. When the animal had a rapid foot-lifting reaction, the time from the start of irradiation to the appearance of the foot-withdrawal escape reaction was recorded, and the measurement was repeated 5 times, with an interval of no less than 10 minutes each time, and the average of the 5 times was taken as the thermal pain latency of the rat.

[0043] Each test was performed at a fixed time between 8:00 and 12:00 every day, and was conducted on the 1st, 3rd, 5th, 7th, 10th, 15th, 20th, 25th, 30th, 35th, and 40th day after radiofrequency thermocoagulation treatment.

[0044] The results showed that HWL of RF55 increased slightly one day after radiofrequency thermocoagulation, decreased significantly from d7 to d15 compared with before radiofrequency thermocoagulation (p<0.05), and returned to the level before radiofrequency thermocoagulation on day 20. HWL of RF65 increased significantly from d1 to d3 after radiofrequency thermocoagulation, decreased significantly from d7 to d30 compared with before radiofrequency thermocoagulation (p<0.05), and returned to the level before radiofrequency thermocoagulation on d35. HWL of rats in the RF75 group was significantly higher than that of the control group and before radiofrequency thermocoagulation during the entire observation period.

[0045] It can be seen that the RF65 group began to develop thermal stimulation neuralgia on the 7th day after radiofrequency thermocoagulation treatment, and gradually disappeared 30 days after radiofrequency thermocoagulation treatment. The duration of thermal stimulation neuralgia was significantly longer than that of the R55 group.

[0046] Table 3 Comparison of thermal stimulation paw withdrawal latency in rats (n=6, unit: sec)

[0047]

[0048] Compared with the control group * p<0.05, ** p<0.01, compared with the same group before radiofrequency thermocoagulation treatment, #p<0.05, ##p<0.01

[0049] The results of Examples 3 and 4 above show that the duration of RF55 neuralgia is short, and the nerve function of rats recovers quickly after radiofrequency thermocoagulation treatment, and the nerve function can be restored to the state before radiofrequency thermocoagulation treatment in about 2 weeks. The nerve damage of rats in the RF75 group was more serious after radiofrequency thermocoagulation treatment, and the plantar MWT and HWL increased significantly, and the sensitivity to mechanical pain stimulation and the sensitivity to thermal pain stimulation were reduced, indicating that 75°C caused obvious damage to the sciatic nerve of rats, which did not meet the clinical manifestations related to pathological neuralgia.

[0050] The duration of neuralgia in the RF65 group was longer, and the sensitivity to mechanical pain stimulation and thermal pain stimulation were increased. The behavioral changes of rats after radiofrequency thermocoagulation treatment were more consistent with the clinical manifestations of the neuropathic neuralgia model. Therefore, the modeling of rats in the RF65 group was successful.

[0051] Example 5 Morphological observation of rat sciatic nerve

[0052] Another 12 rats were randomly divided into two groups: 6 rats in the control group and 6 rats in the 65°C radiofrequency thermocoagulation treatment group (RF65). The model was established according to the method of Example 1. The sciatic nerves of the two groups of rats were obtained 14 days after the radiofrequency thermocoagulation treatment.

[0053] The fixed sciatic nerves of the rats were embedded in paraffin and prepared into 5 μm sections for hematoxylin-eosin staining.

[0054] Sin, HE) staining was performed to observe the pathological changes of nerves and surrounding tissues under a light microscope. The light microscope images are attached. Figure 1 and 2 .

[0055] The results showed that the rats in the RF65 group had nerve rupture, increased vacuoles, tissue edema, and a looser nerve tissue structure, indicating that pathological changes occurred in the sciatic nerve at the RF site.

[0056] Example 6 Expression of Nav1.8 ion channel mRNA in dorsal root ganglia

[0057] 14 days after radiofrequency thermocoagulation, the L4-L5 dorsal root ganglia of rats in the control group and RF65 group of Example 5 were taken for RT-PCR semi-quantitative analysis of Nav1.8 mRNA content. RNA of each group was extracted according to the Trizol (Invitrogen) method, and the extracted RNA was identified by spectrophotometry. cDNA was obtained by reverse transcription, and then the Nav1.8 gene and the internal reference β-Action gene were amplified. After amplification, horizontal electrophoresis was performed and semi-quantitative analysis was performed using an image analysis system. The expression level of Nav1.8 mRNA is shown in the attached Figure 3 .

[0058] The results showed that the expression of Nav1.8 mRNA related to neuropathic pain in the dorsal root ganglion tissue of rats in the RF65 group was significantly increased compared with that in the control group. Thermal damage occurred in the peripheral nerves, but the dorsal root ganglia were involved in the occurrence of neuropathic pain.

Claims

1. A method for constructing an animal model of neuropathic pain induced by thermal injury, characterized in that: The sciatic nerve of adult male SD rats was subjected to radiofrequency thermocoagulation; the radiofrequency thermocoagulation method was to use a bipolar radiofrequency mode, the position was 0.5 cm and 1 cm from the bifurcation of the rat sciatic nerve toward the head end, the radiofrequency temperature was 65°C, the pulse rate was 2Hz, the pulse width was 20ms, the duration was 1min, and the radiofrequency thermocoagulation was performed twice, with an interval of 5 minutes.

2. The method for constructing an animal model of thermal injury-induced neuropathic pain according to claim 1, characterized in that: Before radiofrequency thermocoagulation, the position and depth of the sciatic nerve bifurcation at 0.5 cm and 1 cm toward the head of the rat were located by ultrasound and marked on the skin.

3. The method for constructing an animal model of thermal injury-induced neuropathic pain according to claim 1, characterized in that: The rats were raised for 5 days before radiofrequency thermocoagulation. Adult male SD rats were selected for the feeding method. The temperature of the animal room and laboratory was controlled at 23-26°C, and the relative humidity was controlled at 40-60% to ensure the biological rhythm of the animals. A 12-hour light / dark cycle environment was implemented, and food and water were provided freely. Animal behavioral experiments were all carried out during the day, and the rats were placed in an observation box for 30 minutes every day for 3 days before the experiment to adapt.

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