KR101794203B1 - A discogenic low back pai animal model and method of preparing the same - Google Patents

Abstract

The present invention relates to an intervertebral disc model animal and a method for producing the same. The intervertebral disc model animals prepared according to various embodiments of the present invention accelerate the spontaneous disc herniation for a long period of time and are similar to the symptoms confirmed in patients with acute or chronic pain, It is possible to study on the maintenance period. Therefore, it is confirmed that the prepared model animal has the symptom and onset mechanism of human intervertebral disc back pain unlike the existing animal model, and the model animal has the mechanism of discoloration of back pain And for the development of therapeutic and therapeutic agents for intervertebral disc pain.

Description

A discogenic low back animal model and method of preparing same,

The present invention relates to an intervertebral disc model animal and a method for producing the same.

Generally, spinal disease means backache, lower back pain, or paralysis due to abnormalities or damage of the back, bones, nerves and tissues of the back. Mainly degenerative cases account for 50-70% of spinal diseases, trauma and tumors account for about 10-20%.

Disc degeneration is one of the basic etiologies of various vertebral diseases and is known to be a major cause of patient's pain and disability. It is clinically diagnosed as back pain, herniated disc herniation, neurodegeneration, spinal stenosis, spinal instability It can be expressed in combination with various diseases.

 Degeneration of the intervertebral disc is initiated or developed by multiple factors, such as reduced nutrition, overloaded mechanical force, and genetic factors. As age increases, changes in extracellular matrix composition in the intervertebral disc alter the function of the intervertebral disc cells and increase cell death. There is an increase in proinflammatory cytokines and related degrading enzymes in the microenvironment of intervertebral disc cells. This is the result of a marked reduction in the nutrient diffusion in the chondrocyte plate with thinning and calcification. Mechanical forces also play a direct role in disc degeneration. Both cell survival and extracellular matrix synthesis are susceptible to compressive stress. Some mechanical stimuli are essential for promoting nutrient diffusion and matrix synthesis, but excessive force causes local tissue damage, slowing tissue repair and breaking the inherent strain distribution in extracellular matrix. Finally, genetic factors are also important for individual vulnerability to disc degeneration, supported by results from group studies through twin studies and genome-wide assays.

Discogenic low back pain can occur as the discs lose their normal function of securing motility while adequately dispersing the forces exerted between the vertebrae and vertebrae. It is usually accompanied by a degenerated IVD, and a prolapsed or bulging IVD can cause pain by compressing or irritating nerve root and / or nerve root around the vertebrae , And extruded or herniated IVD may stimulate pain receptors present in the spinal cord dermis or give stretching stress to the posterior spinal cord. This is an indirect pain due to the disc, and recent research has provided experimental evidence of direct pain through the painful nerve fibers of the disc itself. Structural connections of the nucleus pulposus and the fibrous ring, flaws and gaps in the intervertebral discs can not only abnormally increase the proinflammatory molecule particles from the cells present in the nucleus pulposus and fibronules, but also enable activation and invasion of immune cells. In the dorsal root ganglia, neurotrophins induce pain-related cation channels (eg, acid-sensing ion channel 3 (ASIC3)) and nociceptive nerve fibers It has been reported that it can hyperpolarize the pain nerve fibers by promoting the increase of TRPV1.

In this way, the mechanism and the treatment of back pain caused by the intervertebral disc require an abundant research result and results through experimental animal models.

In the case of conventional herniated IVD (SD rats) model, a portion of the nucleus pulposus was extracted after perforating L4 (between lumbar 4-5) with a 25G injection needle, buried in L4 nerve root and posterior root canal, (L4) nerve root and posterior root canal position, or mechanical stimulation to slightly increase the length (Non-Patent Document 1), 21G (as punctured IVD, SD rats) (Non-Patent Document 2). However, in the case of an intervertebral lumbar vertebral model using an experimental animal, the induction condition is difficult, it is difficult to formulate, and most of all, The method (pain behavior test) does not exist and includes results of mutually clashing behavior depending on the study group.

For these reasons, first, mechanical damage and chemical stimulation to the disc may cause disc degeneration, but it may not be a sufficient condition for back pain. Second, It appears that it is properly compensated and may not show a large difference from normal animals.

Accordingly, the inventors of the present invention have found that when an animal model of back pain is produced by an intervertebral disc, a single intervertebral disc is punctured and then the inferior disc herniation is inhaled, and the intervertebral disc pain is reduced by a more reliable, So that the present invention has been accomplished.

Department of Neurosurgery, Department of Neurosurgery, Department of Neurosurgery, Seoul National University, Department of Neurosurgery, Department of Neurosurgery,

The object of the present invention is to (a) drilling lumbar 4 and 5 lumbar vertebrae of an experimental animal; And

(b) sucking the nucleus pulposus in the perforated lumbar vertebrae 4 and 5 using a sound pressure generator, and a method for manufacturing an intervertebral disc backbone model animal.

(1) perforating lumbar 4 and 5 lumbar vertebrae of an experimental animal;

(2) sucking the pulp in the perforated lumbar vertebrae 4 and 5 using a sound pressure generator;

(3) administering a pain treatment candidate to an experimental animal from which disc harrows have been removed in the lumbar vertebrae 4 and 5; And

And (4) measuring the therapeutic effect of the pain treatment candidate administered in the step (3). The present invention also provides a method for screening a candidate for an intervertebral for back pain treatment candidate.

Further, it is an object of the present invention to provide an intervertebral disc backbone model animal which is produced by the above-mentioned production method.

According to an exemplary aspect of the present invention,

(a) puncturing lumbar 4 and 5 lumbar vertebrae of an experimental animal; And

(b) sucking the nucleus pulposus in the perforated lumbar vertebrae 4 and 5 using a sound pressure generator.

In one embodiment of the present invention, the perforating step is performed by keeping the experimental animal in a stomach and stomach position and then approaching the abdomen.

In one embodiment of the present invention, the negative pressure in the sucking step is -765 to -755 mmHg.

In one embodiment of the present invention, the sucking and sucking step is performed by connecting a needle with a blunt end to a negative pressure generating device.

In one embodiment of the present invention, (c) the animal prepared in the step (b) further comprises a step of restoring at least one week after sealing the abdomen.

According to another aspect of the present invention,

(a) puncturing lumbar 4 and 5 lumbar vertebrae of an experimental animal;

(b) sucking the pulp in the perforated lumbar vertebrae 4 and 5 using a sound pressure generator;

(c) administering a pain treatment candidate to an experimental animal from which the nucleus pulposus has been removed in the lumbar vertebrae 4 and 5; And

(d) measuring the therapeutic effect of the pain treatment candidate administered in the step (c).

In one embodiment of the present invention, the step of measuring the therapeutic effect of the pain treatment candidate substance is performed after one week after removing the nucleus pulposus in the lumbar 4 and 5 lumbar spine of the experimental animal.

In one embodiment of the present invention, the step of measuring the therapeutic effect of the pain treatment candidate substance may be a method of measuring the weight load on the quadruple of the experimental animal, or a method of comparing the number of behaviors and duration And comparing the measured values with each other.

In one embodiment of the present invention, the step of measuring the therapeutic effect of the pain treatment candidate substance may include a method of investigating neurotic excitation by applying a mechanical stimulus to an intervertebral disc of an experimental animal, or a method of applying a pressure stimulus to an intervertebral disc of an experimental animal, The method comprising the steps of:

According to another aspect of the present invention, there is provided an intervertebral disc model animal produced by the above-described method.

The intervertebral disc model animal prepared according to various embodiments of the present invention has an advantage of accelerating disc degeneration naturally occurring for a long period of time and controlling the induction timing and maintenance timing of intervertebral disc pain, Has been found to have a symptom and an onset mechanism of human intervertebral disc back pain unlike the existing animal model, and the model animal is useful for the study of the mechanism of intervertebral disc back pain and the treatment of intervertebral disc disease and the development of therapeutic drug .

FIG. 1 is a graph showing the result of repeatedly measuring the weight load on each of four paws for 7 days (80D) to 80 days (80D) after preparing the disc back pain model animal according to an embodiment of the present invention FIG.
FIG. 2 is a graph showing the peak spikes per second and the threshold value of the excitatory action potential per unit time of the disc sensory nerve in the disc back pain model animal according to an embodiment of the present invention Fig.
FIG. 3 is a graph showing the results of the peak spikes per second and the threshold value of excited action potential per unit time of the sensory nerve in the disc back pain model animal according to an embodiment of the present invention Fig.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

The present invention provides a method for producing an intervertebral disc model animal, comprising the steps of: (a) puncturing lumbar vertebrae 4 and 5; And (b) sucking the nucleus pulposus in the perforated lumbar vertebrae 4 and 5 using a negative pressure generating device.

In the present specification, an experimental animal means an animal having no symptoms of intervertebral disc disease. The laboratory animal is not particularly limited and any animal other than human can be used. Preferably, the animal can be a mammal. For example, rodents such as rats, mice, guinea pigs, hamsters, rabbits, dogs And so on.

The discogenic low back pain model animal is an intervertebral disc that serves as a cushion for relieving the impact on the vertebrae between the vertebrae and the vertebrae by appropriately dispersing the force between the vertebrae and the vertebrae The lumbar spine is a symptom that does not occur spontaneously, which means acute or chronic pain, and the disc-like back pain model of the present invention The activity of painful behavior and nervous excitability can be observed in real time through living animals.

In addition, disc lumbar disc herniation is different from degeneration which occurs spontaneously for a long time due to pathological physiological mechanism. Disc lumbar disc herniation should also understand the nerve and peripheral nerves directly connected to the disc.

In one embodiment of the present invention, the perforating step is performed by keeping the experimental animal in a stomach and stomach position and then approaching the abdomen.

In one embodiment of the present invention, the process of puncturing the lumbar 4 and 5 lumbar vertebrae of an experimental animal is performed by anesthetizing the experimental animal, and then performing an abdominal approach (abdomen, supine position, supine position) Without any damage to the nerves running through the muscle, and removing the minimal muscles and exposing the intervertebral disc.

At this time, disc herniation is performed by observing with eyes directly through an optical microscope to avoid nerve damage to the disc.

In one embodiment of the present invention, the negative pressure of the sucking step is -800 to -700 mmHg.

The inhalation process of the recipient of the intervertebral disc backbone model animal manufacturing method is preferably carried out in a state where the negative pressure is maintained at -765 to -755 mmHg, and most preferably at 1 atmospheric pressure (760 mmHg) The negative pressure can be fixed at 760 mmHg.

Empirically, a sound pressure of less than -760 mmHg is time consuming and ineffective in inhalation of the pulmonary tuberosus, and strong sound pressure greater than -760 mmHg is an unnecessary injury to the disc itself or fibrous ring during the inhalation of the disc .

In one embodiment of the present invention, the sucking and sucking step is performed by connecting a needle with a blunt end to a negative pressure generating device.

Meanwhile, when a needle having a sharp end is used in connection with a negative pressure generator, the needle used in performing the suction process is used. When a sharp needle is used or a syringe cylinder which is conventionally used is used, Excessive stimulation to the surrounding tissues or periosteum may change the surrounding tissue, or it may take a long time to inhale the nucleus pulposus or decrease the efficiency.

In particular, the use of a pinhole-shaped needle in connection with a sound pressure generator is suitable for easily puncturing the hole of a conventional syringe tip. However, since the syringe is wound to the femur and penetrates the femur, , Which may arise from tissue stimulation around the intervertebral disc due to some disc herniation or needles draining through the perforated hole rather than disc degeneration itself.

In one embodiment of the present invention, (c) the animal prepared in the step (b) further comprises a step of restoring at least one week after sealing the abdomen.

The experimental animals according to the present invention can be used for the purpose of breeding the experimental animals for a certain period of time after suturing the incised abdomens, performing wound healing, recovering from anesthesia or recovering from anesthesia, It is possible to obtain stability under the conditions suitable for the above.

Preferably for a period of at least a week, but it is possible to dispel a pain component due to the surgical site.

According to another aspect of the present invention,

(a) puncturing lumbar 4 and 5 lumbar vertebrae of an experimental animal;

(b) sucking the pulp in the perforated lumbar vertebrae 4 and 5 using a sound pressure generator;

(c) administering a pain treatment candidate to an experimental animal from which the nucleus pulposus has been removed in the lumbar vertebrae 4 and 5; And

(d) measuring the therapeutic effect of the pain treatment candidate administered in the step (c), and a method of screening a candidate for an intervertebral for back pain treatment candidate.

In one embodiment of the present invention, the step of measuring the therapeutic effect of the pain treatment candidate substance is performed after one week after removing the nucleus pulposus in the lumbar 4 and 5 lumbar spine of the experimental animal.

In one embodiment of the present invention, the step of measuring the therapeutic effect of the pain treatment candidate substance may be a method of measuring the weight load on the quadruple of the experimental animal, or a method of comparing the number of behaviors and duration And comparing the measured values with each other.

In one embodiment of the present invention, the step of measuring the therapeutic effect of the pain treatment candidate substance may include a method of investigating neurotic excitation by applying a mechanical stimulus to an intervertebral disc of an experimental animal, or a method of applying a pressure stimulus to an intervertebral disc of an experimental animal, The method comprising the steps of:

According to another aspect of the present invention, an intervertebral disc backbone model animal produced by the above-described manufacturing method is disclosed.

The intervertebral disc model animal of the present invention accelerates spontaneous disc degeneration over a long period of time and is similar to the symptoms found in patients with acute or chronic pain, Therefore, the model animal prepared above has been found to have a symptom and an onset mechanism of human intervertebral disc back pain unlike the existing animal model. Therefore, the model animal has been studied as a mechanism of intervertebral disc back pain, It can be usefully applied to the development of therapeutic drugs.

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention as set forth in the following claims. It is natural that it belongs to the claims.

Experimental animals and breeding methods

The experimental animals used were Sprague-Dawley rats (Korean Animal Research Center, Korea University, Korea) at 300-350g, and the animal experimentation procedure was conducted by Korea University Animal Experimental Ethics Committee : NIH publication No. 86-23, revised 1985) under the approval of KUIACUC-20131104-2.

Experimental animals were maintained in a standard condition consisting of a 12-hour light-dark cycle, a temperature condition of 20 to 25 DEG C and a humidity condition of 40 to 60%, and animal feed and water were continuously supplied Respectively.

Example 1: Preparation of an intervertebral disc low back pain model (hereinafter referred to as PUNCT)

Anesthesia was induced with ketamine (80-100 mg / kg) and xylazine (5-10 mg / kg) while body temperature of white rats was maintained at 37 ° C. The internal organs were surrounded by sterile gauze soaked with physiological saline through the abdominal approach (abdomen, supine position, supine position) and then rolled to one side, and the minimal muscle was removed without nerve damage running around the disc and the disc was exposed .

After lumbar spine L4 and L5 intervertebral discs were visually observed through an optical microscope, they were punctured with a 22G injection needle (using a pointed needle), except for lumbar spine 2, 3, 6, (CW-300, CHANG WOO) was used to inhale the disc herniation with a sound pressure of -760 mmHg, and then the abdomen was sutured within a short time.

At this time, when sucking the disc in the intervertebral disc, the tip of the injection needle was worn and connected to the negative pressure generator in a blunt state.

He gave time to recover for 7 days after surgery.

In the control group (SHAM), only the abdomen was incised and the lumbar vertebrae 2, 3, 4, 5, and 6 were used as the normal discs. In the non-treated group, Were used.

Experimental Example 1: Measurement of pain-related behaviors of the disc-like back pain model

(1) Measurement of weight load on four feet

The behavior evaluation apparatus (US6629056 B2) developed by the present inventor was used to measure pain in a walking state with a measuring apparatus using a weight load transmitted to the feet when walking.

The experimental apparatus consisted of the origin, the passage, and the destination made of acrylic. The footrest of the passage was attached to a load cell (Dana load cell, working range 0-1000 g, Dana load cell LTD., Korea) The weight being loaded was recorded. Each load cell attached to the bottom of the scaffold is transferred to a computer via an amplifier (DN-AM300, Dana load cell LTD.) And a digital converter (1716, Dana load cell LTD.), The change curve of the weight with the axis was confirmed and analyzed.

Before disc herniation, all animals were habituated to the environment in an isolated room and measuring device, and baseline values were obtained 3 days (-3D) and 1 day (-1D) before puncture. The weight load test was carried out 5-6 times from the start to the end of the measuring device and all weight bearing loads (g) on the left and right front limb and the left and right hind limb were recorded. Percentage (%) of body weight extracted from each foot was compared and analyzed.

The analysis using the mean of each foot pair for further analysis is as follows:

a. FRONT-HIND (left right hind paw mean - (subtract) left right fore front average)

b. RIGHT-LEFT (left front foot average - (minus) right front front foot average)

c. CROSSING (left front leg right hind leg average - (subtract) average of right forelimb and left hind leg).

One-way ANOVA and Tukey post-test were used for statistical significance of the maximum number of firing and threshold values of excited action potentials by individual indicators of pain behavior and mechanical stimuli (or pressure stimuli) Respectively. With respect to all measured data, p <0.05 was applied as a criterion.

As shown in Fig. 1, the weight load on each of the four feet was measured repeatedly from 7 days (7D) to 80 days (80D) after perforation and sucking, and as a result, the control groups (naive and sham ) Did not show the difference between RIGHT-LEFT and CROSSING LIMBS, and showed a tendency to walk in the HIND-FRONT LIMBS when the specific gravity was about 12-13% greater in the hind paw than in the paw.

On the other hand, in the disc herniation group (Example 1), RIGHT-LEFT and CROSSING LIMBS did not show any change similar to the control groups, but HIND-FRONT LIMBS showed a gait that the difference in the specific gravity between the forelimb and hind pals almost disappeared.

(PUNCT and naive, 7D, p <0.01, 10D, p <0.01, 14D, p <0.01, 21D, p <0.01) <0.01, 28D, p <0.01 , 35D, p <0.01, 42D, p <0.01; PUNCT and sham, 7D, p <0.01, 10D, p <0.01, 14D, p <0.01, 21D, p <0.01, 28D , p < 0.01, 35D, p < 0.01, 42D, p < 0.01).

(2) Rearing of the hind legs.

After giving a white mouse a free time (10 min) in a circular space (80 cm in diameter) surrounded by a transparent acrylic wall in an isolated room, the total number of times and total duration Manually measured using a stopwatch. For further analysis, the average duration (total duration / total count) was used to compare.

As shown in FIG. 1, the results of repeated measurements from 7 days to 80 days after puncture were compared with the control groups, and the intervertebral puncture group showed the total duration, the total number of times, the average duration of one time (PUNCT and naive, total duration, 7D, p <0.01, 14D, p <0.01, 35D, p <0.05, 60D, p <0.01, 80D , p <0.01; total number of times, 7D, p <0.01, 14D , p <0.05, 35D, p <0.05, 80D, p <0.01; 1 times the average duration, 14D, p <0.01, 21D , p <0.05; PUNCT and sham; average once; total duration span, 14D, p <0.01, 35D , p <0.01, 80D, p <0.05; total number of times, 14D, p <0.01, 21D , p <0.05, 35D, p <0.01 Duration, 14D, p < 0.01, 42D, p < 0.05) (Fig.

Experimental Example 2: Excitation measurement of single sensory nerve by electrophysiological method in living condition

The white rats were anesthetized with Urethane (1ml / 200g), followed by tracheal intubation to ensure airway, and a polyethylene tube was inserted into the skin vein to facilitate injection of sap and anesthetic during surgery.

In order to monitor blood pressure during surgery, a polyethylene tube was inserted into the left carotid artery and monitored continuously through a pressure transducer (WPI, model BP-1, USA), and the temperature in the esophagus was measured to maintain the body temperature. The body temperature was maintained at 37 占 폚.

In order to remove the noise that may occur in the recording electrode due to muscle movement, muscle relaxant was injected at a certain time to block the nerve-muscle connection, and the end exporatory carbon dioxide was maintained at 4.0 vol%.

Among the afferent fibers known to be connected to the intervertebral disc, single nerve fibers in the sympathetic nerves around the vertebrae were placed on the platinum line. The amplified action potential is continuously monitored by an oscilloscope using an amplifier (DAM 80, WPI), and the amplified action potential is converted into a digital signal by an A / D converter (CED1401, England) spike2) in real time.

(1) Neuro-excitability investigation when mechanical stimuli (von Frey filaments) were applied to the intervertebral disc

The mechanical stimuli were used as the mechanical stimuli (0.4, 0.6, 1, 2, 4, 6, 10, and 26 g; interstimulus interval: 30-40 sec) and the intervertebral disc sensory nerve units The peak spikes per second and the threshold value of the excited action potential per hour were investigated.

As a result, as shown in Fig. 2, the action potential according to the mechanical stimulus intensity (0.4-26 g) was significantly more pronounced than the control groups (sham and naive) in the perforated lumbar intervertebral disc group (35 days after puncture, 35D PUNCT) (Figs. 2A and 2B).

In the stimulus-sensory neuron response curve, 35D PUNCT showed a greater activity potential response pattern at a given stimulus level compared to control groups (35D PUNCT and naive, 1g , p <0.05, 2g, p <0.05, 6g, p <0.05, 26g, p <0.05; 35D PUNCT and sham, 1g, p <0.05, 2g, p <0.05, 6g, p <0.05, 10g, p < 0.05, 26g, p < 0.01) (Fig. 2B). The conduction velocity (CV) of sensory nerves obtained from the electrical stimulation in the intervertebral disc tissue by the group is C-nerve fiber (CV <3 m / s) and the other part is Aδ-nerve fiber (3 <CV <20 m / s ) (Fig. 2C).

(2) Nerve excitability when applied intradiscal pressure in the intervertebral disc

The pressure on the intervertebral discs was calibrated with a PRSIM pressure stimulator (WPI, calibrator) for each experiment, and pressure stimulation in the intervertebral discs (100, 500, 1000, 1500) via a 23G needle and saline- , 2000, 2500, 3000 mmHg; duration of stimulation: 10 sec; interval between stimulations: 70 sec). The measured values were the peak spikes per second and the threshold value of the excited action potential per unit time of the sensory nerve in response to the pressure stimulus.

As a result of the example, when the pressure in the intervertebral disc is increased (100, 500, 1000, 1500, 2000) by saline injection after inserting into the lumbar intervertebral disc with a 23 G needle connected with a syringe syringe (5 ml) , 2500, 3000 mmHg; stimulation time-10 sec; stimulation interval -70 sec), and the number of sensory nerves connected to the intervertebral disc increased per unit time (FIG. 3A). In the case of pressure stimulus-response curves, a group of perforated lumbar intervertebral discs (35 days after puncture, 35D PUNCT, 14 days after puncture, 14D PUNCT) (35D PUNCT and naive, 2000 mmHg, p <0.05, 2500 mmHg, p <0.01, 3000 mmHg, p <0.05; 35D PUNCT and sham, 2000 mmHg, p < 0.05, 2500 mmHg, p <0.01 , 3000 mmHg, p <0.05; 14D PUNCT and naive, 2500 mmHg, p <0.05 ; 14D PUNCT and sham, 1500 mmHg, p <0.05 , 2500 mmHg, p <0.05) ( Figure 3B left). In addition, 35D and 14D PUNCT were lower than the control group (35D PUNCT and sham, p <0.05; 14D PUNCT and naive, p <0.01, 14D PUNCT and sham, p <0.01 (Fig. 3B right). However, in the comparison between 35D and 14D PUNCT, the pressure stimulus-response curves and threshold values did not show statistically significant differences.

Claims (10)

(a) puncturing lumbar 4 and 5 lumbar vertebrae of an experimental animal; And
(b) sucking the nucleus pulposus in the perforated lumbar vertebrae 4 and 5 using a sound pressure generator,
The step (b) is performed by connecting a needle with a blunt end to a negative pressure generator,
Wherein the negative pressure of the negative pressure generating device is from -765 to -755 mmHg.
[2] The method according to claim 1, wherein the puncturing step is performed by keeping the experimental animal in a stomach and stomach position and then approaching the abdomen.
delete delete 2. The method according to claim 1, wherein (c) the animal prepared in step (b) further comprises at least one week of restoration after sealing the abdomen.
(a) puncturing lumbar 4 and 5 lumbar vertebrae of an experimental animal; And
(b) sucking the pulp in the perforated lumbar vertebrae 4 and 5 using a sound pressure generator;
(c) administering a pain treatment candidate to an experimental animal from which the nucleus pulposus has been removed in the lumbar vertebrae 4 and 5; And
(d) measuring the therapeutic effect of the pain treatment candidate administered in step (c)
The step (b) is performed by connecting a needle with a blunt end to a negative pressure generating device,
Wherein the negative pressure of the negative pressure generator is from -765 to -755 mmHg.
[7] The method according to claim 6, wherein the step of measuring the therapeutic effect of the pain treatment candidate substance is performed after one week after removing the nucleus pulposus in the lumbar vertebrae 4 and 5 of the lumbar vertebrae A method for screening a substance.
[7] The method according to claim 6, wherein the step of measuring the therapeutic effect of the pain treatment candidate comprises measuring a weight load on four feet of an experimental animal or comparing the number and duration of an action with the hair of an experimental animal, Wherein said method comprises the steps of:
[7] The method according to claim 6, wherein the step of measuring the therapeutic effect of the pain treatment candidate substance comprises the steps of: applying a mechanical stimulus to an intervertebral disc of an experimental animal to investigate neural excitability; or applying a pressure stimulus to an intervertebral disc of an experimental animal The method comprising the steps of:
5. An intervertebral disc model animal produced by the method of any one of claims 1 to 5.

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