RU2601317C1 - Method of treating endothelial epithelial corneal dystrophy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to ophthalmology, and aims at treating endothelial epithelial corneal dystrophy (EECD). Method involves introducing a suspension of autologous blood mononuclear leukocytes into the posterior chamber of the eye until it replaces the aqueous humor in the anterior chamber by performing paracentesis near the corneal limbus. For this purpose, paracentesis is performed twice at 7 and 11 o'clock with the puncture size of 1-2 mm, wherein an irrigation system in the form of a cannula used to supply sterile air at the pressure of up to 0.2 atm into the anterior chamber is installed into one of the punctures; and 96-98 % cell suspension of autologous blood mononuclear leukocytes is syringed once through the second paracentesis into the anterior chamber between the inner surface of the cornea and air bubble. Then the irrigation system is removed and edges of paracenteses are hydrated. Suspension of autologous blood mononuclear leukocytes is obtained by extraction using the density gradient fractionation method and separating solution of Ficoll-verografin.

EFFECT: use of the invention enables to reduce the length of treatment and complications associated with excessive involvement of ocular tissues, corneal oedema.

1 cl, 10 dwg

Description

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

Currently, various methods have been developed for the surgical treatment of EED of the cornea and early bullous keratopathy: perforated keratoplasty with replacement of the cornea with an allograft (Kopaeva V.G. Subtotal perforated keratoplasty for corneal dystrophy. Optical reconstructive surgery and alloplasty in ophthalmology, M., 1976). keratoplasty with transplantation of the posterior layers of the cornea (Volkov V.V. On the development of a surgical method for the treatment of entothelial-epithelial dystrophy of the cornea. First Congress of Ophthalmologists of the Caucasus: Abstracts of Tbilisi: 1976), intermellar transplantation of the posterior lens capsule (USSR author's certificate N810235, class A61F 9/007, 1981), layered keratoplasty using a gelatin film (Dronov M.M., Karanov K.S. , Bobryr AB A method for the treatment of bullous keratopathy (Russian Patent No. 2082364, priority dated 06/27/1997).

However, the above methods have a number of significant drawbacks: the technical complexity of the operation, the high incidence of complications, the likelihood of an immune conflict with subsequent rejection of donor material, and relapse of the disease.

All of the above served as the basis for the creation of a new method for the surgical treatment of corneal EDF and early bullous keratopathy by intracameral administration of autologous blood mononuclear cells.

Closest to the proposed one is a method of treating corneal edema and other manifestations of early bullous keratopathy by means of intracameral local rapid autocytokine therapy (LEACCT) (Kasparov A.A., Kasparova E.A., Pavlyuk A.S. Russian Patent No. 2357743, priority from 10/13/2009).

According to this method, autologous mononuclear blood cells of a patient activated by a half-dan are introduced into the anterior chamber of a patient’s eye. Blood sampling from a patient is carried out in compliance with the rules of asepsis and antiseptics in a treatment room. Blood is taken from the patient's ulnar vein in an amount of 5.0 ml. The contents of 2 half-vial vials are dissolved in 2.0 ml of water for injection and the aqueous half-vial solution is transferred to a sterile tube with 5.0 ml of blood. The contents of the tube are thoroughly mixed by inverting. Next, the test tube is placed in a thermostat for 2-4 hours. After incubation, the clotted tube is centrifuged for 5-10 minutes at 500-1000 rpm. Then, at the border of serum and clot of coagulated blood, a cell suspension is collected with a pipette. In the operating room, after processing the surgical field and retrobulbar anesthesia of the patient’s patient’s eye, a corneal paracentesis is performed near the limb with a blade. A suspension of autologous blood mononuclear cells in an amount of 1.0-2.0 ml is gradually infused into the anterior chamber until it completely replaces the moisture of the anterior chamber. In the area of corneal paracentesis, a supramid suture is applied. Immediately after surgery, the patient should take the position face down and lie for 1 hour, so that the introduced cells are in contact with the corneal endothelium. LEACCT sessions are carried out with an interval of 3-6 days in an amount of 1 to 4 times.

The disadvantages of this method are: the duration and complexity of obtaining autologous blood mononuclear cells activated by half-don, the need for repetition of cell therapy with an interval of 3-7 days in an amount up to 4 times.

A new technical result is a reduction in treatment time and complications associated with excessive trauma to eye tissue, corneal edema.

To achieve a new technical result in a method for the treatment of endothelial-epithelial dystrophy of the cornea, comprising introducing a suspension of autologous blood mononuclear cells into the inner chamber of the eye through paracentesis performed near the limb until it completely replaces the moisture in the anterior chamber, characterized in that two paracentesis with a size of 1.2 mm are performed , at 7 and 11 hours, in one of which an irrigation system is installed in the form of a cannula for supplying sterile air to the front chamber under a pressure of up to 0.2 atm, while through a second paracent h using a syringe into the anterior chamber at the border between the inner surface of the cornea and the air bubble, a 96-98% autologous blood mononuclear cell suspension, isolated by fractionation in a density gradient on a ficoll-verographin separation solution, is injected once, after which the irrigation system is removed, the edges of the paracentesis are hydrated .

Thanks to a similar method of using autologous blood mononuclear cells, edema of the corneal tissue is significantly reduced, which helps to restore its transparency and improve the visual functions reduced due to the disease.

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 separation 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. Samples are centrifuged at room temperature for 15 minutes at 800 g (2000 rpm). After centrifugation, an interphase layer containing mononuclear cells and located between the plasma and the gradient is taken with a Pasteur pipette. 1.0 ml of isotonic sodium chloride solution was added, and the resulting suspension was again centrifuged for 7 min at 400 g (1550 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 a trypan blue test in the following way: first, a 0.1% eosin solution is prepared on the basis of Ringer's solution. Then, 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 is 97-98%.

In the operating room after anesthesia and treatment of the surgical field in compliance with the rules of aseptic and antiseptic, experimental animals with pre-simulated EED, perform surgery. Two 1.2 mm paracentesis is performed in the cornea at 7 and 11 hours, in one of which an irrigation system is installed in the form of a cannula with sterile air supply with pressure up to 0.2 atm to the anterior chamber (Fig. 1). Using a syringe through a second paracentesis, a cell suspension is layered on the endothelium into the anterior chamber at the border between the inner surface of the cornea and the air bubble (Fig. 2). The irrigation system is removed, the edges of the paracentesis are hydrated.

The method was tested on 16 rats of the breed Wistar weighing 200-250 g

Review of the experimental material: the total duration of the experiment is 21 days. At stage I, each animal in the operating room was preliminarily modeled by EED by mechanical damage and removal of the corneal endothelium of one of the eyes. In stage II, 2 weeks after the development of the pathological process in the cornea, intracameral administration of autologous blood mononuclear cells was carried out as described.

During the experiment, an external examination, photographic recording, histological examination was performed. The material was taken on days 3, 7, 14, 21 from the start of the experiment.

To assess morphological changes, enucleated eyes were fixed in 12% neutral formalin for 24 hours. Then the objects, after 24-hour washing in running water, were subjected to dehydration in alcohols of an ascending concentration, enlightened in O-xylene and embedded in paraffin. After preparation of sections 4-6 μm thick, the preparations were stained with hematoxylin and eosin, as well as hematoxylin and picrofuxin according to Van Gieson.

During morphological studies revealed the following.

3 days after the intracameral administration of a suspension of autologous blood mononuclear cells, the anterior corneal epithelium is preserved and is represented by a layer of stratified squamous non-keratinized epithelium. The anterior border membrane was not clearly defined.

Corneal intrinsic matter is represented by loosely arranged collagen fibers, which in places had an increased convoluted course (Fig. 3). The posterior border membrane was visualized well, but was stained unevenly. The posterior endothelium was absent (Fig. 4).

7 days after the introduction of the cell suspension, the anterior corneal epithelium retained its usual structure, the anterior border membrane was not clearly visualized.

Corneal intrinsic matter was characterized by severe edema, the most common in the posterior third. Collagen fibers of their own substance had an increased tortuous course (Fig. 5).

The posterior border membrane is homogeneous; its boundaries were not clearly defined. When stained with picrofuchsin, the membrane was characterized by a slight violation of tinctorial properties.

From the endothelium, pronounced proliferation of process cells was detected (Fig. 6).

14 days after the introduction of a population of autologous mononuclear cells, the anterior epithelium is not changed, the number of layers is preserved. The anterior border membrane was not clearly visualized, not quite clearly.

The cornea's own substance had a compact structure. Collagen fibers in the posterior third of the main substance, close to the site of exposure, are slightly spaced by the edematous fluid (Fig. 7). When stained with picrofuxin, tinctorial disturbances were not detected.

The posterior border membrane is homogeneous, well visualized throughout. The endothelium is represented by a single layer of flat process cells (Fig. 8).

21 days after the intracameral administration of the cell suspension, the anterior front corneal epithelium retained its usual structure. The anterior border membrane was not clearly defined.

The corneal intrinsic substance is compact, its tinctorial properties are not disturbed (Fig. 9).

The posterior border membrane is well visualized, homogeneous. The endothelium is represented by a single layer of cells (Fig. 10).

Modern ideas about the biological role of mononuclear cells are based on their functional heterogeneity, including phagocytic, regulatory, and effector properties (Zemskov V.M., Nikolaeva E.N., Rodionov S.V. Heterogeneity of mononuclear phagocytes // Successes of modern biology. - 1993. - T. 113, No. 3. - S. 336-349). 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 were 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 (Freidlin I.S. Defects of the cytokine network and principles of their correction // Immunology. - 1998. - No. 6. - P. 23-25). By means of cytokines, mononuclear cells affect the migration and proliferation of granulocytes, endothelial cells, induce chemotaxis and proliferation of fibroblasts, affect collagen production (Mayansky AN, Pikuza OI Clinical aspects of phagocytosis. - Kazan: "Magarif", 1993. - C . 192).

Mononuclear cells are a source of numerous bioregulators - cytokines involved in the formation and regulation of protective reactions of the body (Shubich MG, Avdeeva MG Mediator aspects of the inflammatory process // Arch. Pathology. - 1997. - T. 59, No. 2. - S. 3-8). 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 regeneration processes to repair damaged tissues (Simbirtsev A.S. Cytokines - a new system for regulating the body's defense reactions // Cytokines and inflammation. - 2002. - No. 1. . 9-16).

Local tissue destruction as a result of damage to the endothelial layer of the cornea leads to the synthesis and secretion by cells of the monocytic macrophage series of cytokines such as IL-6, tumor necrosis factor-α (TNF-α), transforming growth factor beta (TGF-beta). IL-6 is the main inducer of the final differentiation of macrophage cells on tissue damage. Transforming Growth Factor Beta (TGF-beta) is a protein that controls the proliferation and cell differentiation of macrophage cells. Tumor necrosis factor-α (TNF-α) affects not only the lipid metabolism and stimulation of IL-6, IL-8, but also the functioning of the endothelium - a single-layer layer of mesenchymal cells responsible for controlling the diffusion of water and ions in the corneal tissue.

The size of paracentesis 1.2 mm does not require the application of a corneal suture due to self-sealing of corneal tissue while reducing the invasiveness of surgical procedures. A cell suspension is obtained on a density gradient of a ficoll-verographin mixture, which helps to obtain 98% purity of the cell culture. Also, the treatment time is reduced due to a single injection of a suspension of mononuclear cells.

The exogenous introduction of autologous peripheral blood mononuclear cells into the anterior chamber producing the cytokines described above during endothelial-epithelial dystrophy of the cornea allows one to create a locally high concentration of cells of this population in the destruction focus and to target the pathological process, thereby promoting the proliferation of the damaged endothelial layer.

Thus, the results of an experimental study showed that the intracameral administration of autologous blood mononuclear cells with their subsequent layering on the inner surface of the cornea during EED and early bullous keratopathy contributes to the regeneration of the endothelial layer of the cornea.

Due to this, the disturbed function of the corneal endothelium is compensated and, as a result, the swelling of the corneal tissue is reduced and its transparency is restored.

Figure 1 - installation of the irrigation system in parasentesis with sterile air supply:

1 - irrigation system;

2 - air bubble.

Figure 2 - the introduction of a suspension of autologous blood mononuclear cells in the anterior chamber at the border of the inner surface of the cornea and an air bubble:

3 - suspension of autologous blood mononuclear cells;

2 - air bubble.

Figure 3 - edema of the cornea’s own substance, increased tortuous course and violation of the tinctorial properties of collagen fibers 3 days after exposure. Stained with hematoxylin and picrofuxin according to Van Gieson, × 400:

4 - edema of the stroma of the cornea.

Figure 4 - desquamation of the endothelium 3 days after exposure. Stained with hematoxylin and eosin, × 400:

5 - corneal endothelium.

Figure 5 - pronounced edema of the own substance of the cornea 7 days after exposure. Stained with hematoxylin and eosin, × 400:

6 - edema of the cornea’s own substance.

Figure 6 - edema of the cornea's own substance, fuzzy borders of the posterior border membrane, proliferation of the posterior corneal epithelium 7 days after exposure. Stained with hematoxylin and picrofuxin according to Van Gieson, × 600:

6 - edema of the cornea’s own substance;

5 - corneal endothelium.

Figure 7 - slight edema of the cornea's own substance 14 days after exposure. Stained with hematoxylin and eosin, × 400:

6 - edema of the cornea’s own substance.

Figure 8 - process cells of the posterior corneal epithelium 14 days after exposure. Stained with hematoxylin and eosin, × 600:

7 - process cells of the endothelium.

Figure 9 - compact arrangement of collagen fibers, uniform perception of the dye in the cornea's own substance 21 days after exposure. Stained with hematoxylin and picrofuxin according to Van Gieson, × 400:

8 - collagen fibers.

Figure 10 - posterior border membrane and posterior corneal epithelium 21 days after exposure. Stained with hematoxylin and eosin, × 600:

9 - posterior border membrane;

10 - posterior corneal epithelium.

Claims (1)

A method of treating endothelial-epithelial corneal dystrophy, which consists in introducing a suspension of autologous blood mononuclear cells through paracentesis near the limbus into the inner chamber of the eye until it completely replaces the moisture in the anterior chamber, characterized in that two paracentesis are performed with a size of 1.2 mm, at 7 and 11 hours, in one of which an irrigation system is installed in the form of a cannula for supplying sterile air into the anterior chamber under a pressure of up to 0.2 atm, while through a second paracentesis using a syringe into the anterior chamber at the boundary between renney corneal surface and the air bubble singly administered 96-98% cell suspension of autologous blood mononuclear cells by fractionation on density gradient ficoll-verografin separating solution, after which the irrigation system is removed, the edges paracentesis hydrated.

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