RU2674237C1 - Method of surgical treatment of endothelial-epithelial dystrophy of cornea - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, specifically to ophthalmology. For the surgical treatment of endothelial-epithelial dystrophy (EED) of the cornea, a local suspension of autologous mononuclear blood leukocytes is administered locally. Preliminarily, 25 G needle, put on 1.0 ml syringe with sterile air, is inserted into the posterior third of the corneal stroma from the top, gradually advanced, the syringe plunger is pressed, 1.0 ml of sterile air is injected, descemet membrane is locally peeled. Then, 0.5–0.6 ml of autologous mononuclear blood leukocyte suspension, separated by fractionation on a density gradient, is injected once into the resulting zone.EFFECT: method allows to reduce the risk of the development of intra- and postoperative complications and to increase the clinical and functional efficacy of the treatment of corneal EED.1 cl, 1 ex, 2 tbl, 8 dwg

Description

The invention relates to medicine, in particular to ophthalmology, and can be used to treat endothelial-epithelial dystrophy (EED) of the cornea.

Corneal EED is currently regarded as a chronic inflammatory-dystrophic disease of the cornea, accompanied by severe pain and leading to a sharp decrease in vision [14]. The pathology is based on the development of chronic edema of the corneal tissue, accompanied by a significant decrease in visual acuity and severe pain [14, 17]. Many aspects of the pathogenesis of this disease remain poorly understood, however, the leading pathogenetic factor is the progressive and irreversible decrease in the number of corneal endothelial cells and, as a result, their loss of barrier and pumping functions [7, 13, 14, 17].

In the future, there is an increase in hydration of the stroma of the cornea, a decrease in the number of keratocytes and glycosaminoglycans of the stroma, which, together, leads to the development of progressive edema of the stroma, and the appearance of edema of the epithelial layer of the cornea. Due to seepage of aqueous humor through the corneal tissue in the surface layer of the cornea, epithelial vesicles filled with fluid are formed - bullae, spontaneous opening of which is accompanied by the development of a pain syndrome of varying severity, expressed by photophobia and lacrimation.

Keratoplasty in various modifications (intralamellar keratoplasty, posterior layer-by-layer keratoplasty or endothelial keratoplasty, intralamellar implantation of keratoprostheses) with replacement of the endothelial layer, which allows to achieve 15, 15 ].

However, as with any surgical intervention, in the postoperative period, the risk of developing purulent complications and transplant rejection is high [16, 18, 19].

The use of cell therapy, in our opinion, is an alternative to radical surgery [1]. In clinical ophthalmology over the past decades, in order to correct reparative processes in the tissues of the eyeball for various pathologies of the organ of vision, cytokines are actively used - immunopeptides produced by cells of a lymphoid and non-lymphoid nature and participating in inflammatory-reparative reactions [5, 6].

Cytokines are a single independent system of regulation of body functions, providing the development of protective reactions and maintaining homeostasis during the introduction of pathogens and violation of tissue integrity [2, 3, 6]. At the local level, cytokines are responsible for all successive stages of the development of an adequate response to damage and for the restoration of the structure of damaged tissue.

Blood mononuclear cells, which are the main source of these biologically active substances, initially have a high secretory potential and the ability to adapt to any microenvironment [2, 3, 6].

Closest to the proposed is a method of treating corneal EDF, developed in an in vivo experiment, namely, that a freshly isolated suspension of autologous blood mononuclear cells is introduced into the anterior chamber of the diseased eye of an experimental animal and layered on the inner surface of the cornea (RF patent No. 2601317).

The method is as follows. Autologous mononuclear cells from the blood of an experimental animal are isolated by density gradient fractionation on a ficoll-verographin separating solution. Blood taken in an amount of 4.0-5.0 ml is placed in a sterile tube containing 1.0 ml of a heparin solution. Heparinized blood is diluted 2 times with isotonic sodium chloride solution. The resulting suspension is layered on 3.0 ml of a mixture of ficoll-verographin. The tube is centrifuged at room temperature for 15 min at 800 g (2000 rpm). After centrifugation, an interphase layer containing mononuclear cells located between the plasma and the gradient is collected by a Pasteur pipette. 1.0 ml of isotonic sodium chloride solution is added, and the resulting suspension is again centrifuged for 7 min at 400 g (1500 rpm). The purity of the mononuclear cells obtained on the ficoll-verographin gradient is up to 96-98%. The viability of the cellular material is evaluated in the test with trypan blue and is 97-98%. In the operating room after anesthesia and treatment of the surgical field in compliance with the rules of aseptic and antiseptic, an experimental intervention is performed in experimental animals with a pre-induced corneal hemorrhaging. In the cornea near the limb, two paracentesis are performed, at 7 and 11 hours, in one of which an irrigation system is installed with sterile air supplied to the anterior chamber under a pressure of up to 0.2 atm. Through a second paracentesis, a cell suspension is injected into the anterior chamber at the border between the inner surface of the cornea and the air bubble using a syringe and layered on the posterior surface of the cornea. The irrigation system is removed, the edges of the paracentesis are hydrated.

The disadvantages of this treatment method are the need to perform manipulations in the anterior chamber, as well as the high probability of insufficient adhesion of mononuclear cells to the inner surface of the cornea, which, on the one hand, increases the risk of developing intra- and postoperative complications (iridocyclitis, damage to the lens capsule, hyphema), and on the other - significantly reduces the effectiveness of the treatment of the disease.

A new technical task is the modification of this method of surgical treatment of corneal EDF associated with the creation of a depot of autologous blood mononuclear cells in the posterior third of the stroma of the cornea in close proximity to the pathological altered endothelial layer.

To solve this problem, a new method has been developed for the surgical treatment of corneal EED, including pneumodescemetoablation followed by the introduction of a suspension of autologous blood mononuclear cells into the posterior third of the cornea stroma.

The method is as follows. Autologous mononuclear cells from the patient’s blood are isolated by density gradient fractionation on a ficoll-verographin separation solution. The selection of autologous mononuclear cells is carried out as follows: preliminary, in the conditions of the treatment room, observing the conditions of aseptic and antiseptic, blood is taken from the patient's ulnar vein in an amount of 4.0-5.0 ml, the blood is placed in a sterile tube containing 1.0 ml of heparin solution. Heparinized blood is diluted 2 times with isotonic sodium chloride solution. The resulting suspension is layered on 3.0 ml of a mixture of ficoll-verographin (12 parts of 9% ficoll and 5 parts of 33.9% verographin with a density of 1.067-1.077 g / ml). The ratio of the volume of the gradient and the shared suspension is 1: 3-1: 4.

The tube is centrifuged at room temperature for 15 min at 800 g (2000 rpm). After centrifugation, an interphase layer containing mononuclear cells located between the plasma and the gradient is collected with a Pasteur pipette. Add 1.0 ml of isotonic sodium chloride solution and the resulting suspension is again centrifuged for 7 min at 400 g (1500 rpm) to wash the cell suspension.

The purity of mononuclear cells obtained on a ficoll-verographin gradient is 96-98%. The viability of the cellular material is evaluated in the trypan blue test in the following way: first, a 0.1% eosin solution is prepared on the basis of Ringer's solution. Next, based on distilled water, a 0.1% trypan blue solution is prepared. After that, 1-2 drops of a fresh mixture of solutions of previously prepared dyes taken in equal volumes are added to a drop of cell suspension. The resulting mixture is placed in the camera Goryaeva. When counting cells, the percentage of stained (dead) elements should be 1.5-2%, which would not exceed the permissible (no more than 3%) amount. The viability of the cellular material in the trypan blue test was 97-98%.

Under operating conditions, after anesthesia, with the help of installations, 1-2 drops of a 0.4% solution of inocaine and a subtenoneal injection of 2.0 ml of a 2% solution of lidocaine, a 25G gauge needle put on a 1.0 ml syringe is inserted prelimbally from above into the corneal stroma at the level of the posterior third with sterile air, and gently push it into the paracentral sections. Then, by pressing on the piston of the syringe, sterile air is introduced, exfoliating the descemet membrane. Next, the obtained cell suspension is introduced into the descemetoablation zone in a volume of up to 0.5-0.6 ml. Antibiotic solution is administered subconjunctively, a monocular dressing is applied for 1 day. In the postoperative period, antibiotic installations are carried out according to the standard scheme.

Clinical studies were conducted on the basis of the ophthalmology clinic of the FSBEI HE Siberian State Medical University of the Ministry of Health of Russia (Tomsk) among 20 patients (11 women and 9 men) with corneal EED, which, depending on the treatment method, were divided into two groups: the main group and the comparison.

Patients of the main group (11 patients, 11 eyes) were treated with the proposed method.

The comparison group is represented by 9 patients (9 eyes) who received conservative treatment (metabolic agents, corticosteroids, keratoprotectors).

Patients of both groups are comparable in sex, age and severity of corneal lesions.

All patients underwent a general phthalmological examination: visometry, perimetry, biomicroscopy of the anterior segment of the eye, reverse binocular ophthalmoscopy, transpalpebral measurement of intraocular pressure, pachymetry, photographic recording.

The criterion of treatment effectiveness was considered to be: elimination of the corneal syndrome, reduction of corneal edema, increased visual acuity.

The total follow-up period was 6 months.

A comparative analysis revealed the significant effectiveness of the new method of surgical treatment of corneal EDF.

So, for example, 5 days after the surgical treatment, patients of the main group showed a positive dynamics of the biomicroscopic picture in the form of a decrease in edema of all layers of the cornea, smoothing of the folds of the descemet sheath.

A gradual increase in the transparency of the cornea in patients of the main group was observed by the 10th day, which is consistent with the data of pachymetry and visometry (table. 1, 2). In patients of the comparison group, by the 10th day from the start of treatment, there was only a slight decrease in corneal edema.

According to the results of a clinical study, the application of the proposed method of treatment has improved the visual function of patients in the main group.

At the time of hospitalization, visual acuity in patients of both groups was 0.04 (Table 1). 5 days after surgery, this indicator in patients of the main group increased by 75% from the initial level, while in the comparison group - by 25%. After 10 days, visual acuity in patients of the main group increased by 42.8% of the achieved value, and in the comparison group - by 20%. After 1 month after the treatment, visual acuity in patients of the main group was 0.2 ± 0.01, after 3 months. - this indicator reached a maximum level of 0.3 ± 0.01 and remained stable throughout the observation period (6 months). In patients of the comparison group, the best indicator of visual acuity was achieved after 1 month. after a course of conservative treatment and amounted to 0.07 ± 0.01, after 3 months. this indicator remained the same, and by the end of the observation (after 6 months), it decreased by 14.2% of the achieved level.

According to the results of pachymetry, the restoration of the thickness of the cornea against the background of the treatment was more significant in the main group (table. 2). So, for example, on the 5th day after surgical treatment, the pachymetry indicator in patients of the main group decreased by 3% from the initial level, while in patients in the comparison group - by 1.2%. 10 days after the treatment, this indicator in patients of the main group decreased by another 5.4% of the achieved level, in patients of the comparison group - by 0.62%. After 1 month after the treatment, in patients of the main group, the pachymetry indicator decreased by 13.9% from the initial level, while in the comparison group - by 2.1%. After 3 months the corneal thickness in patients of the main group decreased by 17.6% from the initial level and remained stable throughout the observation period (6 months), insignificant and unstable dynamics of keratopachymetry indicator was noted in patients of the comparison group - a decrease of 1.9% and 1, 5% after 3 and 6 months. respectively.

The average period of inpatient treatment of patients of the main group was 9 ± 1 days, patients of the comparison group - 12 ± 2 days (p <0.05).

Clinical example for the implementation of the method

Patient M., 76 years old, complained of lacrimation, redness, decreased vision, “fog” in front of her right eye.

Anamnesis of the disease: considers himself ill for about 3 months. after surgical treatment of cataracts of the right eye - phacoemulsification of cataracts with implantation of an intraocular lens (IOL). A month after the treatment, a diagnosis was made of the EED of the cornea of the right eye, about which treatment was carried out twice - crosslinking of collagen of the cornea of the right eye, however, did not notice any improvement against the background of the treatment. Installs the aroma in the right eye 2 times a day.

Objective inspection:

Visus OD = 0.04 n / a

OS = 0.8 n / a

Biomicroscopy OD: easy injection of the bulbar conjunctiva, normal palpation, cornea: diffuse epithelial edema, uneven stromal edema, folds of the descemet sheath, diffuse endothelial edema, 2 small bullae up to 2 mm in diameter are detected in the lower outer quadrant of the cornea. The optical section of the cornea is unevenly thickened, the anterior chamber is of medium depth, the moisture is transparent, the pupil is of medium width, the reaction to light is live, artifact, IOL posterior chamber, centered. Ophthalmoscopy is difficult due to clouding of the cornea (Fig. 1)

OS biomicroscopy: the eye is calm, palpation normal tone, the cornea is transparent, artifact.

Diagnosis: endothelial-epithelial dystrophy of the cornea of the right eye. Artifakia of both eyes.

The treatment was carried out: pneumodescemetoablation of the right eye with the subsequent introduction into the posterior third of the stroma of the cornea of a suspension of autologous blood mononuclear cells. In the postoperative period - antibiotic installations according to the standard scheme.

On the 2nd day after surgical treatment (3rd day of hospital stay):

Vis OD = 0.03 n / a

Status localis OD - the eye is moderately irritated, slight photophobia is observed, palpation is normal, the cornea is rough, the stromal edema gradually decreases, individual thin folds of the descemet shell, endothelial edema persist, the zone of descemetoablation, an air bubble in the anterior chamber, the moisture is clear, the pupil is transparent medium width, live light response, artifact, IOL posterior chamber, centered.

On the 5th day after the treatment:

Vis OD = 0.05 n / a

Status localis OD - the eye is calm, photophobia is absent, palpation is normal, the cornea: the epithelium is calm, stromal edema gradually decreases, descemet sheath adheres throughout, less pronounced endothelial edema, the air bubble in the anterior chamber gradually dissolves, the moisture is clear, the pupil is of medium width , the reaction to light is alive.

On the 7th day after treatment:

Vis OD = 0.07 n / a

Status localis OD - the eye is calm, photophobia is normal, palpation is normal, the cornea: the epithelium is calm, the stromal edema has decreased, single folds of the descemet sheath, the endothelial edema has decreased significantly, the anterior chamber is of medium depth, the moisture is clear, the pupil is of medium width, the response to live light, artifact, IOL posterior chamber, centered.

On the 10th day after treatment:

Vis OD = 0.09 n / a

Status localis OD - the eye is calm, photophobia is normal, palpation is normal, the cornea: the epithelium is calm, the stromal edema has significantly decreased, single thin folds of the descemet shell, moderate endothelial edema, the anterior chamber is of medium depth, the moisture is transparent, the pupil is of medium width, the response to light is live , artifakia, posterior chamber IOL, centered (Fig. 2).

The patient was discharged from the ophthalmological clinic of the Siberian State Medical University in satisfactory condition under the supervision of an ophthalmologist at the place of residence. The total length of hospital stay was 11 days.

After 1 month:

Vis OD = 0.1 n / a

Status localis OD - the eye is calm, photophobia is normal, palpation is normal, the cornea: epithelium is calm, slight swelling of the individual layers of the stroma, single thin folds of the descemet shell, slight endothelial edema, anterior chamber of medium depth, moisture transparent, pupil of medium width, reaction to light live, artifact, IOL posterior chamber, centered (Fig. 3)

After 6 months:

Vis OD = 0.2 n / a

Status localis OD - calm eye, palpation normal tone, cornea: the epithelium is calm, slight and uneven stromal edema, single thin folds of the descemet shell, the endothelium is calm, the anterior chamber is of medium depth, the moisture is clear, the pupil is of medium width, the reaction to live light, artifact, The IOL is posterior, centered (Fig. 4). The positive dynamics of the clinical picture is confirmed by the data of visometry and pachymetry: at the time of hospitalization, visual acuity was 0.04 n / k, the thickness of the cornea was 810 μm (Fig. 5), 10 days after treatment, visual acuity increased to 0.09 n / k , the pachymetry rate was 685 μm (Fig. 6), after 1 month. - visual acuity increased to 0.1, the pachymetry index decreased to 665 μm (Fig. 7), by the end of the observation period the maximum visual acuity was achieved - 0.2 n / k, the corneal thickness decreased to 585 μm (Fig. 8).

Modern ideas about the biological role of mononuclear cells are based on their functional heterogeneity, including phagocytic, regulatory, and effector properties [4]. However, in recent years, along with the extensively studied functions of migration and chemotaxis, endocytosis and intracellular digestion, the secretory function of mononuclear phagocytes has attracted great attention of researchers. Teaching I.I. Mechnikov about macrophage enzymes - cytases grew into the concept of monokines - the secretion products of monocytes / macrophages, which together with lymphokines are called "cytokines". The modern interpretation of mononuclear cells as secreting cells is based on their ability to produce and isolate more than 75 different types of molecules [10]. By means of cytokines, mononuclear cells affect the migration and proliferation of granulocytes and endothelial cells, induce chemotaxis and fibroblast proliferation, and affect the production of collagen [8].

Mononuclear cells are a source of numerous bioregulators - cytokines involved in the formation and regulation of the body's defense reactions [11]. Cytokines represent a complex network of immuno-mediators that regulate and determine the nature of the immune response. The regulatory role of cytokines in the body is limited not only by the immune response: they take part in the regeneration processes to restore damaged tissues [9].

The exogenous introduction of autologous peripheral blood mononuclear cells into the stroma of the cornea producing the above-described cytokines in case of corneal EED allows to create a locally high concentration of cells of this population in the destruction center and to target the pathological process.

Thus, the results of a clinical study indicate the significant effectiveness of the use of a new surgical method for the treatment of corneal EED, including pneumodescemetoablation with intrastromal administration of a suspension of autologous blood mononuclear cells.

Method for surgical treatment of endothelial-epithelial corneal dystrophy

Note: * - level of significance of differences compared with the initial p <0.05; M is a sample mean value; m is the error of the mean.

Note: * - level of significance of differences compared with the initial p <0.05; M is a sample mean value; m is the error of the mean.

application

Table 1

The dynamics of visual acuity in patients with corneal EED, depending on the method of treatment, M +/- m

table 2

The dynamics of the pachymetry indicator in patients with corneal EED, depending on the treatment method, M +/- m

FIG. one

Patient M., 76 years old. Diagnosis: endothelial-epithelial dystrophy of the cornea of the right eye. Biomicroscopy of the anterior segment of the eye before treatment.

FIG. 2

Patient M., 76 years old. Diagnosis: endothelial-epithelial dystrophy of the cornea of the right eye. Biomicroscopy of the anterior segment of the eye on the 10th day after the treatment.

FIG. 3

Patient M., 76 years old. Diagnosis: endothelial-epithelial dystrophy of the cornea of the right eye. Biomicroscopy of the anterior segment of the eye 1 month after the treatment.

FIG. four

Patient M., 76 years old. Diagnosis: endothelial-epithelial dystrophy of the cornea of the right eye. Biomicroscopy of the anterior segment of the eye 6 months after the treatment.

FIG. 5

Patient M., 76 years old. Diagnosis: endothelial-epithelial dystrophy of the cornea of the right eye. Keratopachymetry of the eye before treatment.

FIG. 6

Patient M, 76 years old. Diagnosis: endothelial-epithelial dystrophy of the cornea of the right eye. Keratopachymetry of the eye on the 10th day after the treatment.

FIG. 7

Patient M., 76 years old. Diagnosis: endothelial-epithelial dystrophy of the cornea of the right eye. Keratopachymetry of the eye after 1 month. after the treatment.

FIG. 8

Patient M., 76 years old. Diagnosis: endothelial-epithelial dystrophy of the cornea of the right eye. Keratopachymetry of the eye after 6 months. after the treatment.

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Claims (1)

A method of surgical treatment of endothelial-epithelial corneal dystrophy, which consists in the local administration of a suspension of autologous mononuclear leukocytes of blood, characterized in that a 25G needle inserted in a 1.0 ml syringe with sterile air is gradually inserted into the posterior third of the corneal stroma, gradually moving the needle, press on the piston of the syringe, inject 1.0 ml of sterile air, locally exfoliating the Descemet shell, then 0.5-0.6 ml of a suspension of autologous mononuclear lake is injected into the obtained zone once blood cells isolated by fractionation on a density gradient.

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