RU2555750C1 - Method of intraoperative evaluation of peripheral nerves condition - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method consists in production of cryostat sections of nerve, washing them in two portions of maleate buffer, incubation in incubation medium. Incubation is carried out for 30-60 minutes and incubation medium has the following composition: acetylcholine iodide 10 mg; 0.1 M Na-maleate buffer 2 ml; 0.1 M sodium citrate 4 ml; 0.03 M copper sulphate 4 ml; 0.005M potassium ferricianide 4 ml; medium pH constitutes from 7.4 to 7.6 and temperature is from 50 to 55°C.

EFFECT: method by invention makes it possible to evaluate viability of peripheral nerves in short term, which is necessary during reconstructive-restoring and highly technological neurosurgical operations.

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Description

The invention relates to medicine, namely to reconstructive microsurgery, and is intended for intraoperative assessment of peripheral nerves.

Assessing the condition of peripheral nerves, banking fragments of nerves is of considerable interest to a neurosurgeon, because The outcome of reconstructive and reconstructive operations directly depends on the viability of the hemmed nerves. It is impossible to evaluate whether there is a sprouting of nerve fibers in the nerve, whether the mediator exchange is preserved in the damaged nerve trunk, whether there is an axonal current, without the use of special morphological methods.

Known electrophysiological methods for assessing nerve conduction: electrical stimulation of a nerve, electromyography (Odinak M.M., Zhivolupov S.A. Diseases and injuries of the peripheral nervous system (generalization of clinical and experimental experience): a guide for doctors. - M .: SpetsLit, 2009, 384 s). The impulse caused by electrical stimulation of the nerve is directed along the motor, sensory and mixed nerves, and the characteristics of the impulse are evaluated by recording potentials from the muscles, or directly from the nerve. If the nerve is damaged and contact with the innervation targets has not yet been restored, the value of these methods decreases sharply. In addition, they cannot be used during reconstructive and reconstructive microsurgical operations because of the difficulty in determining the projection of the thin branches of nerves.

The closest technical solution (prototype) of the proposed method is the method Karnovsky MJ, Roots L. (Karnovsky MJ, Roots L., 1964 A ′ ′ direct-coloring ′ ′ Thiocholine method for cholinesterase, J. Histochem. Cytochem., 12, 219- 22), which is used to detect the positive activity of acetylcholinesterase (AChE) in the nervous tissue, to determine the systemic affiliation of nerve conductors and to study the features of the postsynaptic membrane in the neuromuscular synapses of somatic muscles (Nikolaev G.M. Change in acetylcholinesterase activity in the motor ends of striated mus ulatury during hypoxic hypoxia / GM Nikolaev // Archives of Pathology, - 1970. - №3, - s.61-65).

The enzyme acetylcholinesterase is formed in the catfish of the neuron and is transported along the axon to the presynaptic nerve endings or to the neuromuscular synapses (Dixon M., Webb E., Enzymes, transl. From English, vol. I, M., 1982, p.363-364) . A positive reaction to AChE is a sign of the preservation of synthetic and transport processes in the catfish of the neuron and its processes. A positive reaction can occur only in the axial cylinders of the conductors and does not depend directly on the state of the myelin sheaths. A negative reaction to AChE indicates that there are no germinating fibers in this fragment of the nerve.

The determination of the activity of acetylcholinesterase (AChE) according to Karnowski-Roots is carried out as follows: take the material, then make frozen sections, fix the samples in 10% formalin for up to a day, wash in two portions of sodium maleate buffer for 5 minutes each, incubate in a special medium: acetylcholine iodide 10 mg; 0.1 M Na-maleate buffer 2 ml; 0.1 M sodium citrate 4 ml; 0.03 M copper sulfate 4 ml; 0.005 M potassium ferricyanide 4 ml, nonspecific cholinesterase inhibitor (1.0 ml 10-4 M iso-OMF) at a temperature of 4 ° C, pH 6.0-24 hours, washed with distilled water, enclosed in a balm, microscopic. The total reaction time is 18-24 hours.

This method is specific for detecting a specific fraction of acetylcholinesterase.

The disadvantages of this method are: the complexity and duration of the procedure, as a result of which the inability to use it intraoperatively.

The aim of the proposed method is to simplify the procedure and reduce the time to determine the state of the peripheral nerve.

This goal is achieved by changing the incubation mode: incubation time from 30 to 60 minutes, and the composition of the incubation medium: there is no inhibitor of nonspecific cholinesterase, the pH of the medium is from 7.4 to 7.6, and the temperature is from 50 to 55 ° C.

The proposed method for assessing the condition of peripheral nerves by AChE activity allows for a short period of time to conduct a study of their viability, which makes it possible to use it intraoperatively.

The novelty of the proposed method lies in the fact that the incubation time is significantly reduced, the incubation medium has a simpler composition: acetylcholine iodide 10 mg; 0.1 M Na-maleate buffer 2 ml; 0.1 M sodium citrate 4 ml; 0.03 M copper sulfate 4 ml; 0.005 M potassium ferricyanide 4 ml, and its pH is from 7.4 to 7.6; temperature from 50 to 55 ° C.

Technical solutions that have features that match the distinguishing features of our proposed method are not identified, which allows us to conclude that the proposed method meets the criterion of "inventive step".

The proposed method is as follows: during the operation, a nerve fragment is taken, then cryostatic sections are made. The obtained sections are glued onto slides, washed in two portions of maleate buffer, placed in an incubation medium of the following composition: acetylcholine iodide 10 mg; 0.1 M Na-maleate buffer 2 ml; 0.1 M sodium citrate 4 ml; 0.03 M copper sulfate 4 ml; 0.005 M potassium ferricyanide 4 ml, at a temperature of 50 to 55 ° C, pH 7.4-7.6. The incubation time is 30-60 minutes. After incubation, microscopy of the sections is carried out to detect the activity of acetylcholinesterase (AChE). If the reaction to acetylcholinesterase is positive, the nerve is considered viable; if negative, the nerve is assessed as nonviable. Slices are dehydrated, enclosed in a balm, photographed for documentation.

Example 1. Patient T., 27 years old, diagnosis: partial left-sided paralysis of the facial nerve. During the operation, a 0.3 cm length of a nerve was taken to determine the condition of the facial nerve trunk. A nerve segment was delivered to the laboratory, 40 μm cryostat sections were prepared, washed in two portions of maleate buffer, and placed in an incubation medium with a pH of 7.4 and temperature 52 ° C, incubation time 45 minutes After 45 minutes, the sections were washed in distilled water and microscopy was performed. As a result, it was found that the activity of acetylcholinesterase in the trunk is positive, the nerve trunk is viable. Then, the damaged facial nerve was restored using a nerve autograft sewn between the trunk of the facial nerve and its zygomatic branch. The calf nerve was used as a nerve autograft. A restoration of facial expressions of the left side of the face is achieved.

Example 2. Patient M., 35 years old, was admitted to the hospital with a diagnosis of damage to the right brachial plexus. An operation was performed to restore the integrity of damaged nerves. During the operation, damage to the median nerve and neuroma throughout was detected. The part of the nerve that contained the neuroma was removed. After removal of a part of the damaged nerve, there was a need for plastic surgery of the nerve with gastrocnemius nerve autografts, since there was a nerve defect of 7 cm. Then the question arose: is the nerve more proximal? To assess the state of the proximal part of the median nerve, a 0.3 cm part of the nerve was taken for intraoperative histochemical assessment of the condition. This sample was delivered to the laboratory, where nerve samples were frozen in a cryostat and cryostatic sections, 40 μm, were made. Sections were washed in two portions of a buffer solution (0.1 M Na-maleate buffer, pH = 7.6). Next, the sections were placed in an incubation medium with pH = 7.6. Incubated in a thermostat at 55 ° C for 30 minutes. After 30 minutes, the sections were washed in distilled water and microscopy was performed. As a result, it was found that the nerve at this level has positive acetylcholinesterase activity, which made it possible to reduce the distance between the damaged nerves and thereby reduce the size of the calf nerve autografts to replace the nerve defect. As a result of surgery, the mobility of the right hand appeared.

The proposed method was used in a surgical clinic for operations of autologous transplantation of peripheral nerves.

Claims (1)

The method of intraoperative assessment of the condition of peripheral nerves by identifying the positive activity of acetylcholinesterase, which consists in the manufacture of cryostatic sections of the nerve, washing them in two portions of maleate buffer, incubation in an incubation medium, characterized in that the incubation is carried out for 30-60 minutes in an incubation medium that has following composition: acetylcholine iodide 10 mg; 0.1 M Na-maleate buffer 2 ml; 0.1 M sodium citrate 4 ml; 0.03 M copper sulfate 4 ml; 0.005 M potassium ferricyanide 4 ml, pH 7.4 to 7.6 and temperature 50 to 55 ° C.