RU2702735C1 - Method for determining the pro-inflammatory and anti-inflammatory interleukins in the anterior chamber of the eye in the patients with presenile cataract of the eye for diagnosing the postoperative complications - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to ophthalmology, and aims at determining the risk of developing postoperative complications in surgical treatment of presenile cataract of eye. Method for determining risk of developing postoperative complications in surgical treatment of presenile cataract eye involves collecting anamnesis based on hereditary factors, presence of chronic somatic diseases, eye injuries and pathologies, infectious diseases, as well as moisture intake of the anterior eye chamber during surgical treatment. In the anterior eye chamber moisture level of tumor necrosis factor alpha (TNF-α) is determined by immunoassay. If the TNF-α level is more than 2 times higher than its normal levels of 0.8–3.1 pg/ml, a risk of postoperative complications is identified.

EFFECT: use of the invention enables more effective determination of the risk of developing postoperative complications in surgical treatment of presenile cataract of the eye.

1 cl, 2 ex

Description

The invention relates to medicine, namely to ophthalmology and can be used to diagnose features of the inflammatory process in the anterior chamber of the eye in patients undergoing cataract phacoemulsification.

Interleukins are universal mediators that regulate all stages of the inflammatory process, from the moment of its induction to resolution. In light of the current advances in molecular medicine, the priority role of pro- and anti-inflammatory interleukins in regulating the local inflammatory process, including after tissue damage, has been established (Shaimova V.A. Role of pro-inflammatory cytokines in eye diseases. Cytokines and inflammation 2005; 2 (4): 13-15.). It is proved that the local production of cytokines in the focus of inflammation exceeds their systemic synthesis (Simbirtsev A.S. Cytokines, a new system for regulating the body's defense reactions // Cytokines and inflammation - 2002 - No. 1 - P. 9-16). Inflammatory processes may involve eye structures that limit the anterior chamber after surgical treatment or eye injury (Anderson DH, et al. A role for local inflammation in the formation of drusenin the aging eye. Am. J. Ophthalmol. 2002. No. 134 P. 411-431). At the same time, an imbalance in the synthesis of anti-inflammatory and pro-inflammatory interleukins by epithelial cells of the cornea of the eye has been shown to be important in the pathogenesis of glaucoma, cataracts and degenerative eye diseases (Bikbov M.M., Shevchuk N.E., Fayzrakhmanov PP, Yarmukhametova A.L. , Gilmanshin TR Local level of cytokines in various morphological variants of the neovascular membrane in patients with a wet form of age-related macular degeneration Medical almanac No. 1 (31) March 2014, 66-68; Shpak AA, Geht AB, Druzhkova T.A., Kozlo and K.I. Ratio of neurotrophic factors in the lacrimal fluid and moisture of the anterior chamber in patients with age-related cataract.Ophthalmic surgery. 2017; (1): 16-20; Chua J., Vania M., Cheung CM., Ang M. et al Expression profile of inflammatory cytokines in aqueous from glaucomatous eyes. Molecular Vision 2012; 18: 431-438; Sawada H., Fukuchi T., Tanaka T., Abe H. Tumor necrosis factor-alpha concentration in the aqueous humor of patients with glaucoma. Invest Ophthalmol Vis Sci 2010). From these positions, the question remains open about local production of cytokines in the uncomplicated and complicated course of the postoperative process in the surgical treatment of presinyl eye cataract. On the other hand, it has been proved that the interleukin profile has constitutional variability even in somatically healthy people, which can determine the course and outcome of cataract extraction surgery using phacoemulsification (Burilov KB, Kostenev SV, Trunov AN pro-inflammatory cytokines in the lacrimal fluid after the formation of the corneal valve with a mechanical microkeratome and a femtosecond laser, Ophthalmology, 2017, 6, 66-79). Thus, the preoperative level of pro-inflammatory cytokines, such as interleukin 1 beta (IL-1b) and tumor necrosis factor alpha (TNF-alpha), can predict surgical and postoperative complications.

Studies of systemic and topical concentrations of pro-inflammatory and anti-inflammatory interleukins for additional diagnosis of postoperative complications in the surgical treatment of eye cataracts have been proposed repeatedly.

The work of P.P. Abdullina (2004), who studied the levels of IL-1b and TNF-alpha in the tear and blood serum of patients before extraction of traumatic cataract with IOL implantation. The author found that the study of these interleukins allows you to control the degree of postoperative inflammatory response. So, according to the results of the work, it was found that the development of traumatic cataract is associated with a significant increase in levels (IL-1b, TNF-alpha) in the lacrimal fluid and serum. In addition, it was shown that surgical intervention leads to an increase in the production of pro-inflammatory cytokines in patients, regardless of the duration of the injury and surgery, and the subsequent course of the postoperative period (P.P. Abulina 2004). The main disadvantage of this method is that only pro-inflammatory interleukins were determined without taking into account the anti-inflammatory potential and in the group of patients with traumatic cataract in which the blood-ophthalmic barrier is violated.

А,

Е,

J, Bessenyei Е, Kardos L, Berta A. Enhanced release of IL-6 and IL-8 into tears in various anterior segment eye diseases. Ophthalmic Res. 2006; 38 (4): 182-8. Epub 2006 May 5). Кроме того, авторы указывают, что определение уровня данного цитокина может быть использовано в качестве индикатора воспалительных реакций в раннем послеоперационном периоде. Данные исследования были выполнены на когорте пациентов из Франции и результаты этого исследования не могут быть транспорированы на популяции России. Кроме того исследовалась слезная жидкость, которая продуцируется на поверхность роговицы и объем жидкости может иметь выраженные индивидуальные особенности, а значит и концентрация интерлейкинов будет широко варьировать.In relation to the development of non-traumatic age-related cataracts, an associative positive relationship was established with the level of IL-6 in the tear fluid (Fodor M,

BUT,

E

J, Bessenyei E, Kardos L, Berta A. Enhanced release of IL-6 and IL-8 into tears in various anterior segment eye diseases. Ophthalmic Res. 2006; 38 (4): 182-8. Epub 2006 May 5). In addition, the authors indicate that determining the level of this cytokine can be used as an indicator of inflammatory reactions in the early postoperative period. These studies were performed on a cohort of patients from France and the results of this study cannot be transported to the Russian population. In addition, a lacrimal fluid was studied, which is produced on the surface of the cornea and the volume of fluid may have distinct individual characteristics, which means that the concentration of interleukins will vary widely.

In addition, the study of topical interleukins in the eye is associated with the analysis of more than five analytes in a small amount of biological material. However, with the development of technologies for multiplex analysis and capillary electrophoresis, this problem fades into the background (Sharma RK, Rogojina AT, Chalam KV Multiplex immunoassay analysis of biomarkers in clinically accessible quantities of human water humor. Molecular Vis 2009; 15: 60-69; Tzovolou DN , Lamari F., Mela EK, Gartaganis SP, Karamanos NK Capillary electrophoretic analysis of brimonidine in aqueous humor of the eye and blood sera and relation of its levels with intraocular pressure. Biomedical chromatography: BMC 2000; 14 (5): 301-305 . doi: 10.1002 / 1099-0801 (200008) 14: 5 <301 :: AID-BMC4> 3.0.CO; 2-Q). At the same time, there is no widespread use of these technologies in practical healthcare of the Russian Federation due to the lack of domestic devices and consumables and the high cost of research.

А,

Е,

J, Bessenyei Е, Kardos L, Berta A. Enhanced release of IL-6 and IL-8 into tears in various anterior segment eye diseases. Ophthalmic Res. 2006;38(4): 182-8. Epub 2006 May 5), при котором исследовались провоспалительные цитокины в слезной жидкости при не травматической возрастной катаракте. Недостатки метода представлены выше.Closest to the claimed method in terms of technical nature and the achieved result is the methodological approach used by Fodor M. (Fodor M,

BUT,

E

J, Bessenyei E, Kardos L, Berta A. Enhanced release of IL-6 and IL-8 into tears in various anterior segment eye diseases. Ophthalmic Res. 2006; 38 (4): 182-8. Epub 2006 May 5), in which pro-inflammatory cytokines in the lacrimal fluid were studied in non-traumatic age-related cataracts. The disadvantages of the method are presented above.

The technical result of the invention is to improve the effectiveness of the diagnosis of postoperative complications in the surgical treatment of presynil cataracts of the eye by detecting an imbalance of pro-inflammatory and anti-inflammatory interleukins in the moisture of the anterior chamber of the eye.

It is known that the corneal epithelium and corneal stromal cells of the eye constantly produce interleukins primarily with suppressor qualities (interleukin 10 - IL1), but with damage to the structures of the anterior chamber of the eye and with the physiological process of formation of intraocular antigens in these cells, the synthesis of pro-inflammatory interleukins, such as interleukin, is induced 1 - IL-1, interleukin 6 - IL-6, interleukin 8 - IL-8, tumor necrosis factor alpha - TNF-a and interferon gamma - INF-g (Yerichev V.P., Petrov S.Yu., Saturday AM , Volzhanin A.V., Germanova V.N., Karl VA, E.V., Role of cytokines in the pathogenesis of eye diseases, National Journal of Glaucoma 2017, T. 16, No. 1, pp. 85-99; Russian Journal of glaucoma 2017, Vol.16, No. 1, pp. 85-99), so the connection between the severity of the inflammatory process in the anterior chamber of the eye and the activation of pro-inflammatory cytokines (Nao X., Yi C., Wang Y., Li J. et al. Identification of intraocular inflammatory mediators in patients with endophthalmitis. Molecular Vision 2016; 22: 563-574). These data served as a starting point for a new method for the diagnosis of postoperative complications in the surgical treatment of presinyl eye cataract.

SUMMARY OF THE INVENTION

A method is proposed for determining pro-inflammatory and anti-inflammatory interleukins in the moisture of the anterior chamber of the eye in patients with presynil cataracts of the eye for the diagnosis of postoperative complications, including collecting an anamnesis taking into account hereditary factors, the presence of chronic somatic diseases, injuries and pathologies of the eyes, infectious diseases, moisture collection of the anterior chamber of the eye and research pro-inflammatory and anti-inflammatory interleukins in the moisture of the anterior chamber of the eye.

The difference is that the study of moisture in the anterior chamber of the eye is carried out using a polymerase chain reaction with DNA extraction, the detection of topical cytokines and cytokines of their own, acquired as a result of hereditary diseases, determining their ratio and quantifying them.

The operation to remove the cataract is abdominal, occurs with the opening of the lining of the eye. The operation ejects a certain amount of topical cytokines. But, due to the presence of hereditary diseases, the moisture of the anterior chamber of the eye contains its own cytokines, as a result of hereditary diseases. Previously, the diagnosis was carried out by tear fluid, which washes the eye. And as practice has shown, previously used diagnostics gave a significant error. Lacrimal fluid is an indicator of inflammation of the surface of the eye, and not internal structures.

Previously, PCR (polymerase chain reaction) was not used to study cytokines. Using PCR, we investigated possible antigens that could be in moisture before surgery as a result of hereditary, acquired diseases, and chronic infections. That is, the infections that preceded the operation, they were already inside in the moisture of the anterior chamber, they served as the unfavorable background that worked when opening the anterior chamber during surgery.

DNA is a carrier of information about cytokines, its (this acid) amount is measured according to the description of the application in pg / ml. The increased content in the moisture of the anterior chamber of the tumor necrosis factor in pg / ml was a marker of inflammation.

The claimed method examined 24 patients aged 38-55 years (average age 45.28 ± 0.21 years) who were hospitalized as planned in KOKOV from February 2016 to December 2016 for the surgical treatment of presenile eye cataract. In this group there were 14 men and 10 women.

The criteria for inclusion in the group were the presence in the patient of an uncomplicated presenile cataract of one eye; the absence of acute infectious diseases, as well as chronic hepatitis C, hepatitis B, syphilis and HIV infection; patient age from 38 to 55 years; patient consent to participate in a research study.

Upon admission of patients to the hospital, an anamnesis was drawn up according to the developed questionnaire, which took into account hereditary factors, the presence of chronic somatic diseases, eye injuries, previously undergone surgical treatment of eye cataracts or other eye pathologies, as well as infectious acute and chronic diseases (hepatitis, tuberculosis, syphilis, HIV). When examined by an ophthalmologist, intraocular pressure and visual acuity were taken into account.

During surgical treatment of eye cataracts, all patients underwent moisture collection of the anterior chamber to study cytokine concentrations in it and conduct PCR diagnostics for herpetic viruses.

In the postoperative period, complications from the internal organs, eyes and transplanted lens were evaluated, as well as the degree of increase in visual acuity. To assess postoperative complications, a point system was used (presence of complications - 1 point, absence - About points).

The study of interleukins and in the moisture of the anterior chamber. The concentration of interleukin 1 (IL-1b), the receptor antagonist of interleukin 1 (IL-IRa), interleukin 4 (IL-4), tumor necrosis factor alpha (TNF-a) and interferon alpha (INF-a) in the blood and in the anterior the eye chambers were examined by enzyme-linked immunosorbent assay on commercial kits of LLC Tsitokin in St. Petersburg in accordance with the attached instructions. Regarding the concentration of cytokines in secrets in the literature, the question arose repeatedly about the interpretation of the concentrations of one or another analyte. One of the proposed methods for balancing concentrations is associated with the conversion of the amount of analyte per gram of protein contained in the test secret. Considering the possibility of different dilutions of moisture in the anterior chamber of the eye, studies were conducted in each sample of the total protein by the colorimetric method using the pyrogallol red dye. Received a concentration of total protein of 0.5 g / l in each sample, the value was constant. Based on the fact that the corresponding conversion factor for cytokine concentrations per gram of protein also turned out to be constant, this method of alignment was abandoned and subsequently, the concentration of cytokines obtained in ELISA was analyzed in pg / ml.

All patients who took moisture from the anterior chamber of the eye were diagnosed with PCR for the presence of genomes of the herpes simplex virus type 1 and 2 (HSV), as well as cytomegalovirus (CMV), in the moisture of the anterior chamber of the eye. Isolation of DNA from the moisture of the anterior chamber of the eye and PCR itself was carried out on commercial sets of NPF DNA Diagnostics, Moscow, according to the attached instructions. We used kits for real-time PCR on a real-time amplifier DT-322 NPF DNA Technology, Moscow.

The study of the concentration of cytokines and PCR diagnostics was performed on the basis of LLC Modern Medical Technologies (chief doctor - MD, associate professor Shabaldina Elena Viktorovna).

Statistical processing of the obtained data was carried out using the software package MS Excel 2007, "Statistica for Windows v. 7.0" and "SPSS v. 22.0 for Windows. " For each quantitative characteristic, the nature of the distribution was studied. For this, tests of Kolmogorov-Smirnov and Shapiro-Wilk were used. The paper used descriptive statistics approaches. In the normal distribution of indicators, descriptive statistics were used, presented in the form of the arithmetic mean value (M) and standard error of the mean (m). In the absence of a normal distribution, descriptive statistics are presented in the form of a median (Me), as well as 25 and 75 quartiles (Q25 - UQ and Q75 - LQ). The significance of differences between normally distributed indicators in the compared groups was determined using Student's t-test (t-test) at a level of statistical significance less than 0.05. When comparing nonparametric indicators, the Wilcoxon test was used. The Mann-Whitney test was used to determine the significance of differences for pairwise comparisons. The strength of the connection between the studied characters in the case of a parametric distribution of the characteristics was determined using the Pearson correlation coefficients, and with a nonparametric distribution, Spearman.

The reliability of changes in indicators was considered with an error of less than 5%, which corresponds to biomedical research (Lakin V.M., 1991 Biometry).

The study showed that in the examined patients, all the studied interleukins were found in the moisture of the anterior chamber of the eye. The most pronounced concentration was in IL-IRa in the moisture of the anterior chamber (100.91 ± 0.48 pg / ml), as well as for IL-1b (10.56 + 0.22 pg / ml) and IL-4 (2, 67 ± 0.11 pg / ml). The minimum concentrations in the studied biological fluid were in INF-a and TNF-a. Benchmarks of interleukin moisture in the anterior chamber of the eye are taken from the work of V.P. Yerichev (Yerichev V.P., Petrov S.Yu., Saturday AM, Volzhanin A.V., Germanova V.N., Karlova E.V. Role of cytokines in the pathogenesis of eye diseases. National Journal of Glaucoma 2017, T. 16, No. 1, pp. 85-99 Russian journal of glaucoma 2017, Vol.16, No. 1, pp. 85-99), where for TNF-a the normal range of 0.8-3.1 pg / ml is indicated. These normative indicators were taken for the developed method for the diagnosis of postoperative complications in the surgical treatment of eye cataracts.

A positive reliable correlation was shown between the severity of postoperative complications (scored) and the level of TNF-α in the moisture of the anterior chamber of the eye (pg / ml). It should be noted that the level of TNF-a in the moisture of the anterior chamber of the eye had a positive correlation with the reaction of erythrocyte sedimentation (ROE) according to the general blood test. These data indicate the involvement of both local and systemic inflammation in TNF-a production and its role in the formation of postoperative complications in the surgical treatment of eye cataracts.

Thus, in a preferred embodiment of the method, it is technically not a complicated procedure for collecting moisture from the anterior chamber of the eye for examination, a time-consuming method that does not require additional control series of studies.

In another preferred embodiment, pathogenetically substantiated studies of the local secretion of a set of pro-inflammatory and anti-inflammatory interleukins that provide immunoregulatory potential in the anterior chamber of the eye are carried out.

In a further preferred embodiment, the research method is screening, but specific enough for the diagnosis of postoperative complications in the surgical treatment of eye cataracts.

Examples

Example 1. Patient G., 47 years old (medical card GBUZ KO KOKOV No. 2317), turned to an ophthalmologist with complaints of decreased vision of the right eye. On examination, presynil cataract of the left eye was revealed. To diagnose possible postoperative complications, it was recommended that the anterior chamber moisture be taken during surgical treatment and the study of pro-inflammatory, anti-inflammatory interleukins in it. 100 ml of moisture was taken during the operation; no complications were obtained.

A study of interleukin moisture in the anterior chamber of the eye revealed an increase in the level of tumor necrosis factor alpha to 13.34 pg / ml, which was more than 2 times the normal values of this analyte. PCR moisture testing for CMV and HSV viruses showed the expression of CMV virus.

Upon examination of the patient after 6 days, a fibrin film on the artificial lens was revealed.

Based on the results obtained, it was found that the development of postoperative complications in the form of a fibrin film on an artificial lens is associated with the activation of virus-induced inflammation, the marker of which was an increased level of TNF-a in the anterior chamber of the eye and CMV expression.

Example 2. Patient C, 52 years old (medical card GBUZ KO KOKOV No. 2317) turned to an ophthalmologist with complaints of decreased vision. On examination, presynil cataract of the right eye was revealed. To diagnose possible postoperative complications, it was recommended that the anterior chamber moisture be taken during surgical treatment and the study of pro-inflammatory, anti-inflammatory interleukins in it. Moisture collection in a volume of 10 μl was performed during the operation; no complications were obtained.

A study of interleukin moisture in the anterior chamber of the eye revealed a normal level of tumor necrosis factor alpha 2.6 pg / ml. A study of the moisture of the anterior chamber of the eye by PCR for CMV and HSV viruses showed a lack of expression of viral genomes. When examining the patient after 2 weeks, vision was restored, the artificial lens was transparent.

Based on the results obtained, it was found that the absence of postoperative is associated with normal levels of TNF-a in the moisture of the anterior chamber of the eye by suppression of CMV expression.

The essence of the differences of the invention is as follows. In a method for determining the levels of a specific set of pro-inflammatory and anti-inflammatory interleukins in the moisture of the anterior chamber of the eye: interleukin-1-beta, interleukin-4, receptor antagonist interleukin-1, interferon-alpha, tumor necrosis factor-alpha - in patients with presinal eye cataract during surgical treatment. An increase in the concentration in the moisture of the anterior chamber of the eye: tumor necrosis factor - alpha more than 2 times, in relation to normal values, indicates a risk of postoperative complications.

The method was tested on the basis of GBUZ KO KOKOV in 24 patients with presenile cataract of the eye. Complications in the collection of moisture in the anterior chamber of the eye were not obtained. The examination confirmed the importance of increasing TNF-a in the moisture of the anterior chamber of the eye in the diagnosis of postoperative complications.

Claims (1)

A method for determining the risk of developing postoperative complications in the surgical treatment of presynil cataract of the eye, including taking an anamnesis taking into account hereditary factors, the presence of chronic somatic diseases, injuries and pathologies of the eyes, infectious diseases, taking moisture from the anterior chamber of the eye, characterized in that during surgical treatment moisture collection of the anterior chamber of the eye, in the moisture of the anterior chamber of the eye determine the level of tumor necrosis factor alpha (TNF-α) by enzyme-linked immunosorbent assay lysis and with an increase in the level of TNF-α by more than 2 times compared with its normal levels of 0.8-3.1 pg / ml, the risk of developing postoperative complications is revealed.