RU2483306C1 - Method for prediction of primary open-angle glaucoma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: lachrymal fluid and blood serum are examined by enzyme immunoassay using specific test systems. Higher levels of metalloproteinase-9 (MMP-9) exceeding 52.5 ng/ml in lachrymal fluid and 274.49 ng/ml in blood serum; higher levels of a complex of metalloproteinase-9 and its tissue inhibitor (MMP-9/TIMP-1) exceeding 0.19 ng/ml in lachrymal fluid and 4.93 ng/ml in blood serum, and higher levels of secretory immunoglobulin A (slgA) exceeding 47.38 mg/l in lachrymal fluid and 2.1 g/l in blood serum are the criteria to diagnose primary open-angle glaucoma.

EFFECT: use of the declared method can effectively diagnosing primary open-angle glaucoma.

6 dwg, 2 tbl, 1 ex

Description

The invention relates to medicine, in particular to ophthalmology, and can be used for early diagnosis of primary open-angle glaucoma (POAG) in the population.

Glaucoma is one of the most severe forms of ophthalmopathy, occupying a leading place among the causes of blindness and low vision. Primary open-angle glaucoma (POAG) accounts for up to 90% of the total number of all its forms [3]. Currently, there are more than 70 million people in the world who are losing sight from glaucoma. It is known that the diagnosis of the early stages of glaucoma, when it is possible to suspend and control the disease, is extremely underdeveloped. The traditional criterion for increased intraocular pressure (IOP) in patients with POAG is currently not mandatory, the proportion of glaucoma with normal or reduced IOP is from 20 to 35%. Almost every second patient learns about his disease in advanced late stages, without attaching importance to signs of visual impairment in previous years.

In recent decades, thanks to the intensive development of biochemical, histochemical, molecular genetic, morphological, immunological methods for the diagnosis of various pathological processes, great strides have been made towards understanding the mechanisms of the pathogenesis of the disease. Despite the active study of glaucoma by scientists of various specialties, ophthalmologists, morphologists, endocrinologists, therapists, immunologists around the world, the etiology and pathogenesis of the disease are still unknown, indicating significant difficulties in early diagnosis and low effectiveness of treatment measures [1, 2, 5, 6, 8, 9].

In the pathogenesis of glaucoma optic neuropathy, the main mechanisms are considered to be vascular factors of optic disk nerve damage (optic nerve disc), apoptosis of retinal ganglion cells, and metabolic shift. Recently, more and more attention has been paid to the molecular mechanisms of damage to the retina, optic nerve and optic tract in glaucoma. However, such studies, especially concerning the effect of immune processes in the pathogenesis of glaucoma, are few, fragmentary, and very controversial [8].

Diagnosis of glaucoma presents significant difficulties. To date, there is no universally accepted clear definition of glaucoma. Glaucoma is defined as a multifactorial disease with a threshold effect, which in an advanced stage is characterized by the development of glaucoma optical neuropathy (GON), with typical changes in the optic head (GZN) and the inner layers of the retina. The diagnosis is at an early stage and is established taking into account risk factors and pathogenic factors. Glaucoma lesion, as a rule, consists of several links - stages: violation of the outflow of aqueous humor from the eye, increased intraocular pressure (IOP), optic head ischemia (GZN), atrophy of the optic nerve and ganglion cells of the retina (GCS). These links are not always sequential, do not depend on each other and are not always found in a complete set.

The initial causes of such a variety of changes affecting various departments and tissues of the eye are still not fully established. Some scientists consider the dominance of biomechanical factors as leading, others as vascular, metabolic, genetic, immune disorders, the result of which is progressive optic atrophy and blindness.

There are many theories explaining these processes, the dominant of which are the ophthalmic hypertensive (biomechanical) theory of glaucomatous excavation of optic disc, neurotrophic, vascular and others.

The basis of the biomechanical concept of glaucomatous excavation is considered to be the failure of the supporting structures of the sclera and ethmoid membrane, which is the initial and decisive phase of the pathogenesis of POAG, and glaucomatous excavation of the optic disc is a compression injury. Moreover, the deflection of the lattice membrane of the optic nerve disc that occurred even with normal IOP leads to a violation of the axoplasmic current, deficiency of neurotrophic factors, and death of neurons [2; 3]. The essence of the neurotrophic theory (optical neuropathy) is the decisive importance of the pathology of the deep layers of the retina, where the corticosteroids are located, as well as their axons that make up the optic nerve [8]. Another important theory of the pathogenesis of glaucoma in terms of the number of works and convincing evidence is the vascular, hemodynamic, or the theory of primary ischemia of optic disc with subsequent atrophy of the optic nerve and visual field defects [1, 6, 8]. However, the processes of ischemia, discirculatory disorders, and vasospasm, while not leading in the early period of the disease, play the role of process progression factors.

An important discovery was the proof of the role of apoptotic processes in the development of the glaucomatous process. It is assumed that as a result of increased IOP and / or a violation of local microcirculation, programmed pathological tissue processes that cause progressive cell death, the so-called ischemic apoptosis, can be activated.

In the last decade, the role of immunological mechanisms in the pathogenesis of the glaucomatous process has been clarified. The study of immune factors revealed a variety of immune defense disorders in POAG patients. Immunological studies in patients are few and multidirectional results. It has been established that with the glaucomatous process there are significant changes in both the cellular and humoral parts of the immune system [5, 8, 10, 11]. It is characteristic that the identified changes were detected in the early stages of the disease.

It is known that a number of pathological conditions in humans are precursors of the development of glaucoma. Identification of the main risk factors in the development of glaucoma and their interactions is of great importance in the early diagnosis of the glaucomatous process [2, 3, 10]. The high probability of a frequent combination of the glaucomatous process in patients with myopia, as well as those suffering from hypertension and diabetes mellitus, is proved. The mechanisms of development of such a combined pathology are unclear.

The work of S. Rönkkö [11] is known where the significance of the complex of matrix metalloproteinases (MMPs) with their tissue inhibitors (TIMPs) in the pathogenesis of POAG has been established. Elevated levels of MMP-1, 2, 3, 9 and imbalance with their tissue inhibitors (TIMP-1, 2, 3) were found in the tissues of the Schlemm canal obtained intraoperatively. The authors believe that high concentrations of MMPs cause remodeling of the extracellular matrix of the structures of the anterior chamber of the eye, impaired outflow of aqueous humor and increased levels of intraocular pressure.

However, only one link of the immunopathogenesis of glaucoma is reflected in the work, without taking into account other immunological parameters and their interaction. In addition, an invasive diagnostic method is used, which is difficult to apply in practical health care.

A known method for the diagnosis of clinical variants of glaucoma in people with myopic refraction [12], in which the prevalence of pathology in the arterial and venous link of intraocular pressure is differentiated by the blood flow velocity in the central retinal artery, orbital vein and short ciliary arteries, determining the dominant hemodynamic link in the pathogenesis of POAG in persons with myopia. However, the author does not take into account the importance of immunological aspects in the development of these vascular changes, although it is known that the pathogenesis of POAG is multifactorial.

A known method for the early diagnosis of primary open-angle glaucoma, which uses the method of infrared spectrometry of the lacrimal fluid with the calculation of the Mahalanobis criterion, with a value of 34 srvc. units and more are diagnosed with POAG [13].

However, the known method does not take into account the immunological mechanisms of the pathogenesis of glaucoma, does not provide for the examination of groups of patients with diseases - predictors of glaucoma.

A known method for the early diagnosis of glaucoma based on the determination of autoantibodies against antigens of the retina and optic nerve in two biosubstrates - blood serum and lacrimal fluid [14].

However, this method has several disadvantages. The authors use the biological fluids of healthy people and patients with glaucoma as a control without examining the risk group for the development of the glaucomatous process. In addition, autoimmune processes are not prevalent in the pathogenesis of glaucoma, unlike systemic mediators such as cytokines, metalloproteinases and immunoglobulins.

A known method for the diagnosis of glaucoma [15], which suggests the identification of genetic disorders of Wnt signaling pathways, namely, changes in the level of bioactivity of frizzled related protein.

However, the known method only affects the genetic and biochemical aspects of the diagnosis of glaucoma, without considering the multifactorial development of the disease.

A known method for diagnosing the initial stage of open-angle glaucoma, taken as a prototype [16], the essence of which is to determine the content of autoantibodies to antigens of native and denatured DNA and the content of circulating immune complexes in the patient’s lacrimal fluid, at a certain level of which the initial stage of glaucoma was diagnosed.

However, the known method has several disadvantages:

1) the study of only the local level of autoantibodies, without determining these parameters in the systemic circulation;

2) the study of only one marker for the diagnosis of POAG;

3) the comparison group did not include patients who constitute a risk category for the development of the disease;

4) the study of only the autoimmune link of the pathogenesis of POAG with known significance and other factors of the development of this disease.

The task of the invention is to eliminate the disadvantages of the prototype; to develop an effective way to predict the development of POAG.

The problem is achieved by the claimed method, which consists in determining the concentration of early diagnostic markers of primary open-angle glaucoma: secretory immunoglobulin (sIgA) and matrix metalloproteinase-9 (MMP-9), as well as its complex with a tissue inhibitor (MMP-9 / TIMP-1) in patients with primary open-angle glaucoma and in risk groups for its development.

The technical result of the proposed method is as follows:

1. The complexity of the studied parameters, reflecting the molecular mechanisms of the pathogenesis of primary open-angle glaucoma.

2. The studied markers were determined simultaneously in two biosubstrates (systemic content in the blood serum, local in the lacrimal fluid).

3. Comparison of indicators in patients with POAG was carried out not only with the control group, but also with the group of patients with POAG in combination with myopia, hypertension, and diabetes mellitus.

4. The studied indicators were studied in patients at risk of developing primary glaucoma, suffering from myopia, hypertension, and diabetes mellitus.

5. Using methods of modern statistics (ROC analysis), a reliable connection between the existing immune disorders and the presence of the disease has been established.

The essence of the claimed invention.

The study was conducted in blood serum and lacrimal fluid by enzyme-linked immunosorbent assay (ELISA) using specific test systems.

Venous blood and lacrimal fluid were used as biological materials for immunological studies. Venous blood samples were taken in the morning, on an empty stomach, in compliance with the rules of asepsis and antiseptics from the ulnar vein by venipuncture in 1 tube without anticoagulant in an amount of 5 ml. Then, by centrifugation (3000 rpm), serum was obtained, poured into epindorfs and placed in a refrigerator (-76 ° C). The levels of immunoglobulin (IgA), matrix metalloproteinase-9 (MMP-9) of the human secretory type and its complex with a tissue inhibitor (MMP-9 / TIMP-1) were determined in blood serum. The tear fluid was taken after irritation with ammonia vapors, an insulin syringe (the needle was previously removed) from the inner corner of the eye and was used to study secretory immunoglobulin A (sIgA), matrix secretory metalloproteinase-9 (MMP-9) and its complex with a tissue inhibitor ( MMP-9 / TIMP-1).

Determination of the concentration of secretory immunoglobulin class A (IgA).

The determination method is based on enzyme-linked immunosorbent assay using the kit "IgA secretory - ELISA - BEST". 80 μl of serum dilution solution (PPC) was added to the wells of the tablet intended for calibration and control samples, 90 μl of PPC was added to the wells used for the analyzed samples. Then, 20 μl of calibration and control (with a known concentration of sIgA) solutions in duplicates were added. The contents of the wells were mixed. In the wells intended for analysis and containing 90 μl of PPC, 10 μl of pre-diluted samples of biological fluids were added. Incubated for 30 min in an incubator at 37 ° C, and then washed the wells of the plate with washing buffer. 100 μl of conjugate were added to all wells. Incubated for 30 min in an incubator at 37 ° C. After incubation, the wells were washed, then 100 μl of a solution of tetramethylbenzidine was added to them, incubated in the dark for 25 min in an incubator at 37 ° C, and 50 μl of a solution of stop reagent (5% H ₂ SO ₄ ) was added. The results were recorded using a plate spectrophotometer (µQuant Bio-Tek Instruments, USA) at a wavelength of 450 nm. According to the measurement results, the arithmetic mean value of the optical density in the duplicate wells was calculated, and the concentration of sIgA in the analyzed samples was determined.

Determination of the concentration of matrix metalloproteinase 9 (MMP-9).

The determination of the concentration of MMP-9 was carried out using a set of "R&D Systems, Inc. USA. " The determination method is based on enzyme-linked immunosorbent assay.

100 μl of a solution of mouse antibodies against human MMP-9 at a concentration of 1.0 μg / ml in phosphate-buffered saline (FSB-137) was added to the wells of a tablet (Costar, corhing Inc., USA). Incubated overnight at room temperature, after which the contents of the plate were removed, the wells were washed with wash buffer. Then, 300 μl of serum dilution solution (PPC) was added to each well, incubated for 2 hours at room temperature, then 100 μl of calibration samples in duplicates were added. 100 μl of the analyzed samples were added to the remaining wells of the plate (blood serum was diluted 1: 200 using PPC, tear fluid was added without dilution). After incubation for 2 hours at room temperature, the wells were washed. 100 μl of a solution of biotinylated anti-MMP-9 antibodies at a concentration of 100 ng / ml in PPC was added to each well. Incubated for 2 hours at room temperature, followed by washing of the wells. 100 μl of streptavidin peroxidase conjugate were diluted 1: 200 in PPC into each well. Then incubation at room temperature and washing the wells. Then, 100 μl of tetramethylbenzidine solution was added to each well. Then incubated in the dark for 20 minutes. 50 μl stop reagent solution (5% H ₂ SO ₄ ) was added to all wells of the plate.

The results were recorded using a tablet spectrophotometer (µQuant Bio-Tek Instruments, USA) at a wavelength of 450 nm. According to the measurement results, the arithmetic mean value of the optical density in the duplicate wells was calculated and the concentration of MMP-9 in the analyzed samples was determined using a calibration graph.

Determination of the concentration of the complex of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 (MMP-9 / TIMP-1).

The concentration of the MMP-9 / TIMP-1 complex was determined using the R&D Systems, Inc. kit. USA. " The determination method is based on enzyme-linked immunosorbent assay.

In the wells of the plate (Costar, corhing Inc., USA), 100 μl of a solution of murine antibodies against human MMP-9 at a concentration of 4.0 μg / ml in phosphate-buffered saline (PBS) was added. After overnight incubation at room temperature, the contents of the plate were removed, and the wells were washed with wash buffer. Then 300 μl of blocking buffer (1% bovine serum albumin in PBS) was added to each well. After two hours of incubation at room temperature, the wells were washed and 100 μl of duplicate calibration samples were added. 100 μl of the analyzed samples were added to the remaining wells of the plate, incubated at room temperature, then the wells were washed. 100 μl of a solution of biotinylated anti-human TIMP-1 antibodies at a concentration of 50 ng / ml in PPC was added to each well. Then, 100 μl of streptavidin peroxidase conjugate at a dilution of 1: 200 in PPC were added to each well. Then incubation at room temperature and washing the wells. 100 μl of tetramethylbenzidine solution was added to each well, incubated in the dark for 20 minutes at room temperature. 50 μl of stop reagent solution (5% H ₂ SO ₄ ) was added to all wells of the plate.

The results were recorded using a plate spectrophotometer (µQuant Bio-Tek Instruments, USA) at a wavelength of 450 nm. The arithmetic mean value of the optical density in duplicate wells was calculated from the measurement results and the concentration of the MMP-9 / TIMP-1 complex in the analyzed samples was determined.

A distinctive feature of the proposed method from the prototype is the conduct of extended comparative immunological studies in different groups of patients with POAG and chronic diseases - predictors of glaucoma, as well as in two biological substrates (blood serum and lacrimal fluid).

The inventive method differs from the known solutions in the expanded application of immunological tests for the diagnosis of glaucoma in different groups of patients: a healthy population, patients with hypertension, type 2 diabetes, myopia, in patients with initial and advanced stages of POAG. In addition, tests used include characterization of both cellular and humoral immunity.

As shown by the analysis of the available scientific and patent information, information related to the use of this complex of immunological tests to predict the development of glaucoma was not found.

A similar set of immunological diagnostic tests in patients with POAG and risk groups for the development of this disease was not found in the available literature.

The essence of the invention is illustrated by graphic images, where figure 1 shows the structure of the combined pathology in patients with POAG; figure 2 shows the structure of the pathology in patients at risk of developing POAG; figure 3 shows the coefficients of the ratio of the levels of MMP-9 and MMP-9 / TIMP-1 in patients with POAG and at risk of developing glaucoma in serum and lacrimal fluid; figure 4 shows the level of secretory immunoglobulin A (mg / l) in the lacrimal fluid in patients with POAG and at risk for glaucoma; figure 5 shows the ROC curve of the evaluation of local production sIgA; figure 6 shows the ROC curve of the evaluation of local products MMP-9.

An example implementation of the method.

We observed 80 patients with POAG (the main group) as well as 35 people with chronic diseases belonging to the risk group for developing POAG, that is, they are possible predictors of POAG - hypertension II-III degree (GB), type 2 diabetes mellitus (DM) , as well as patients with POAG in combination with myopia. The age of patients ranged from 45 to 69 years. The control group consisted of 30 healthy individuals of a comparable age to the group of patients. In addition, the main group of patients with POAG is divided into 2 subgroups: 42 people without the presence of concomitant chronic diseases included in the list of risk groups, and 38 patients suffering from these diseases before the manifestation of POAG (Figure 1).

The diagnosis of primary open-angle glaucoma (POAG) was established on the basis of clinical and instrumental data, in accordance with the requirements of the National Guide to Glaucoma. All patients underwent a general clinical examination and a set of methods for ophthalmological research: visometry, biomicroscopy, autorefractometry, ophthalmoscopy, tonometry, topography, perimetry, confocal laser scanning ophthalmoscopy, ultrasound examination of the eyes, electrophysiological examination of the eyes.

According to the progression of the glaucomatous process, the structure of the examined was as follows: 11 patients with the initial stage of glaucoma (13.7%), 31 - with advanced (38.75%), 28 - far-reaching (35%), 10 - terminal stages (12.5% )

To assess intraocular pressure (IOP), the following generally accepted classification was used:

And - normal pressure (does not exceed 21 mm Hg),

B - moderately elevated pressure (from 22 to 32 mm Hg),

C - high pressure (exceeds 32 mm Hg).

IOP within the normal range was determined in 54 (67.5%) patients, moderately elevated in 22 patients (27.5%), and high intraocular pressure was recorded in 4 patients (5%).

A separate risk group for the development of glaucoma (35 people) was made up of patients suffering from GB II-III art., Type 2 diabetes and myopia (Figure 2). Myopia was observed in 40.5%, of which 21.4% suffered from a weak degree of myopia, 50% from an average degree, and 28.6% from a high degree. Hypertension II-III Art. in the structure of the risk group amounted to 35.7%. Type 2 diabetes mellitus was diagnosed in 23.8% of patients. It should be noted that the ratio of concomitant pathology in the main group of patients with POAG and in the risk group was comparable.

The study of the level of immunoglobulin A (IgA) in patients with POAG and at-risk for the development of POAG

An IgA study was performed in serum and lacrimal fluid in 80 patients with primary open-angle glaucoma, 31 patients at risk and 30 healthy individuals (control group). Immunoglobulin A (IgA) is known to exist in two forms; secretory and serum.

Serum IgA values were less significantly different from the control in all groups of patients (Figure 4). The study of secretory immunoglobulin A (sIgA) in the lacrimal fluid showed an increase in its levels in patients with POAG (main group) by 3 times compared with the control (table 1). SIgA is known to be the main type of immunoglobulin involved in the local immune response. Selective accumulation of it in the tear fluid indicates an increase in the protective function of the mucous membranes relative to any microbial antigen, toxin, autoantigen, as well as the desire to limit the development of the pathological process. A high degree of reliability (p <0.001) between the secretory IgA values was observed in patients with POAG of two subgroups. The highest values (4-5 times higher than normal) were observed with a combination of POAG with myopia.

SIgA levels in patients at risk of diabetes mellitus, both in combination with POAG and without it, did not differ much from each other (p> 0.05). These results make it possible to consider the contingent of patients with diabetes less vulnerable to the risk of developing glaucoma, in contrast to patients with myopia and hypertension.

In the risk group for the development of glaucoma, the level of secretory IgA was increased compared with the control and the main group of patients with POAG. It is important to note that the values of this parameter in patients at risk were close to those of patients with POAG without concomitant chronic pathology (p> 0.05).

Table 1 The level of immunoglobulin A (IgA) in patients with POAG and at risk for glaucoma Groups examined n Blood serum Tear fluid g / l mg / l The control thirty 2.05 ± 0.5 293.1 ± 39.6 POAG general 80 2.46 ± 0.41 * 1040.3 ± 117.0 * # POAG without soput. pathology 38 2.2 ± 0.3 105.31 ± 19.8 POAG with the presence of soput. pathology 42 2.71 ± 0.2 * 1111.4 ± 303.0 * # POAG + myopia eighteen 2.8 ± 0.33 * 1408.4 ± 168.0 * # POAG + GB fourteen 2.9 ± 0.2 * 524.0 ± 98.5 * # POAG + SD 10 2.2 ± 0.1 504.5 ± 95.5 * # Risk group 35 2.2 ± 0.2 842.8 ± 94.8 * Myopia fourteen 2.3 ± 0.3 997.9 ± 183.1 * GB eleven 2.2 ± 0.1 758.6 ± 46.6 * SD 10 2.0 ± 0.2 518.0 ± 72.3 * Note: * reliability of indicators of groups of patients with control, # groups of patients with POAG and risk groups.

When conducting ROC analysis (Figure 5) revealed a high specificity and sensitivity of local secretion of IgA in patients with POAG in comparison with the control (Sensitivity 77.8%, Specificity 100%, Criterion 47.38). This indicates the great importance of sIgA as a criterion for early diagnosis of the glaucomatous process.

The study of the level of metalloproteinase - 9 (MMP-9) and its complex with a tissue inhibitor (TIMP-1) in patients with POAG and at-risk groups for the development of POAG

Matrix metalloproteinases (MMPs) are endopeptidases that regulate the nature of the structural and functional properties of the extracellular matrix in normal and pathological conditions. The nature of the expression of metalloproteinases and their active participation in the processes of remodeling of biological structures are very similar to the expression of classical acute phase proteins. The synthesis of metalloproteinases is controlled by natural tissue inhibitors - TIMP, which are produced by the same cells simultaneously and side by side. The regulation of the functions of MMPs and their tissue inhibitors is a finely coordinated process. The determination of the activity of the MMP / TIMP complex is important, since the imbalance between the synthesis and degradation of the components of the extracellular matrix leads to fibrosis, tissue sclerosis.

An elevated level of MMP-9 was recorded in the lacrimal fluid and blood serum of patients with POAG (Table 2), and significantly significant compared with the control concentrations were noted in the POAG group with the presence of concomitant chronic pathology. Patients with POAG combined with myopia and hypertension were distinguished by the highest MMP-9 values.

The most informative was the local expression of MMP-9; in the lacrimal fluid, protease levels exceeded the level of healthy people by 7-8 times. It is interesting that changes in protease indices showed direct unidirectional correlation in blood serum and lacrimal fluid in patients with POAG combined with hypertension (p = 0.002, U-test, r = 0.9).

When conducting ROC analysis (Fig.6) revealed the highest specificity and sensitivity of local secretion of MMP-9 in patients with POAG in comparison with the control (Sensitivity 100%, Specificity 100%, Criterion> 52.5). This indicates the great importance of MMP-9, as a marker for the diagnosis of glaucomatous process.

table 2 The activity of MMP-9 and its complex with an inhibitor of MMP-9 / TIMP-1 in patients with POAG and at risk for glaucoma Groups examined n Blood serum Tear fluid MMP-9 ng / ml MMP-9 / TIMP-1 ng / ml MMP-9 ng / ml MMP-9 / TIMP-1 ng / ml The control thirty 192.2 ± 19.05 4.34 ± 0.72 34.8 ± 2.86 0.15 ± 0.01 POAG general 80 252.3 ± 19.2 * 7.0 ± 0.3 * # 206.5 ± 27.8 * # 0.19 ± 0.01 POAG without soput. pathology 38 215.4 ± 17.4 5.6 ± 0.34 * 221.5 ± 42.6 * # 0.2 ± 0.03 POAG with the presence of soput. pathology 42 285.1 ± 28.8 * 7.3 ± 0.4 * 184.0 ± 31.7 * # 0.2 ± 0.01 * POAG + myopia eighteen 234.4 ± 25.5 * 6.8 ± 0.5 * 184.3 ± 50.5 * # 0.15 ± 0.002 POAG + GB fourteen 335.9 ± 55.1 * 8.3 ± 0.8 * 258.3 ± 79.9 * # 0.23 ± 0.03 * POAG + SD 10 268.1 ± 41.4 8.8 ± 0.6 * 145.2 ± 13.4 * # 0.22 ± 0.02 * # Risk group 35 262.2 ± 26.8 7.4 ± 0.9 * 108.7 ± 21.3 * 0.19 ± 0.02 Myopia fourteen 214.5 ± 36.6 4,5 ± 0,1 96.3 ± 26.6 * 0.16 ± 0.007 GB eleven 372.8 ± 53.1 10.9 ± 1.4 * 155.6 ± 47.9 * 0.23 ± 0.04 SD 10 213.0 ± 18.5 7.3 ± 1.6 * 57.0 ± 2.5 * 0.18 ± 0.004 * Note: * reliability of indicators of groups of patients with control, # groups of patients with POAG and risk groups.

Serum levels of the protease complex with the inhibitor (MMP-9 / TIMP-1) were also significantly increased almost twice. When analyzing the values of MMP-9 / TIMP-1 in the tear fluid, their significant increase was found only in patients with POAG with the presence of a combined pathology.

Separately, it is worth noting the revealed changes in the levels of MMP-9 and MMP-9 / TIMP-1 in patients at risk. In the lacrimal fluid of the examined patients, the level of MMP-9 / TIMP-1 did not differ much from the control group. At the same time, the level of protease in serum was increased only 1.5 times (p <0.05).

The range of fluctuations of indicators in all the studied biomaterials in this category of patients was noteworthy. So, the median (Me) of MMP-9 content in the tear fluid was 136.8 ng / ml (min. 66.8 ng / ml, max. 570.5 ng / ml), and in the blood serum 259.2 ng / ml (min. 110.4 ng / ml, max. 510.5 ng / ml). The median concentration of MMP-9 / TIMP-1 in the tear fluid was 0.18 ng / ml (min. 0.1 ng / ml, max. 0.42 ng / ml), in serum - 7.5 ng / ml (min 2.3 ng / ml, max. 11.58 ng / ml).

Given the varying degrees of increase in metalloproteinase-9 and its complex with an inhibitor and the features of their expression at the system and local levels, the coefficients of the ratio of these indicators in the studied biological fluids were calculated (Figure 3). In the control group, this coefficient was 47.4 ± 1.6; in tear fluid 489.6 ± 74.0. In all patients with serum glaucoma, this indicator was close to normal. However, in the lacrimal fluid, its significant increase was more than 3 times. Moreover, higher values of the coefficient MMP-9 / TIMP-1 were noted in a subgroup of patients with combined chronic pathology. In the group of patients at risk of developing glaucoma (with myopia, hypertension, diabetes mellitus with a normal serum coefficient, its value in the tear fluid was high (p <0.001) compared to the control group. Its level was close to that in patients with glaucoma (p > 0.05). This indicates the high information content of this indicator, which allows to identify the initial signs of the glaucoma process in people with myopia, hypertension, and diabetes mellitus.

Thus, changes in the concentration of metalloproteinase-9 and its complex with the MMP-9 / TIMP-1 inhibitor in patients with glaucoma indicate a violation of tissue barrier equilibrium, both at the local and systemic levels. So the concentration of MMP-9 in the tear fluid exceeded the level of its complex with the inhibitor by almost 3 times.

The revealed immune imbalance, especially at the local level, can be a key factor in the mechanisms of dysregulation of remodeling processes in the structures of the trabecular network, the trellis plate of the sclera and the optic nerve disc. At the systemic level, the identified MMP-9 hyperfunction, especially in patients with combined pathology (POAG + Myopia, GB, DM), is compensated by the activation of tissue inhibitors of metalloproteinases.

The study of the local concentration of the studied parameters (lacrimal fluid) compared with the systemic (blood serum) was more informative and revealed high concentrations not only in patients with glaucoma, but also at risk of developing the disease. When analyzing MMP-9 indices in patients at risk of developing primary open-angle glaucoma, on the contrary, the highest and most reliable values were obtained in the systemic circulation, which may indicate the dominance of vascular disorders in the initial mechanisms of pathogenesis.

The data obtained indicate the significant role of these mediators in the pathogenesis of POAG, being markers of inflammatory activity, factors in the development of extracellular matrix degradation, cell destabilization and tissue damage. Excessive synthesis of extracellular matrix, perhaps, is the basis for reducing the outflow of intraocular fluid, degenerative changes in the head of the optic nerve (GBN).

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Claims (1)

A method for predicting primary open-angle glaucoma disease, characterized in that, for the purpose of early diagnosis of the disease, lacrimal fluid and blood serum are examined by enzyme-linked immunosorbent assay using specific test systems, where elevated levels of metalloproteinase-9 (MMP-9), which exceed 52 5 ng / ml in tear fluid and 274.49 ng / ml in serum; elevated levels of the metalloproteinase-9 complex with its tissue inhibitor (MMP-9 / TIMP-1), whose values exceed 0.19 ng / ml in tear fluid and 4.93 ng / ml in blood serum, and increased levels of secretory immunoglobulin A ( sIgA), whose values exceed 47.38 mg / l in the lacrimal fluid and 2.1 g / l in the blood serum, are the criteria for diagnosing primary open-angle glaucoma.

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