RU2850368C1 - Method for modelling radiculopathy in experiments - Google Patents

Abstract

FIELD: medicine; neurology.

SUBSTANCE: invention relates to medicine, namely neurology, and can be used to reproduce a model of radiculopathy in experiments on laboratory animals, as well as to develop new methods of treating radiculopathy in experiments. Under general anaesthesia, a pre-straightened surgical cutting needle with surgical thread is inserted at the level of the intervertebral foramen. The needle is passed transforaminally at the level of the dorsal part of the fifth lumbar intervertebral foramen so that the thread remains in the intervertebral foramen, creating physical compression of the spinal nerve root. The thread is then fixed to the rat's back with a surgical knot. After the operation, the experimental groups of animals are assessed for behaviour, pain sensitivity and appearance, and then removed from the experiment. The surgical thread is impregnated with a radiopaque solution, the needle is inserted, and the thread is passed and fixed under fluoroscopic control. The behaviour, pain sensitivity and appearance of the animals are assessed daily, starting on the second day after the operation.

EFFECT: minimal invasiveness due to the absence of incisions and the passage of the thread through the intervertebral foramen of the rat.

4 cl, 11 dwg, 3 tbl, 2 ex

Description

The invention relates to medicine, namely to neurology, and can be used to reproduce a model of radiculopathy in an experiment on laboratory animals, as well as to develop new methods of treating radiculopathy in an experiment.

Worldwide, low back pain is a leading cause of disability and is difficult to treat. Among adults, low back pain ranks first among 291 conditions for disability in the Global Burden of Disease study and sixth in overall socioeconomic burden.

Several rodent models have been described to study the mechanisms underlying intervertebral disc degeneration and disc-radicular conflict. Among these models, the most widely used method for inducing low back pain is the intervertebral disc puncture model, as it results in progressive and reproducible degeneration similar to changes observed in humans (Daly C, Ghosh P, Jenkin G, Oehme D, Goldschlager T. A review of animal models of intervertebral disc degeneration: pathophysiology, regeneration, and translation to the clinic. Blamed Res Int 2016). The pattern of low back pain is partly due to the removal or leakage of the nucleus pulposus from the intervertebral disc, resulting in lumbar radicular pain (Takahashi K, Aoki Y, Ohtori S. Resolving discogenic pain. Eur Spine J. 2008 Dec; 17 Suppl 4(Suppl 4):428-31.).

Another set of models involves ligation of spinal nerve roots with a ligature, through surgical access to the root. For these models of radiculopathy using ligation of the dorsal and ventral L5 nerve roots, it was quantitatively demonstrated that the severity of the initial mechanical tissue damage directly correlates with both the amount of mechanical allodynia that occurs and the degree of increase in cytokine levels in the spinal cord after injury (Kawakami M, Weinstein JN, Chatani K, et al. 1994. Experimental lumbar radiculopathy. Behavioral and histologic changes in a model of radicular pain after spinal nerve root irritation with chromic gut ligatures in the rat. Spine 19: 1795-1802.). This method of forming the clinical picture of radiculopathy was taken as a prototype in this study.

The advantage of the proposed method is the less invasiveness of the procedure for the formation of a radiculopathy clinic than in the model taken as a prototype, in which the laboratory animal was subjected to direct surgical intervention.

Our goal was to create a new, effective model of radiculopathy in rats that would eliminate direct surgical intervention and be a minimally invasive method. This model could be used to test new treatment methods or to determine the effectiveness of drugs for treating radiculopathy associated with direct disc-radicular conflict in laboratory animals.

The proposed method of radiculopathy model involves mechanical compression of spinal nerve roots with a surgical thread passed through the intervertebral foramina of a rat.

The objective of the proposed method is to develop a model of radiculopathy in rats without a surgical incision in the area of the spinal nerve root.

The method is carried out as follows.

Ten adult (2 months old) male mongrel laboratory white rats were used in the experiment. The rats were divided into three groups: two experimental groups (No. 1 and No. 2) of four rats each, and one control group of two rats. The rats' initial weight ranged from 280 to 310 g (Table 1). Before the experiment, pain levels were determined in all groups (Table 2) and a hot plate test was performed (Table 3). During the experiment, the rats were kept in individual cages with free access to food and water. The light cycle was 12 hours of light and 12 hours of darkness.

Under general anesthesia (ether vapor inhalation) and fluoroscopic guidance, a cutting surgical needle (No. 36) was inserted at the level of the intervertebral foramina. The needle was straightened beforehand. A surgical suture (metric 6 lavsan) impregnated with Omnipaque radiopaque solution was inserted into the needle. The needle was inserted transforaminally at the level of the dorsal aspect of the fifth lumbar intervertebral foramen, through the foramen, such that the suture remained within the foramen, creating physical compression of the spinal nerve root. The suture was secured to the rat's back with a surgical knot (Figs. 1, 2, 3).

After surgery, rats were given one day to acclimatize before any procedures. Standard pain model testing was then performed using the hot plate test and pain assessment using the Rat Grimace Scale (Figs. 6, 9). The mechanical paw withdrawal threshold (PWT) was used as the criterion.

Rats in the control group (without spinal nerve root compression) underwent a hot plate test daily to determine reference values, determine body weight, and assess pain levels (Tables 1, 2, Figs. 5, 6, 7, 9).

The rats participating in the experiment (8 rats) also underwent a hot plate test starting from the second day after surgery to confirm signs of radiculopathy (decreased sensitivity at the damaged level, manifested by a higher temperature value at which the rat withdrew its paw).

Next, in experimental group of animals No. 1 (4 rats), the thread was removed from the intervertebral foramen to determine spontaneous resolution of radiculopathy; in experimental group of animals No. 2 (4 rats), the thread was left.

For 5 days after the operation, both groups were assessed for behavior, pain sensitivity (hot plate test), rat appearance (neatness/ruffled fur), and body weight (Figs. 5, 6, 9).

Observation demonstrated that rats in the control group withdrew their paws when the plate was heated to 45-47°C. Meanwhile, rats in the experimental groups withdrew their paws at a temperature of 62-65°C at the level of injury (Table 3, Fig. 9). Observation also revealed that after mechanical stimulation of the spinal nerve root, persistent manifestations of radiculopathy were observed without spontaneous resolution: the withdrawal temperature was the same in both experimental groups.

The phenomena of radiculopathy in rats of the experimental groups persisted until withdrawal from the experiment.

Five days after surgery, all animals were anesthetized with deep isoflurane. At autopsy, the ligature site was identified in rats from experimental group 2, and compression of the nerve root (1) by the thread (2) was visually confirmed (Fig. 4).

Histological examination confirmed damage to the spinal nerve roots. In the specimens from animals in groups 1 and 2, nerve fibers exhibited necrotic changes and sparse, scattered lymphocytic infiltration with isolated plasma cells (Figs. 10 and 11). Hematoxylin and eosin staining was used.

Examples of the method implementation.

All manipulations were performed in compliance with the requirements for humane treatment of animals (Strasbourg, France, 1986).

Example 1.

A healthy 2-month-old male laboratory rat, weighing 310 g at baseline, was inserted into the fifth lumbar intervertebral foramen using a 36-gauge cutting needle under fluoroscopic guidance under general anesthesia (ether). The needle was withdrawn from the opposite intervertebral foramen. A USP 3-4 metric 6 polyester (lavsan) suture, pre-impregnated with Omnipaque contrast agent, was passed through the intervertebral foramina. The suture was tied in a knot at the back.

One day was given to the animal for acclimatization after the operation.

Beginning on the second day after surgery, the rat was removed from the cage and subjected to a hot plate test and assessed using the Rat Grimace Scale. During the experiment, the rat's appearance, behavior, activity, appetite, and cage condition were observed. Mild pain was assessed using the Rat Grimace Scale throughout the experiment. According to the hot plate test, the hind paw on the side of the damaged root was withdrawn at a temperature of 62°C, compared to 45-47°C in the control group. Before the rat was removed from the experiment, control weight measurements were taken. Before removal, the rat's weight had decreased slightly to 300 g. Radiculopathy symptoms, such as hypoesthesia and analgesia, persisted until the rat was removed from the experiment 5 days after surgery.

Example 2.

A healthy 2-month-old male laboratory rat, weighing 290 g initially, was inserted under general anesthesia (ether) using a 36-gauge cutting surgical needle under fluoroscopic guidance at the level of the fifth lumbar intervertebral foramen. The needle was withdrawn from the opposite intervertebral foramen. A USP 3-4 metric 6 polyester (lavsan) suture, pre-impregnated with Omnipaque contrast agent, was passed through the intervertebral foramina. The suture was tied in a knot at the back.

One day was given to the animal for acclimatization after the operation.

On the second day, after the hot plate test was performed and the presence of radiculopathy in the form of hypoesthesia and analgesia was confirmed, the thread was removed from the rat.

Beginning on the third postoperative day, the rat was removed from the cage and subjected to a hot plate test and assessment using the Rat Grimace Scale. During the experiment, the rat's appearance (Fig. 8), behavior, activity, appetite, and cage condition were observed. Mild pain was assessed using the Rat Grimace Scale throughout the experiment. According to the hot plate test, the hind paw on the side of the damaged root withdrew at a temperature of 64°C, whereas in the control group of rats, the paw withdrew at a temperature of 45-47°C. Observations showed that spontaneous resolution of radiculopathy was absent upon thread removal. Control weight measurements were taken before the rat was removed from the experiment. Before withdrawal, the rat's weight had decreased slightly to 285 g. The phenomena of radiculopathy in the form of hypoesthesia and analgesia persisted until the rat was withdrawn from the experiment 5 days after the operation.

The advantages of the proposed method include the absence of a surgical incision and tissue trauma to the rat. The procedure is less time-consuming compared to radiculopathy models with a surgical incision.

The technical result of the invention is the development of an effective experimental model of radiculopathy, confirmed by the presence of hypoesthesia and analgesia without surgical invasiveness and with faster implementation. This radiculopathy model will be used in the future to study new experimental treatment methods for radiculopathy, as well as to evaluate the effectiveness of existing and newly discovered drugs.

The effectiveness of the created model was proven both with preserved ligatures and in the case where the thread is removed 24 hours after modeling radiculopathy.

Claims (4)

1. A method for experimentally modeling radiculopathy that involves ligating nerve roots, characterized in that, under general anesthesia, a previously straightened cutting surgical needle with surgical thread is inserted at the level of the intervertebral foramina; the needle is inserted transforaminally at the level of the dorsal part of the fifth lumbar intervertebral foramen through and through in such a way that the thread remains in the intervertebral foramen, creating physical compression of the spinal nerve root; the thread is then fixed on the back of the rat using a surgical knot; after the operation, the behavior, pain sensitivity, and appearance of the animals in the experimental groups are assessed, and then they are removed from the experiment. 2. The method according to paragraph 1, characterized in that the surgical thread is impregnated with a radiopaque solution. 3. The method according to paragraph 1, characterized in that the insertion of the surgical needle, the insertion and fixation of the thread are carried out under the control of a fluoroscope. 4. The method according to paragraph 1, characterized in that the assessment of the behavior, pain sensitivity and appearance of animals is carried out every day, starting from the second day after the operation.