SU1204197A1 - Method of recovery of nerve trunk - Google Patents

Description

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The invention relates to medicament, namely neurosurgery, and can be used in the treatment of injuries of the peripheral nervous system.

The aim of the invention is to accelerate the restoration of the integrity of the damaged nerve trunk and to achieve complete regeneration of its nerve fibers.

The drawing shows a diagram of the procedure for connecting the nerve ends to the transplant ohm.

The essence of the proposed method is as follows.

Before surgery, a homogenate of lemmocytes was prepared from nerve trunks from embryonic or adult animals. The aorta is isolated from the animal donor, pieces of vessels are cut out from it, corresponding to the caliber of the diameter of the operated nerve trunk, and placed in a nutrient medium before the operation. In the operated animal, a piece of nerve is removed, thereby creating a defect. One of the ends of the nerve is drawn into the lumen of the vessel prepared for transplantation and fixed with glue, and a previously prepared homogenate of lemmocytes is introduced into the opposite opening of the transplant. Thereafter, the other end of the damaged nerve is inserted into a transgendered vessel and also fixed with glue.

Due to the fact that an arterial vessel with sufficient elasticity and a thick musculoskeletal gland with a teat is used as a graft, its lumen is maintained and the diameter remains uniform throughout, which promotes free and unhindered growth of nerve fibers from the proximal to the distal end. Correspondence of the diameter of the vessel lumen to the caliber of the nerve trunk also ensures complete restoration of the nerve trunk. Studies have shown that the presence of a lemmocyte homogenate in the arterial lumen accelerates the formation of lemmocyte-axon relationships and stimulates the faster formation of nerve fibers. The absence of a suture promotes the formation of microneurus, and also accelerates the growth of regenerating nerve fibers along the lumen of the vessel.

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Example, For the successful implementation of the method, it is necessary to prepare a homogenate of lemmocytes and arterial vessels for transplantation, as well as

5 to have the technique of seamless connection of the ends of the damaged nerve with the transplanted vessel,

Lemmocyte homogenate is prepared as follows. Highlighted

10 nerve trunks from 20-day embryos or from newborn animals (linear or outbred rats) were placed under sterile conditions in a small volume (1 ml) of Hanks solution.

15 or Wednesday Needle on the watch glass and subjected to mechanical dissociation. Under a MBS-1 binocular microscope, the nerves were divided into two small thin toothpipes and two safety razors broken at an acute angle, and then the nerves were crushed to small conglomerates, and then multiple pipetting was performed to obtain a cell suspension.

For the same purpose in another case

25 under sterile conditions, the nerve trunks of the brachial plexus and the sciatic nerves from newborn animals were taken, cultivated for 4-6 days on a fibrin substrate in

30 medium Needle at 37 ° C, and then described. mechanically, the dissociation was crushed to a cell suspension and left in a thermostat at

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37 C in a small volume of Hanks solution or Eagle's medium before the operation.

. For the preparation of arterial vessels for transplantation, the descending aorta from 1-2 donor rats was prepared up to the iliac arteries,. placed in Hanks solution in Petri dishes and cleaned from the adventitia sheath there (with sufficient experience with 2 tweezers, the latter can be easily removed with a stocking). After that, the aorta, cleaned of rye connective tissue and adipose tissue, was transferred to Hanks fresh solution, where it was dissected into 3-4 parts

- 1.0-1.5 cm. The larger the separated pieces, the easier it is for the operation to select from them a vessel of the desired diameter corresponding to the diameter of the sciatic nerve, which is important for successful implementation of the method and obtaining good results. The vessels prepared in this way for transplantation remained in growth until the operation.

37 C in a small volume of Hanks solution or Eagle's medium before the operation.

. For the preparation of arterial vessels for transplantation, the descending aorta from 1-2 donor rats was prepared up to the iliac arteries,. placed in Hanks solution in Petri dishes and cleaned from the adventitia sheath there (with sufficient experience with 2 tweezers, the latter can be easily removed with a stocking). After that, the aorta, cleaned of rye connective tissue and adipose tissue, was transferred to Hanks fresh solution, where it was dissected into 3-4 parts

1.0-1.5 cm. The more pieces that have been isolated, the easier it is for the operation to select from them a vessel of the desired diameter corresponding to the diameter of the sciatic nerve, which is important for successful implementation of the method and obtaining good results. The vessels prepared in this way for transplantation remained in the Hanks solution or the Eagle medium in the thermostat before the operation. Transplants were used not only from linear and outbred rats, but also from rabbits. In all cases, positive results were obtained.

The technique of seamless connection of the ends of the damaged nerve with the transplanted vessel is as follows.

In a recipient animal, under numbutal anesthesia on the right side of the limb, more than one third of the knee joint, epilation and skin incision were made. The blunt side of the scalpel was moved apart by connective tissue fascias and thighs, the sciatic nerve was exposed and a safety razor was extirpated with a 5–8 mm long razor blade. The section was made on a flat surface of a Pasteur wooden rod, connected under the nerve. Next, a seamless connection of the ends of the damaged nerve trunk was carried out using a transplanted blood vessel of the donor according to an established procedure.

A thin nylon thread with a straight needle was threaded to the proximal end of the nerve, passed through the lumen of the vessel and retractedly put the vessel on the proximal segment so that its end was sticking out from the opposite side. This small piece was cut off along with the bundle, and the vessel was pulled out so that only the tip of the nerve 1.5-2.0 mm in length remained in its lumen. The rest of the lumen was free, and into this open end of the vessel with a pipette or syringe. lemmocyte homogenate injected from all nerves. Then the thread with the needle was tied (only slightly) to the distal segment of the nerve, the needle was pierced through the wall of the vessel near the rim, and, pulling the thread, the distal end of the nerve was pulled into the lumen. After tensioning, the thread was easily broken and pulled out through the wall without any damage to the latter. Thus, the small ends of the nerve 1.5-2.0 mm each turned out to be constricted from both sides in the lumen of the vessel. The distance between them was 5-8 mm. In order to avoid spontaneous loss or exit of the ends from the transplanted vessel, the edges of the vessel wall are fixed with epineural membranes with surgical cyanacrylate glue MK-7. It is important that the glue does not flow into the lumen of the vessel. Therefore, it is necessary to select in advance vessels with a diameter corresponding to the diameter of the operated nerve. Glue was applied directly from the tube to the joint in a small amount (1-2 small drops). It takes 1-2 minutes to dry the glue, after which the wound was stitched in layers, treated with iodine and covered with streptocide.

The experiments were carried out on .150 rats - Wistar males and outbreds with animals weighing 150-250 g. Experimental animals were killed with ethyl ether

in terms of 20.30, 180, and 270 days

after operation. The nerves, together with the transplanted vessels, were removed and examined with the help of neurohistological and general histological

methods (silver impregnation according to Bilshovsky-Gros, hematoxylin-eosin, azure 11-eosin). Histochemical techniques for phospholipids were used to detect the myelin sheaths, reflecting the degree of maturity and the level of differentiation of nerve fibers: the method of Becker and Sudan Cherni.

Research results showed that nerve regeneration through transplanted arterial vessels, in contrast to venous ones, according to a known method, proceeded differently and much faster. After 20 days between

A continuous transition of the forming connective tissue from the surface of the nerve to the surface of the vessel wall was observed by the suft transplant and the nerve cults. Histological analysis in most cases showed good preservation of the smooth muscle wall of the vessel and the absence of inflammatory elements in it. Minor lymphoid infiltration of the elements was detected mainly only in the places where the nerve and the graft were connected, where glue was applied during the operation. A large number of various cellular elements (lemmocytes, macrophages, endothelial cells) were observed in the lumen of the vessel. The most well-known lemmocytes,

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which were located in the lumen of the vessel between the proximal and distal ends of the nerve in the form of thick and thin wires and nets. Among the cell masses, closer to the vascular wall, there were 5 newly formed capillary sinusoidal cells with wide gaps. With the help of silver impregnation, within this period, within the shells of lemocytes, there was a large amount of gold.

a lot of fine baseballs are diffusely distributed

regenerated nerve fibers with a diameter of 0.5-1.0 microns. Many of them are oriented along the vessel lumen. Part of the fibers dichotomously 15 shared. In the course of some conductors, varicose veins, characteristic of growing young fibers, were revealed. The density of regenerated nerve fibers in the lumen of the vessel is not the same and decreased in the distal direction. Already on this early line, part of the thin bundles of cordless-nerve fibers germinated through the entire lumen of the graft and, entering the 5th distal trunk, was traced in it for some time (by a known method after 20 days there was no growth of nervous fibers)

After 30 days, the transplanted vessel had the same diameter as before the operation and did not subside. In its wall, viable smooth cone cells and 35 elastic fibers were clearly distinguished. In the lumen, evenly, along the entire length from the proximal to the distal parts of the nerve, there were numerous, rarefied bundles of nerve hairs — a con — some of which grew in a spiral, and some in a straight line. Between the bundles, there were already thin newly formed vessels: arterioles, venules, and capillaries. The diameter of the main mass of nerve fibers during this period as compared to the previous one (it increased to 1.5-2.0 µm due to the onset of formation of myelin sheaths around axons, which were clearly revealed using histochemical methods according to Becker and Sudan black Thin myelinated nerve fibers were observed for a considerable period in p; the true segment of the nerve trunk (by a known method, only the beginning of growth

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nerve fibers, and myelination still completely absent).

90 days after the operation, continued regeneration processes were observed inside the transplanted vessel. The entire lumen of the vessel from the proximal to the distal end of the nerve is filled with a mass of myelin fibers. Their diameter was 2–4 µm. As shown by microscopic studies, the nerve fibers are collected in numerous bundles that are located closer to the vessel wall in a spiral, and in the central part of the vessel - in the forward direction. Between the bundles of myelin fibers during this period there were still wide spaces, which indicated a lack of completeness of regeneration.

180 days after the operation, the mass of regenerating nerve bundles increased dramatically and significantly condensed due to the formation of new and myelinated nerve fibers, as well as due to the increase in the diameter of the forming myelin sheaths, the overwhelming number of which reached 4 µm ( only on the 10th month.).

After 270 days (which corresponds to 9 months after the operation), the entire volume of the lumen of the transplanted vessel is so densely filled with numerous bunches of thick myelinated: nerve fibers that between them were optically difficult to detect the free spaces that occurred in the early stages.

Between the bundles, well-developed capillaries, arterioles, and venules were often encountered, which indicated good vascular repair of nerve regeneration. Macroscopically, the regenerate was an organic unity with proximal and distal ends. The nerve trunk and regenerate became one. Thus, by the 9th month, the integrity of the damaged nerve trunk was fully restored. However, microscopic, histological, and study revealed certain structural differences between the areas of the proximal and distal stem and the regenerates in the lumen of the vessel. They consisted in the fact that in the proximal and distal trunks to me

The new nerve fibers had a slightly wavy parallel course, characteristic of the norm, while the regenerate had a beam structure: the numerous myelinated jas were collected in separate dense bundles arranged in the form of a braided spit. However, the act is that after leaving the graft, all this mass of beams in the distal 10

The nerve segment of the nerve united into a single trunk with a characteristic fibrous structure, as was normal, with convincing evidence of successful regeneration and restoration of the integrity of the entire nerve.

The transplanted arterial vessel, in the lumen of which the regeneration of nerve fibers occurred, did not resolve and did not die even 20 months after transplantation. In most of its walls, viable smooth-muscle cells and an elastic stroma were revealed. Starting from the 3rd month the transplanted vessel is surrounded by an epitheliomorphic lining similar to the perineural envelope.

HepBa. In the later periods, thin venous and arterial vessels, which did not penetrate into the smooth muscle wall one or 30 ka, were formed in this obochka.

Comparing the obtained results with the data of a known method, it is noted that the regeneration of the injured 35th nerve in the lumen of a transplanted arterial vessel, in contrast to autotransplanted

The nasal vessel occurs much faster and achieves more complete recovery. As a result of the collapse of the walls of the venous autograft, the diameter of the regenerate compared with the proximal and distal trunks is much smaller, which limits the completeness of the restoration of the integrity of the nerve. When using the proposed method, as a result of resistance to failure of the arterial vessel wall, the diameter of the nerve and the lumen of the vessel remain the same, which ensures the complete restoration of the integrity of the nerve trunk. In addition, a seamless connection is used which does not cause the formation of a microneurus in the lumen of the arterial vessel. In order to accelerate and regulate the direction of growth of regenerating nerve fibers in experiments, the Transplanted vessel was injected with a homogenate of cells of the lemmocytes isolated from embryos and newborn animals, which also contributed to the stimulation of the regenerative process. When the nerve of the extremity is formed using autograft of the femoral vein, the beginning of the growth of regenerating nerve fibers through the lumen of the vessel was observed only after a month, while the results of studies using the proposed method showed germination of the nerve fibers through the entire lumen of the arterial vessel from the proximal to the distal end after 20 operations.

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Claims (1)

METHOD FOR RESTORING THE NERVOUS STEM, including connecting the proximal and distal ends of the damaged trunk with a transplanted vessel, characterized in that, in order to accelerate the restoration of integrity and achieve complete regeneration, a donor arterial vessel filled with lemocyte homogenate is used as a graft in its lumen on both sides the ends of the nerve trunk are introduced and glue their epineural membranes with the edges of the graft. β SU ,,,. 1204197>