RU2440801C1 - Method of treating deep stromal keratites - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and can be used for treatment of deep stromal keratites. Treatment is resalised by traditional pharmacotherapy, including specific antiviral and/or antibacterial medications, non-steroid anti-inflammatory and anti-allergic medications, as well as keratoprotectors. Additionally into layer of ill eye corneal stroma around pathologic nidus introduced are 0.5 ml of cell suspension, containing autologous mononuclear cells (AM). Suspension is introduced immediately after its preparation. For this purpose AM are isolated from heparinised patient's blood by method of fractioning in density gradient on separating ficoll-verografin solution with ratio of gradient and separated suspension volumes 1:3-1:4, at 2000 rev/min, at room temperature during 15 minutes. After that, 1.0 ml of isotonic sodium chloride solution is added and obtained suspension is re-centrifuged during 7 minutes at 1500 rev/min. Then, obtained cell suspension is placed into sterile vial with addition of 3.0 ml of isotonic sodium chloride solution and is used for introduction into cornea stromal layer. In the process of treatment procedure is repeated 1-2 times with 4-6 day interval depending on disease severity degree and clinical picture dynamics.

EFFECT: method ensures elimination of corneal syndrome, disappearance of cornea edema, resorption of necrotic masses, cornea epithelisation and increase of visual acuity, thus reducing treatment terms and number of disease complications.

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Description

The invention relates to medicine and can be used to treat deep stromal keratitis.

The need to develop new treatments for deep stromal keratitis is due to the lack of effectiveness of traditional pharmacotherapy and the high incidence of complications. According to published data [3, 7, 13, 14, 15], in 8–9% of cases, the pathological process ends with anatomical death of the eye, enucleation is performed in 17% due to the failure of treatment, and a complicated course of the disease is observed in 23–25%.

Among viral eye diseases, herpetic lesions are most common. As recognized worldwide, herpes virus is the most common cause of corneal damage [7]. Herpes virus keratitis is recognized as the first cause of corneal blindness. According to the Moscow Research Institute of Eye Diseases. Helmholtz, herpetic keratitis accounts for 66.6% of the total number of patients with corneal pathology, 55.4% of the number of patients with corneal ulcers. Herpetic keratitis is characterized by a severe prolonged course and a tendency to relapse, which occur in approximately 25% of patients after the first episode of the disease, in 50% after the second and up to 75% after repeated ones. A typical manifestation of corneal lesions is treelike keratitis with a characteristic form of infiltrate. Other varieties are less common: epithelial keratitis, interstitial keratitis, necrotizing stromal keratitis, disk-like stromal.

Currently, the negative effects of the use of antibacterial drugs have become apparent: suppression of immunity, increased resistance of microorganisms [3, 7, 18]. As a rule, the use of keratoprotectors for these types of pathologies is ineffective, especially in patients with gross changes in the cornea, and surgery is performed with appropriate indications (layered or through keratoplasty). These operations are technically complex and require the presence of transplants, which is currently one of the most important problems in ophthalmology. Serious postoperative complications associated with the development of immunological reactions leading to destruction or rejection of the graft are possible.

In recent years, studies have appeared that indicate the possibility of accelerating regenerative processes in the damaged cornea under the action of cytokines produced by mononuclear blood cells [5, 6, 11].

Closest to the proposed is a method of intracameral local rapid autocytokine therapy (LEACCT), in which, under operating conditions, autologous mononuclear blood cells of the patient activated by Poludan are introduced into the anterior chamber of the 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 vials of Poludan are dissolved in 2.0 ml of water for injection and the aqueous solution of Poludan is transferred into 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. Further, 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 near the limb with a blade, a corneal paracentesis of 2-3 mm is performed. 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. A supramid suture is applied in the area of corneal paracentesis. 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.

However, the widespread use of this method in clinical practice is limited due to the duration and the complexity of obtaining autologous blood mononuclear cells activated with the help of Poludan in the preparatory period.

A new technical task is to increase the effectiveness of treatment by reducing the time and reducing the number of complications.

To solve the problem in a method of treating deep stromal keratitis by introducing autologous blood mononuclear cells against the background of traditional pharmacotherapy, including specific antiviral and / or antibacterial drugs, non-steroidal anti-inflammatory and anti-allergic drugs, as well as keratoprotectors, after local anesthesia, 0.5 ml of cell suspension AM is injected into the stroma of the cornea of the diseased eye around the pathological focus immediately after its preparation, for which AM is isolated from arinized blood of a patient by density gradient fractionation on a ficoll-verographin separation solution with a ratio of the gradient volumes and the suspension divided by 1: 3-1: 4 by centrifugation at 2000 rpm at room temperature for 15 minutes, add 1.0 ml of isotonic solution sodium chloride, the resulting suspension is washed by centrifugation for 7 min at 1500 rpm and placed in a sterile vial, and during the treatment the procedure is repeated 1-2 times with an interval of 4-6 days, depending on the severity diseases and dynamics of the clinical picture.

The method is as follows.

After the patient arrives, they are examined and pharmacotherapy is prescribed at the same time, including specific antiviral and / or antibacterial drugs, non-steroidal anti-inflammatory and anti-allergic drugs, as well as keratoprotectors. On the second day after the start of treatment, the procedure according to the proposed method is carried out, which consists in the isolation of autologous mononuclear cells (AM) from the patient’s blood and the subsequent introduction of the resulting cell suspension into the pathological focus of the patient’s eye. For this, autologous mononuclear cells (AM) of the patient’s blood are isolated by density gradient fractionation on a ficoll-verographin separation solution [2] and, after washing and placing the cell suspension in a sterile vial with the addition of 3.0 ml of an isotonic sodium chloride solution, the procedure begins directly.

The selection of autologous mononuclear cells (AM) is carried out as follows: in the conditions of the treatment room in compliance with aseptic and antiseptic conditions, blood is taken from the patient's ulnar vein in an amount of 4.0-5.0 ml, which is placed in a sterile tube containing 1.0 ml of solution heparin. 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 autologous 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 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 should be between 97-98%.

Under operating conditions, after preliminary instillation of 1-2 drops of a 0.4% solution of inocaine and a subtenoneal injection of 2.0 ml of a 2% solution of lidocaine, the resulting cell suspension in a volume of 0.5 ml is injected into the thickness of the corneal stroma around the pathological focus. The mode of the method is based on the analysis of the results of clinical observations: the introduction of 1-2 times with an interval of 4-6 days is enough to achieve positive dynamics of the disease (cleansing the ulcer from necrotic masses, stopping inflammation, epithelization of the cornea), while excessive trauma is prevented .

Treatment by the proposed method is carried out against the background of traditional pharmacotherapy using specific (antiviral and / or antibacterial) drugs, non-steroidal anti-inflammatory and anti-allergic drugs, as well as keratoprotectors.

Clinical studies were conducted in a group of 50 patients (50 eyes), which, depending on the method of treatment, were divided into 2 groups - the main and comparisons. All patients of the main group of 27 people (27 eyes) under the background of traditional pharmacotherapy, including specific (antiviral or antibacterial) drugs, non-steroidal anti-inflammatory and anti-allergic drugs, as well as keratoprotectors, were given intrastromal injections of autologous blood mononuclear cells 1-2 times per treatment course with an interval of 4 -6 days depending on the severity of the disease and the dynamics of the clinical picture.

The comparison group is represented by 23 patients (23 eyes) who received conventional treatment without intrastromal administration of autologous blood mononuclear cells.

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

All patients underwent general phthalmological examination, including visometry, perimetry, biomicroscopy, ophthalmoscopy, determination of corneal sensitivity, fluorescein test, photorecording, examination of conjunctival smears. The intensity of corneal opacity was evaluated on a 10-point scale Voino-Yasenetsky.

The criterion of recovery was considered to be: elimination of the corneal syndrome, the disappearance of corneal edema, resorption of necrotic masses, corneal epithelization, increased visual acuity.

A comparative analysis of the treatment results for deep stromal keratitis revealed a significant effectiveness of intrastromal administration of autologous blood mononuclear cells, the data are presented in table 1.

The period of cleansing of a ulcer defect from necrotic detritus in patients of the main group decreased by 1.5-2 times in contrast to the comparison group (p <0.05). The resorption of perifocal edema and corneal stroma infiltration accelerated by 40-42%. In the main group, earlier than in the comparison group, complete corneal ulcer epithelization occurred (7.2 ± 0.17 days and 11.0 ± 0.16 days, respectively).

Thus, the application of the proposed method accelerates 1.5 times the healing of corneal ulcer in patients of the main group, and also reduced the number of complications of this disease (formation of descemetocele, perforation of the cornea, development of endophthalmitis).

The corneal opacity formed in the outcome of the disease was lower in the patients of the main group than in the patients of the comparison group (on the Vojno-Yasenetsky scale - 4.5 and 6.0 points, respectively).

Moreover, in most patients of the comparison group, corneal opacity went beyond the defect by 3-4 mm.

The use of the proposed method in the complex treatment of patients with deep stromal keratitis positively affected the state of visual functions. At the time of treatment, visual acuity in patients of both groups was 0.01-0.02. By the end of treatment, an increase in visual acuity to 0.4-0.5 was observed in 62% of cases (17 people) in the main group and in 47.8% (11 people) in the comparison group.

Also important in the medical and social aspect was the reduction in the stay of patients of the main group in the hospital by an average of 5.5 ± 0.3 hospital days. The average period of inpatient treatment of patients of the main group was 13.0 ± 0.5 days, patients of the comparison group 19.0 ± 0.2 days.

A clinical example on the implementation of the method.

Example 1. Patient T., 22 years old, complained of redness, lacrimation, decreased vision of the left eye.

Medical history: about 1 month ago, during the repair, a foreign body fell into the left eye. The patient turned to an ophthalmologist after 2 days. The foreign body was removed, antibiotics and keratoprotectors were locally prescribed. However, the patient took the treatment irregularly, and therefore a few days later noted a deterioration in the form of sharp redness, photophobia, pain, lacrimation and decreased vision in the left eye. The ambulance team was hospitalized in the ophthalmology department of MSC No. 2 with a diagnosis of a corneal ulcer of the left eye, where anti-inflammatory treatment was prescribed and a therapeutic contact lens was applied. However, the treatment carried out did not give positive results, OS corneal ulcer progressed with the development of descemetocele, in connection with which the patient was transferred for further treatment to the ophthalmological clinic of the Siberian State Medical University.

Objective inspection:

Vis OD = 1.0

OS = 0.5 n / a

On examination, the palpebral fissure OS is already OD due to blepharospasm, lacrimation was noted.

Biomicroscopy OD - the eye is calm. Conductive media are transparent.

OS - a mixed injection of the bulbar conjunctiva, on the cornea, paracentrally from below, an oval ulcerative defect, in d≈4.0 mm the edges are loose, saped, and significantly thinned. The bottom of the ulcer is lined with necrotic detritus. Edema and infiltration of the corneal stroma were perifocal. Parts of the anterior chamber are difficult to see due to corneal edema. Ophthalmoscopy is difficult due to clouding of the cornea. Sensitivity of the cornea is absent.

Diagnosis: herpetic corneal ulcer with a threat of perforation of the left eye.

The prescribed treatment: general - tablets Acyclovir-Akos 200 mg 4 times a day, intramuscularly - Ascorbic acid solution 5% - 2 ml No. 10, Suprastin solution 1.0 ml No. 5, Diclofenac solution 75 mg No. 5; intravenously - cefazolin solution 1.0 No. 5; local-subconjunctival solution of Poludan 0.5 ml, eye ointment Acyclovir 3% - for the lower eyelid after 1 hour, installations - Poludan, Ophthalmoferon every hour.

In addition to local treatment for 2 days in the operating room, the patient underwent treatment according to the proposed method, including the selection and subsequent intrastromal administration of autologous blood mononuclear cells. In the treatment room, blood taken from the patient’s elbow vein was placed in a sterile tube containing 1.0 ml of heparin solution. Heparinized blood was diluted twice in isotonic sodium chloride solution. The resulting suspension was layered in 3.0 ml of fecoll-verographin mixture. The tube was centrifuged at room temperature for 15 min at 800 g (2000 rpm). After centrifugation, the interphase layer containing mononuclear cells was taken with a Pasteur pipette and after adding 1.0 ml of physiological saline, the resulting suspension was again centrifuged for 7 min at 400 g (1500 rpm). In the operating room, after processing the surgical field in compliance with aseptic and antiseptic rules, local anesthesia was performed (installations of a 0.4% inocaine solution, subtenoneally 2% - 2.0 ml lidocaine solution) of the sick eye and a suspension of autologous blood mononuclear cells was injected into the corneal stroma in an amount of 0 5 ml.

The dynamics of the disease:

On the 2nd day after the procedure (respectively, the 3rd day after hospitalization and the start of treatment), a positive dynamics of the clinical picture was noted.

Vis OS = 0.6c (+) 1.0 ^d = 1,0

Status localis OS: slight photophobia, moderate ciliary soreness. Ophthalmotonus is normal. A peptic ulcer in diameter decreased slightly due to epithelization, the bottom cleared of necrotic masses, perifocal corneal edema also decreased slightly. The anterior chamber is small, the moisture is transparent, the pattern of the iris is somewhat smoothed out, the pupil is of medium width, rounded, the reaction to light is weakened.

On the 5th day after the intrastromal injection of cell suspension:

Vis OS = 0.7c (+) 0.5 ^d = 1,0

Status localis OS: palpation is painless. The edges of the ulcerative defect became less friable and sap, however, they were slightly thinned in the zone from 1 h to 5 h. Minor edema of the corneal stroma remained perifocal. The growth of vascular branches in the stroma of the cornea around the ulcer defect was noted. The bottom of the ulcer is almost completely lined with epithelium (nevertheless, when conducting a fluorescein test, point sites of de-epithelization are detected). The anterior chamber is uniform, the moisture is transparent, the pupil is round in shape, of medium width, the reaction to light is well defined.

On the 7th day after the intrastromal injection of cell suspension:

Vis OS = 0.9

Status localis: the edges of the ulcer became homogeneous, smooth, the bottom is completely lined with epithelium, which is confirmed by the results of the fluorescein test.

On the 12th day:

Vis OS = 0.9-1.0

Status localis: complete epithelization of a peptic ulcer. The surrounding cornea is smooth, shiny, transparent. Bottom outside surface vascularization. The anterior chamber is of medium depth, the moisture is transparent, the pupil is of regular shape, medium width, the reaction to light is well defined. 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 duration of the patient’s hospital stay was 14 days.

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. The teachings of II Mechnikov on macrophage cytase enzymes developed into the concept of monokines, products of the secretion 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 [4, 11]. Using 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, 9, 11].

Once in the focus of inflammation, mononuclear cells are influenced by many local factors, including both the inducer of inflammation (for example, a microorganism) and endogenous phlogogens — complement components, prostaglandins, etc. As a result, the activation of mononuclear phagocytes takes place, which is combined with the growth of the microbiocidal and cytotoxic potential of inflammatory effector cells.

The desire to restore the disturbed balance in the system of autofibrinolysis and repair is embodied in the method of treating ulcerative keratitis by injecting the patient’s autologous blood directly into the corneal infiltrate (Poromonova I.Yu., Myusin F.A., 2005), which contains antiprotease substances necessary for terminating the process destruction of corneal tissue, as well as keratoplastic and trophic components, allowing to optimize the resorption and healing of the ulcer defect [10].

Intrastromal administration of autologous blood mononuclear cells in inflammatory and dystrophic diseases of the cornea allows you to create a locally high concentration of cells of this population in the focus of exudative-destructive inflammation and to target specific stages of the pathological process.

It can be assumed that due to the action of proinflammatory cytokines such as interleukins 1, 6, 8, tumor necrosis factor and interferon, polymorphonuclear leukocytes are recruited to the site of inflammation with the development of a respiratory explosion, stimulation of degranulation, induction of synthesis and secretion of lysosomal enzymes, and the formation of an autocrine regulation pathway cells [1, 8, 9].

Due to the functional cooperation of exogenously introduced mononuclear cells and polynuclear phagocytes migrating under their influence in the focus of exudative-destructive inflammation, accelerated cleansing of necrotic masses occurs, which is confirmed by the results of a clinical study, and the inflammatory process goes into the repair phase.

It should be noted that the fibroplastic process, the course of which is controlled by many mechanisms, originates in the bowels of cell infiltration in the focus of inflammation. First of all, the fibroplastic process depends on the structural and functional parameters of the fibroblasts themselves, as well as on the emerging complex cascades of intercellular interactions. An important role is played by the tandem “macrophage-fibroblast” and the triad “lymphocyte-macrophage-fibroblast” [8, 9, 16]. The connecting figure in these interactions is the macrophage.

Mononuclear cells, as noted above, are a source of numerous bioregulators, including factors that stimulate proliferation and other functions of fibroblasts [8, 9, 16, 17]. This provides one of the central mechanisms for connecting fibroblasts to reparative processes.

Of course, in addition to monokines, fibroblasts perceive the products of lymphocytes, neutrophils, platelets, and epithelium. Thrombin and a number of proteinases trigger proliferation of fibroblasts [8, 9]. However, there is more evidence that it is macrophages that lead the main dialogue with fibroblasts, transforming through them part of their own pathogenetic potential, as well as the effector capabilities of a number of other cells and humoral factors [16]. In other words, activated macrophages are considered as the most important link between fibroblasts and damage, fibrogenesis and homeostasis.

Morphologically macrophage-fibroblastic interactions lead to migration and accelerated proliferation of fibroblasts, their differentiation and production of extracellular matrix components [9, 16], which ensures the closure of the ulcerous defect by connective tissue and the formation of a rough corneal opacity in the outcome of the disease.

However, depending on the changing conditions of the inflammatory process, mononuclear cells, due to collagenolytic enzymes and other hydrolases, can also participate in the resorption of interstitial substances, are able to secrete factors that stimulate collagenase production in fibroblasts, and factors that enhance fibril phagocytosis by fibroclasts [16].

In this regard, it can be assumed that additional exogenous intake of mononuclear cells into the pathological focus contributes to the inclusion of mechanisms to suppress the excessive proliferation of connective tissue, providing a combination of inflammation and regeneration. Under the influence of humoral stimuli and microenvironment factors, most of the fibroblasts differentiate into low-activity fibrocytes. At the same time, the phenomenon of fibroclasia is also enhanced, which ensures remodeling and involution of newly formed connective tissue. Clinically, this is manifested by the formation of a smaller area and intensity of corneal opacity.

Thus, the results of clinical studies indicate the high efficiency of the proposed method in the complex treatment of deep stromal keratitis.

Information sources

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Table 1 The dynamics of the course of deep stromal keratitis depending on the treatment methods Clinical sign Group of patients the main comparisons The disappearance of corneal edema Start 3.5 ± 0.17 5.2 ± 0.16 completion 6.2 ± 0.17 8.5 ± 0.16 Ulcerative epithelization Start 3.7 ± 0.17 5.6 ± 0.16 completion 7.2 ± 0.17 11.0 ± 0.16 Resorption of infiltration Start 2.8 ± 0.17 5.1 ± 0.17 completion 5.1 ± 0.15 10.2 ± 0.16 Relief of inflammation 7.9 ± 0.17 14.5 ± 0.16

Claims (1)

A method of treating deep stromal keratitis by administering autologous blood mononuclear cells (AM) against the background of traditional pharmacotherapy, including specific antiviral and / or antibacterial drugs, non-steroidal anti-inflammatory and anti-allergic drugs, and keratoprotectors, characterized in that after local anesthesia is performed, AM cell suspension in volume 0.5 ml is injected into the stroma of the cornea of the diseased eye around the pathological focus immediately after its preparation, for which AM is isolated from the patient’s parinized blood by density gradient fractionation on a ficoll-verographin separation solution with a ratio of the gradient volumes and the divided suspension of 1: 3-1: 4, at 2000 rpm, at room temperature for 15 minutes, add 1.0 ml of isotonic solution sodium chloride and the resulting suspension are again centrifuged for 7 minutes at 1500 rpm, then the resulting cell suspension is placed in a sterile bottle with the addition of 3.0 ml of isotonic sodium chloride solution, and during the treatment process they bite 1-2 times with an interval of 4-6 days, depending on the severity of the disease and the dynamics of the clinical picture.