Method of intraoperative evaluation of peripheral nerves condition

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.

2 ex

 

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

Assessment of peripheral nerves, bankirovanii fragments of nerves is of considerable interest to the neurosurgeon, because the outcome of the restorative and reconstructive surgery depends on the viability stitched the nerves. To assess whether the nerve sprouting of nerve fibers, stored in damaged nerve trunk exchange of the mediator, whether axonal current, is impossible without use of special morphological methods.

Known electrophysiological methods of assessing conduction in the nerve: electrical stimulation of the nerve, electromyography (Odinak M. M., Zhivolupova S. A. Diseases and injuries of the peripheral nervous system (generalization of clinical and experimental experience): a guide for physicians. - M.: spec lit, 2009, 384). The pulse caused by electrical stimulation of the nerve, is sent by motor, sensory and mixed nerves, and the characteristics of the pulse are measured by recording potentials from the muscles, either directly from the nerve. If the nerve is damaged and has not happened yet restore the contact with the innervation targets, the value of these methods decreases dramatically. In addition, they cannot be used in �burden restorative and reconstructive microsurgical procedures due to the complexity of determining the projection of the thin branches of the nerves.

The closest technical solution (prototype) of the proposed method is the method of Karnovsky M. J., L. Roots (Karnovsky M. J., L. Roots, 1964 A "direct-coloring" Thiocholine method for cholinesterase, J. Histochem. Cytochem., 12, 219-22), which is used to identify the positive activity of acetylcholinesterase (ache) in nervous tissue, definition nervous system accessories guides and study the characteristics of the postsynaptic membrane in the neuromuscular synapses of somatic muscles (Nikolaev, G. M. Changes of acetylcholinesterase activity in motor endings in striated muscle during hypoxic hypoxia / G. M. Nikolaev // Archives of pathology, - 1970. - No. 3, pp. 61 - 65).

The enzyme acetylcholinesterase is formed in the neuron soma and transported along the axon to the presynaptic nerve endings or neuromuscular synapses (Dixon M., Webb E., Enzymes, TRANS. from English., T. I, M., 1982, pp. 363-364). Positive reaction to ache is a symptom of conservation synthetic and transport processes in soma of the neuron and its processes. A positive reaction can occur only in the axial cylinders of conductors and does not directly depend on the state of the myelin sheaths. Negative reaction to ache suggests that in this fragment of the nerve yet sprouting fibers.

Determination of the activity of acetylcholinesterase (ache) by Karnowski-roots trail runs�explained as follows: pick up the stuff, then make frozen sections, fixed samples in 10% formalin to one day, washed with two portions of sodium malatova buffer for 5 minutes each, incubated in a special environment: acetylcholine iodide (10 mg; 0.1 M Na-malaty buffer 2 ml; 0.1 M sodium citrate 4 ml; 0.03 M copper sulfate (4 ml; 0.005 M potassium ferricyanide 4 ml, an inhibitor of non-specific cholinesterase (1.0 ml 10-4 M ISO-OMPA) at 4°C, pH 6.0 for 24 hours, washed with distilled water, put in conditioner mikroskopiruût. The total duration of the reaction is 18-24 hours.

This method is specific for detection of a certain faction acetylcholinesterase.

The disadvantages of this method are as follows: the complexity and duration of the procedure, resulting in the inability to use it intraoperatively.

The proposed method is to simplify procedures and reduce the time for determining the state of the peripheral nerve.

This object is achieved in that changes the mode of incubation: the incubation time from 30 to 60 min, and the composition of the incubation medium: no nonspecific inhibitor of cholinesterase, pH ranges from 7.4 to 7.6, temperature between 50 to 55°C.

The proposed method for the assessment of peripheral nerve ache activity allows for a short period of time to conduct a study of their viability, which gives in�possibility to use it intraoperatively.

The novelty of the proposed method lies in the fact that significantly reduced the incubation time, the incubation medium has a simpler composition: acetylcholine iodide (10 mg; 0.1 M Na-malaty 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 ranges from 7.4 to 7.6, a temperature of 50 to 55°C.

The technical solutions that have the signs consistent with the distinctive features of our proposed method, not identified that allows to make a conclusion about conformity of the proposed method the criterion of "inventive step".

The proposed method is as follows: during the operation, take a piece of nerve, then make frozen sections. The resulting paste slices on glass slides, washed in two portions malatova buffer, are placed in the incubation medium of the following composition: acetylcholine iodide (10 mg; 0.1 M Na-malaty 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 and 7.6. The incubation time of 30-60 minutes. After incubation spend microscopy of slices for the detection of acetylcholinesterase activity (ache). If the response to acetylcholinesterase positive, the nerve is considered to be viable; if negative - the nerve is assessed as non-viable. Sections were dehydrated, sign in balm, photographed DL� documentation.

Example 1. Patient T. 27 years old, diagnosis: partial left-sided paralysis of the facial nerve. During the operation to determine the status of the facial nerve was taken cut of the nerve length 0.3 cm Segment of the nerve was taken to the laboratory, prepared frozen sections of 40 µm, washed them in two portions malatova buffer, were placed in incubation medium with a pH of 7.4, a temperature of 52°C, incubation time 45 min. After 45 minutes, the sections were washed in distilled water and made microscope. As a result, it was found that the activity of acetylcholinesterase in the barrel is positive, the trunk of the nerve is viable. Then there began the restoration of the damaged facial nerve using a nerve graft is sewn between the trunk of the facial nerve and its zygomatic branch. As nerve autograft was used sural nerve. Achieved restoration of facial expressions left side of his face.

Example 2. Patient M., 35 years was admitted to the hospital with a diagnosis of injury of the right brachial plexus. The operation was performed to restore the integrity of damaged nerves. During surgery it was discovered damage to the median nerve and neuroma throughout. Removed part of the nerve, which contained a neuroma. After the removal of the damaged nerve arose the need for grafting nerve sural nerve autotr�spuntata, since there was a defect nerve 7 see, then the question arose: not damaged nerve proximal to? To assess the status of the proximal part of the median nerve was taken as part of the nerve 0.3 cm for intraoperative histological assessment of the state. This sample was delivered to the laboratory, where samples of the nerve is frozen in the cryostat and frozen sections were made, 40 μm. The sections were washed in two portions of buffer solution (0.1 M Na-malaty buffer, pH=7,6). Subsequently, the slices were placed in incubation medium with pH of 7.6. Incubated in a thermostat at 55°C for 30 minutes. After 30 minutes, the sections were washed in distilled water and made microscope. As a result, it was found that the nerve at this level is positive acetylcholinesterase activity that gave the opportunity to reduce the distance between damaged nerves and thereby reduce the size of the sural nerve autografts to replace the defect of the nerve. As a result of operative intervention there is mobility of my right hand.

The proposed method was used in the surgical clinic during operations autologous peripheral nerves.

Method of intraoperative assessment of the state of the peripheral nerves by identifying positive acetylcholinesterase activity, which is manufacturing cryostat�x slices of the nerve, washing them in two portions malatova buffer, incubation in the incubation medium, characterized in that incubation is carried out in 30-60 minutes in the incubation medium, which has the following composition: acetylcholine iodide (10 mg; 0.1 M Na-malaty 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 from 7.4 to 7.6 and a temperature of 50 to 55°C.



 

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