RU2485518C1 - Method of integrated assessment of various levels of involvement of vegetative nervous system structures - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: for the purpose of assessing the levels of involvement of the vegetative nervous system structures, blood serum of the patient showing the signs of peripheral vegetative failure is analysed as follows: by ELISA test, blood serum samples are analysed for α3-acetylcholine receptor (α3-ACR) gangliac subunit, β2-adrenoceptor (β2-AR) and nerve growth factor (NGF) antibodies; an EIA analyser is used to measure light absorption in the samples at wave length 450 nm; and if the level of α3-ACR autoantibodies 0.24 and more, the level of β2-AR autoantibodies less than 0.24, and the level of NGF autoantibodies less than 0.37, the gangliac involvement of the vegetative nervous system structures is stated; if the level of α3-ACR autoantibodies less than 0.24, the level of β2-AR autoantibodies 0.24 and more, and the level of NGF autoantibodies 0.37 and more, the effector involvement of the vegetative nervous system structures is stated; no α3-ACR, β2-AR and NGF antibodies shows stating the suprasegmentary involvement of the vegetative nervous system structures.

EFFECT: possibility of using the method to identify the gangliac, effector and suprasegmentary involvement of the peripheral structures of the vegetative nervous system in a broad range of patients.

3 ex

Description

The invention relates to medicine, namely to neurology, and can be used to detect damage to the peripheral structures of the sympathetic and parasympathetic departments of the autonomic nervous system at the ganglionic level and at the level of the effector organ in a wide range of patients, as well as at the supra-segmentary level.

Information about autonomic disorders and their localization can help resolve the issue of the nature and location of the pathological process. Sometimes the identification of signs of autonomic imbalance is of particular importance. Changes in the functions of the hypothalamus and other suprasegmental structures of the autonomic nervous system lead to generalized autonomic disorders. The defeat of the vegetative nuclei in the brain stem and spinal cord, as well as the peripheral parts of the autonomic nervous system, is usually accompanied by the development of segmental autonomic disorders in a more or less limited part of the body. Studying the state of autonomic functions can provide important information about the presence of a functional or organic lesion of the nervous system in a patient, often contributing to solving the issue of topical and nosological diagnosis.

Known clinical (dermographism, pupillometry, pilomotor reflex, dynamics of blood pressure and heart rate in ortho-, clinostatic and reflex tests with the calculation of various coefficients, etc.) and electrophysiological (measurement of the RR interval at various loads, evaluation of the skin-galvanic reflex etc.) research methods allow us to establish the predominance of the sympathetic or parasympathetic department of the autonomic nervous system (ANS) in various pathological conditions. There is also a method of assessing the state of the ANS with pharmacological tests (adrenergic and anticholinergic drugs) for the presence of autonomic dysfunction and, in some cases, localization of the level of its damage. However, the introduction of these (mainly potent group A) drugs is associated with a risk of developing pronounced side effects, especially in patients with organic damage to the vital structures of the peripheral ANS. (In the book. Vegetative disorders. Clinic, diagnosis, treatment. Edited by A.M. Wayne. - M .: Medical Information Agency LLC, 2003, pp. 44-102). However, these methods do not allow to establish the cause and level of affection of the ANS.

Closest to the invention are a method for detecting damage to individual structures of the peripheral ANS by determining antibodies to the ganglionic subunit of the α3-acetylcholine receptor (α3-AChR) and antibodies to other antigenic targets by radioimmunoprecipitation in patients with neurological and oncological diseases. At the same time, 1% of patients were seropositive (the level of antibodies to α3-AChR was from 0.03 to 18.8 nmol / L, in the control ≤0.02 nmol / L), the majority were patients with pandizavtonomy, limited dysautonomy, peripheral polyneuropathy, encephalopathy as well as some oncological (carcinomas and adenocarcinomas of various localization, thymoma, thyroid cancer, etc.) diseases (Ms. Keon A., Lennon VA, Lachance DH, Fealcy RD, Pittock SJ Ganglionic Acetylcholine Receptor Autoantibody. Oncological, Neurological, and Serological Accompaniments, Arch. Neurol. 2009. Vol.66 (No. 6), 735-741).

This method allows you to set the level of affection of the ANS due to the radioimmunological determination of antibodies in blood serum, but it is laborious to perform, as it involves working with radioactive isotopes, cell cultures, biological material obtained from animals, which requires special equipment, facilities and, accordingly, significant investment .

Our proposed method is cheaper and safer to determine specific antibodies in the blood serum by interacting with the following antigens: α3-AChR, β2-AP, NGF, by enzyme-linked immunosorbent assay. In addition, this method allows for the same specificity in a simpler and more economical way (in particular, which does not require measures of protection against radiation and work with cells) to diagnose various levels of damage to the structures of the ANS.

The technical result is achieved by the fact that in order to assess various levels of damage to the autonomic nervous system, a blood serum test is performed, and in the blood serum samples, the a3-acetylcholine receptor (α3-AChR), β2-adrenoreceptor autoantibodies to the ganglion subunit are determined by enzyme-linked immunosorbent assay (TIFA) (β2-AR) and nerve growth factor (NGF), optical absorption of light (OD) is measured on an ELISA analyzer at a wavelength of 450 nm and when the level of autoantibodies to α3-AChR is 0.24 or more, the level of ay antibodies to β2-AP less than 0.24 and levels of autoantibodies to NGF less than 0.37 assess damage to the structures of the autonomic nervous system at the ganglion level, when the level of antibodies to α3-ACh is less than 0.24, the level of antibodies to β2-AR 0.24 and more and the level of autoantibodies to NGF of 0.37 or more assess the damage to the structures of the autonomic nervous system at the level of the effector organ, in the absence of autoantibodies to α3-AChR, β2-AR and NGF, the damage to the structures of the autonomic nervous system is assessed at the suprasegmental level.

The method is as follows.

The patient is sampled from the cubital vein, then the serum is separated from the blood clot by centrifugation for 15 minutes at 3000 rpm.

Serum is tested for the presence of antibodies to α3-AChR, β2-AR, NGF in ELISA.

Recombinant proteins: α3-AChR, β2-AR, NGF obtained by genetic engineering in the laboratory of finished dosage forms of the Center for Bioengineering of the Russian Academy of Sciences (head of laboratory - candidate of biological sciences Zeynalov O) are used as antigen for the determination of autoantibodies by the ELISA method. .BUT.).

To quantify the level of autoantibodies to α3-AChR, β2-AR, NGF, the next step of ELISA is carried out.

On 96-well plates from Costar, the α3-AChR, β2-AP, NGF domain is adsorbed at a concentration of 2 μg / well, 4 μg / well, 4 μg / well and incubated for 60 minutes at 37 ° C. The first row of wells is left free for conjugate control. To block nonspecific binding, a 2% solution of BSA (Sigma) in PBS / Tween-20 at 200 μl / well was added to the wells and incubated for 60 minutes at 37 ° C. After incubation, control donor sera are added to the second row, 1: 100 dilutions of the patient sera are added to all subsequent rows and incubated for 60 min at 37 ° С followed by 3-fold washing (0.2% BSA in PBS / Tween-20 200 μl / well) for 3 minutes. The presence of antibodies is determined by staining using a conjugate of peroxidase with human anti-IgG (Sigma), which is added at 100 μl / well at a dilution of 1: 1000 and incubated for 60 minutes at a temperature of 37 ° C, followed by a 3-fold washing (0, 2% BSA in PBS / Tween-20 at 200 μl / well) for 3 minutes. Tetramethylenebenzidine (Sigma) is used as a chromogen: 10 mg is dissolved in 5 ml of citrate buffer (pH 4.5) and 15 μl of hydrogen peroxide and 100 μl / well are added. Staining developed within 20 minutes. Inhibition of the reaction is carried out with a 0.25 M sulfuric acid solution - 100 μl / well. The measurement of optical light absorption at a wavelength of 450 nm is carried out on an ELISA analyzer.

Results are expressed in units of optical density (OD). The following indicators were considered a high level of antibodies: for α3-AChR - more than 0.24 OD450 nm, for β2-AP also more than 0.24 OD450 nm and for NGF - more than 0.37 OD450 nm.

Moreover, when the level of autoantibodies to α3-AChR is 0.24 or more, the level of autoantibodies to β2-AP is less than 0.24 and the level of autoantibodies to NGF is less than 0.37, damage to the structures of the autonomic nervous system of autoimmune genesis is assessed at the ganglion level. When the level of autoantibodies to α3-AChR is less than 0.24, the level of autoantibodies to β2-AP 0.24 or more and the level of autoantibodies to NGF of 0.37 or more, damage to the structures of the autonomic nervous system of autoimmune genesis is assessed at the level of the effector organ. In the complete absence of autoantibodies to α3-AChR, β2-AR, and NGF, damage to the structures of the autonomic nervous system is assessed at the suprasegmental level.

Thus, the method proposed by us differs from the prior art in the following:

1) allows you to evaluate the ANS with high accuracy by examining the blood of patients by our method of ELISA in serum by interacting with the three antigens α3-AChR, β2-AR, NGF;

2) allows you to assess the level of damage to the ANS;

3) allows you to abandon the use of radioactive isotopes as reagents when performing the methodology;

4) allows you to diagnose, differential diagnosis, objectify the dynamics of the pathological process, and also facilitates the selection of adequate therapy in patients with signs of peripheral autonomic insufficiency (PVN);

5) is more economical and no less sensitive than other methods;

6) does not require the introduction into the body of patients of potent drugs that are specially registered;

7) allows you to quantify the concentration of autoantibodies to α3-AChR, β2-AR, NGF and allows you to diagnose autoimmune damage to the peripheral ANS at the level of damage to the peripheral structures of the sympathetic and parasympathetic parts of the autonomic nervous system at the ganglion, effector organ levels, as well as the supra-segmented level, not associated with autoimmune damage.

Method implementation examples

Example 1

Patient B., 58 years old, was observed in the clinic of nervous diseases named after Kozhevnikov I MGMU them. Sechenova I. with signs of peripheral autonomic insufficiency, progressing over 8 years: Horner's syndrome on the right, disturbed accommodation with a distorted reaction of the pupils in the sample with adrenaline, the opposite reaction of blood pressure in clinostatic and orthostatic samples (increase in the supine position and a significant decrease in standing position), lack of dynamics of heart rate when changing posture, change of constipation with frequent loose stools (up to 13-16 times per night), weakness in standing and sitting, inability to move to be played without dragging clothes, fatigue, dry skin and mucous membranes with proximal hyperhidrosis. She received symptomatic therapy (enhanced water regimen, mineralocorticoids, and dragging clothes) with an insignificant temporary effect against the background of disease progression. A study of the patient's blood serum by the proposed method revealed a significant increase in the level of autoantibodies to α3-AChR - 1.79 OD _{450 nm} . As well as a decrease in the level of autoantibodies to β2-AP - 0.14 OD _{450 nm} and the level of autoantibodies to NGF - 0.17 OD _{450 nm} . This indicates damage to the peripheral structures of the autonomic nervous system of autoimmune genesis at the ganglion level.

Based on the analysis, a diagnosis of autoimmune autonomic vegetative gangliopathy was made, mineralocorticoids were replaced with glucocorticoids, the patient's condition gradually improved with immunosuppressive therapy.

Example 2

Patient U., 36 years old, is observed in the clinic of nervous diseases named after Kozhevnikov 1 MGMU them. I.M.Sechenova with signs of peripheral autonomic insufficiency: orthostatic hypotension, erectile dysfunction, skin hypohidrosis with dry mucous membranes. A study of the patient’s blood serum by enzyme-linked immunosorbent assay revealed an increase in the level of autoantibodies to β2-AP - 1.3 OD _{450 nm} and the level of autoantibodies to

NGF - 0.87 OD _{450 nm} and a decrease in the level of autoantibodies to α3-AChR - 0.21 OD _{450 nm} . The insufficiency of the sympathetic ANS at the level of autoimmune effector organs was revealed, immunosuppressive therapy was recommended, against which the patient's condition improved.

Thus, an increase in autoantibodies to β2-AP, NGF and their decrease to α3-AChR indicates a deficiency of the structures of the sympathetic link at the level of effector organs.

Example 3

Patient I., 46 years old, is observed in the SC Neurology RAMS with a diagnosis of myasthenia gravis, generalized form. The patient showed symptoms of autonomic dysfunction in the form of attacks of tachycardia, sweating with dry mucous membranes, visual impairment (in addition to diplopia associated with myasthenic external ophthalmoparesis, periodic “defocusing” of the contours of objects at an average distance). In addition, in addition to weakness and fatigue of voluntary muscles, general weakness, fatigue, not responding to acetylcholinesterase inhibitors (AChE). The study of blood serum by the proposed method did not reveal autoantibodies in the blood serum, which made it possible to attribute these disorders to the suprasegmental level of ANS dysfunction against the background of dishormonal disorders. Four years ago, she was operated on for thyroid cancer, there are irregularities in the menstrual cycle with alternating uterine bleeding and long periods of amenorrhea. This allowed us to adjust the patient therapy.

Thus, the determination of autoantibodies to α3-AChR, β2-AR, NGF by the method of ELISA in blood serum reflects the suprasegmental level of damage to the ANS of hormonal origin.

The inventive method for assessing ANS allows to improve the diagnostic process in patients with signs of autonomic insufficiency in a short time, to trace the dynamics of the pathological process and choose the right treatment tactics.

42 patients were examined. Of these, 14 patients with myasthenia gravis and damage to the structures of the peripheral autonomic nervous system at the ganglion level revealed autoantibodies to α3-AChR 1.5 ± 1.26 (0.24-2.76), that is, 0.24 or more; β2-AR 0.18 ± 0.05 (0.13-0.23) - less than 0.24; to NGF 0.27 ± 0.09 (0.18-0.36) - less than 0.37. In 16 patients with myasthenia gravis and damage to the structures of the peripheral autonomic nervous system at the level of effector organs, autoantibodies to α3-AChR 0.21 ± 0.02 (0.19-0.23), that is, less than 0.24; β2-AR 1.3 ± 1.06 (0.24-2.36) 0.24 or more and NGF 1.9 ± 1.53 (0.37-3.43) 0.37 and more. In this case, the presence of antibodies to β2-AR also makes it possible to distinguish a group of patients resistant to therapy with β2-adrenergic agonists. In 12 patients with a suprasegmental level of damage to the structures of the peripheral nervous system, autoantibodies were absent.

Thus, based on the above data, it is possible to assess ANS deficiency both at the level of systemic effects and on cells that do not have direct autonomic innervation (cells of the immune system), which gives an idea of the mechanisms that underlie clinical symptoms and allows differential diagnosis organic lesions of the ANS.

On the basis of differential diagnosis data, adequate therapy is prescribed to patients not only neurological, but also cardiological, gastroenterological, urological and ophthalmological clinics.

Thus, the proposed method has a high specificity for detecting the presence of antibodies to α3-AChR, β2-AR, NGF in ELISA and allows to detect with high accuracy an increased and decreased number of antibodies in which various levels of damage to the autonomic nervous system are diagnosed.

This allows you to start timely adequate treatment aimed at suppressing the causes of affection of the ANS and improving the quality of life of patients with PVN.

Claims (1)

A method for assessing various levels of damage to the structures of the autonomic nervous system, including the study of the patient’s blood serum with signs of peripheral autonomic insufficiency, characterized in that blood serum samples are determined by the enzyme-linked immunosorbent assay (TIFA) of the anti-ganglion subunit of the α3-acetylcholine receptor (α3-АX) , β2-adrenergic receptor (β2-AR) and nerve growth factor (NGF), optical absorption of light (OD) in the samples is measured on an ELISA analyzer at a wavelength of 450 nm and at If the level of autoantibodies to α3-AChR is 0.24 or more, the level of autoantibodies to β2-AP is less than 0.24 and the level of autoantibodies to NGF is less than 0.37, damage to the structures of the autonomic nervous system is assessed at the ganglion level, with the value of the level of autoantibodies to α3-AChR less than 0.24, the level of autoantibodies to β2-AP 0.24 or more, and the level of autoantibodies to NGF 0.37 or more assess the damage to the structures of the autonomic nervous system at the level of the effector organ, in the absence of autoantibodies to α3-AChR, β2-AP and NGF assess the damage to the structures of the autonomic nervous system at the suprasegmental level.