RU2324488C1 - Method of treatment of corneal ulcers - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: separation of the mononuclear leukocyte cell suspension from patient's blood is performed by density fractionation. The cell suspension is used immediately after centrifugation of 4 mL blood. The suspension is washed out and diluted by 3.0 mL isotonic sodium chloride. The suspension is instilled 6-8 times daily with 3-4 days break, the course consists of 2-3 procedures.

EFFECT: method increases efficiency of treatment by reduction of treatment time.

1 tbl, 1 ex

Description

The invention relates to medicine, ophthalmology, and specifically to methods for treating corneal ulcers.

Closest to the proposed is a method of treating corneal ulcers by autolymphokine therapy, based on the use of a complex of cytokines secreted by peripheral blood mononuclear cells [5, 6, 11]. The essence of the method is as follows: mononuclear cells are isolated from the patient’s peripheral blood, placed in a culture dish, and incubated with laboratory diagnosticum phytohemagglutinin for 3 hours. Then the cells are washed twice with Hanks solution and re-cultured for 24 hours. Next, the cells are precipitated by centrifugation and the supernatant is taken. After sterilization, the supernatant is packaged in sterile vials and stored in the freezer compartment of the refrigerator at -20 ° C. Treatment with autocytokines is carried out in combination with basic therapy in the form of installations 2 drops 5 times a day for 10 days.

However, the widespread use of this method in clinical practice is limited due to the duration and complexity of obtaining the supernatant in the preparatory period, the effectiveness of the method is not high enough, and the course of treatment is quite long.

A new technical task is to increase the efficiency of the method by reducing the healing time of a peptic ulcer, reducing the number of complications with a significant simplification of the method.

To solve the problem in a method of treating corneal ulcers, including the introduction of antibacterial and anti-inflammatory drugs and the installation in the conjunctival cavity of the eye of a cell suspension of mononuclear cells isolated from the patient’s peripheral blood by fractionation in a density gradient, a cell suspension obtained directly after centrifugation is used for installations, which washed and diluted at least three times with an isotonic sodium chloride solution, and installations are carried out at frequencies oh 6-8 times a day and with an interval of 3-4 days, the course includes at least 2 procedures.

The method is carried out in the following way. The blood taken from the patient's ulnar vein in an amount of 4.0-5.0 ml is placed in a sterile tube containing 1.0 ml of heparin. Autologous mononuclear cells are isolated by density gradient fractionation (1.067-1.077 g / ml) on a ficoll-verographin separation solution [2]. The taken blood is 2 times diluted with isotonic sodium chloride solution and layered on 3.0 ml of a mixture of ficoll-verographin. Samples are centrifuged for 15 minutes at 800 g (2000 rpm). Next, the interphase layer containing mononuclear cells and located between the plasma and the gradient is taken with a pipette. 1.0 ml of isotonic sodium chloride solution was added and the resulting suspension was again centrifuged for 7 minutes at 400 g (1500 rpm) to wash the cells. The purity of mononuclear cells isolated on a ficoll-verographin gradient is 95-98%. After placement in a sterile vial and adding 3.0 ml of an isotonic sodium chloride solution, the cell suspension is installed in the conjunctival cavity of the sick eye with a frequency of 6-8 times a day every 3-4 days, depending on the severity of the disease and the dynamics of the clinical picture, performing at least 2 treatments.

The treatment of the proposed method is carried out against the background of traditional pharmacotherapy of corneal ulcers using antibacterial and anti-inflammatory drugs.

The need to develop new methods for the treatment of corneal ulcers is due to the insufficient effectiveness of the traditional pharmacoteralia of this disease and the high incidence of complications. According to published data [3, 7, 12, 13, 14], in 8–9% of cases, the pathological process ends with anatomical death of the eye, enucleation is performed in 17% due to treatment failure, and a complicated course of the disease is observed in 23–25%. In addition, to date, the negative effects of the use of antibacterial drugs have become apparent: the development of dysbiosis, suppression of immunity, increased resistance of microorganisms [3, 7, 17].

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 peripheral blood mononuclear cells [5, 11].

The mode of the proposed method is selected based on the analysis of clinical observations of a group of 56 patients (56 eyes) with corneal ulcers of varying severity. In this case, a severe pathological process was observed in 67% of patients, moderate severity - in 33%. At the time of going to the hospital, visual acuity in most cases (59%) was below 0.1, in 41% - 0.1-0.2. The terms of circulation ranged from 1 to 3-4 days.

A bacteriological study of a separated ulcerative defect revealed an increase in microflora in 78.9% of cases. Pathogenic microflora were found in 63.4% and were represented by Pseud. aeruginosa - 42.1%, Staph. aureus - 21.1%. Conditionally pathogenic microflora was detected in 15.5%, among which Staph prevailed. epidermidis - 5.2%, E. coli - 10.5%.

Along with the traditional treatment (antibacterial and anti-inflammatory drugs), all patients of this group underwent the installation of freshly prepared cell suspension of autologous peripheral blood mononuclear cells diluted with isotonic sodium chloride solution with a frequency of 6-8 times a day. During treatment, the procedure was repeated 2-3 times with an interval of 3-4 days, depending on the severity of the disease and the dynamics of the clinical picture.

The control group was represented by 40 patients (40 eyes) who received similar treatment without the use of installations 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, conjunctival smear examination.

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, resorption of necrotic masses and infiltration of the corneal stroma, resorption of hypopion, corneal epithelization, increased visual acuity.

A comparative analysis (Table 1) of the results of the treatment of corneal ulcers revealed the significant effectiveness of the use of installations of autologous blood mononuclear cells.

So, for example, the period of cleansing of a ulcerative defect from necrotic masses decreased by 1.5-2 times compared with the control group (p <0.01). Resorption of perifocal edema and corneal stroma infiltration accelerated by 35-40%. As a result, corneal epithelization occurred earlier than in the control group (in the main group by an average of 6.2 ± 0.17 days from the start of treatment, in the control group by 11.0 ± 0.16 days). The number of complications (the formation of descemetocele, perforation of the cornea, the development of endophthalmitis) decreased in the study group by 2.5 times.

The corneal clouding formed in the outcome of the ulcer was significantly lower in the patients of the main group than in the patients in the control group (according to the Voino-Yasenetsky scale - 4.5 and 6.0 points, respectively). Moreover, in most patients of the control group, corneal clouding exceeded the limits of the ulcer by 1.5-2.0 mm.

The use of installations of autologous peripheral blood mononuclear cells according to the proposed method in the complex treatment of patients with ulcerative lesions of the cornea had a positive effect on the state of visual functions. At the beginning of treatment, visual acuity in patients of both groups was 0.01-0.02. By the end of treatment, in patients of the main group, visual acuity averaged 0.2-0.3, in patients of the control group - 0.09-0.1. Moreover, an increase in visual acuity to 0.4-0.5 was observed in 46% of cases (25 people) in the main group and in 27% (10 people) in the control.

Of great importance in the medical and social aspect is the fact that the duration of the stay of patients of the main group in the hospital is reduced by an average of 5.5 ± 0.6 bed days.

The proposed treatment regimen is based on clinical observation, a lower frequency of installations of autologous peripheral blood mononuclear cells (3-4 times a day) is insufficient to achieve positive dynamics of the disease (cleansing the ulcer defect from necrotic masses, stopping inflammation, corneal epithelization). In order to consolidate the achieved therapeutic effect, the procedure must be performed at least 2 times with an interval of 3-4 days under the mandatory control of the clinical picture. However, it should be noted that the procedure more than 3 times is undesirable, because in some cases, it can lead to negative consequences (increase in peptic ulcer in area and depth).

Based on previously conducted experimental and clinical studies, it was found that the functional activity of freshly prepared mononuclear cells is comparable to that of culturing cells under stationary conditions. Given the proposed new treatment regimen for pathology with a frequency of instillation of cell suspension 6-8 times a day, in a short course, the possibility of using autologous blood mononuclear cells obtained immediately after isolation and washing with saline, previously diluted at least three times to obtain a cell suspension was confirmed with sufficient density.

Clinical example for the implementation of the method

Patient M., 54 years old, complained of acute pain in the right eye, photophobia, lacrimation, decreased vision of the right eye.

Anamnesis of the disease: during a car repair a foreign body fell into the right eye. He turned to the optometrist only after 2 days. The foreign body was removed, antibacterial and anti-inflammatory treatment was prescribed. However, the patient made appointments irregularly, and after 6-7 days, he again noted deterioration in the right eye, and therefore turned to the eye trauma center of the city emergency hospital.

Objective inspection:

Visus OD = 0.03

OS = 1.0

Biomicroscopy OD - mixed injection, lacrimation, blepharospasm. Palpation is determined by ciliary soreness. On the cornea, paracentrally from below, an ulcerative defect of irregular shape d = 3.0 mm with loose, undercut edges. The bottom of the ulcer is filled with necrotic masses. Swelling and infiltration of the corneal stroma are perifocal. Front camera of medium depth. Hypopion 2.0 mm. The pupil is narrow, the reaction to light is sluggish. The iris pattern is faded.

Ophthalmoscopy OD is difficult.

Diagnosis: post-traumatic purulent ulcer of the cornea of the right eye. Hypopionuveitis of the right eye.

Treatment was prescribed: general - intramuscularly Cefazolin 0.5 g 2 r. per day, Suprastin 1.0 ml 1 r. in a day. Intravenously Hemodez 250.0 ml 1 r. per day, inside Indomethacin, Nystatin; local - subconjunctival and parabulbar Gentamicin, Streptomycin, Mesatone, 5% sodium chloride solution, installations - Ciprolet, Sulfacil sodium.

In addition to local treatment, installations of autologous peripheral blood mononuclear cells of 6-8 p. in a day. With an interval of 3 days, the procedure was performed three times.

The dynamics of the disease

On the 2nd day after the installation of cell suspension and, accordingly, the 3rd day after hospitalization and the start of treatment, a positive dynamics of the clinical picture was noted.

Visus OD = 0.06-0.07

Status localis OD: the bottom of the ulcerative defect cleared of necrotic masses, the effects of perifocal corneal stroma infiltration decreased. The level of hypopion decreased to 1.0 mm.

After the second procedure (8th day after hospitalization and the start of treatment):

Visus OD = 0.1-0.2

Status localis OD: slight photophobia, moderate ciliary soreness. The edges of the ulcer became significantly less loose and saped. Minor edema of the corneal stroma is perifocal. The bottom of the ulcer is almost completely covered with epithelium. The moisture of the front camera is transparent. The iris pattern is more prominent. The pupil is of medium width, round, the reaction to light is somewhat weakened.

After the 3rd procedure (12th day after hospitalization and the start of treatment):

Visus OD = 0.5-0.6

Status localis OD: a gentle cloud-like clouding at the level of the anterior third of the stroma is formed paracentrally from below on the cornea. Perifocal stroma is transparent. The moisture of the front camera is transparent. The pattern of the iris is embossed. The reaction of the pupil to the light is "live".

Upon discharge from the hospital (the length of stay in the hospital was 16 beds / days):

Visus OD = 0.6-0.7

Status localis OD: on the cornea, paracentrally from below, whitish cloudiness. The remaining parts of the cornea are transparent. The anterior chamber is of medium depth, the moisture is transparent. The iris pattern is embossed, the pupil’s reaction to the light is “live”.

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 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 [4, 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, 9, 10].

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.

Exogenous introduction of autologous peripheral blood mononuclear cells into the conjunctival cavity with corneal ulcers 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 bactericidal factors, the formation of an autocrine cell regulation pathway [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, 15]. 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, 15, 16]. 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 [15]. 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, 15], which ensures closure of the ulcerous defect by connective tissue and the formation of a rough corneal opacity in 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 [15].

In this regard, it can be assumed that additional exogenous intake of freshly prepared suspension of mononuclear cells into the pathological focus in the proposed mode contributes to the inclusion of mechanisms for suppressing excessive overgrowth 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 proposed method allows for the effective treatment of corneal ulcers in a shorter time, the results while reducing the number of complications.

Table 1
The dynamics of the course of a bacterial corneal ulcer depending on the method of treatment Clinical sign Group of patients the main control Corneal epithelization Start 3.7 ± 0.15 5.6 ± 0.16 completion 6.2 ± 0.17 11.0 ± 0.16 Resorption of infiltration Start 2.9 ± 0.16 5.1 ± 0.17 completion 5.2 ± 0.15 8.5 ± 0.16 Hypopion resorption Start 1.9 ± 0.17 3.8 ± 0.17 completion 3.7 ± 0.17 5.5 ± 0.16 Relief of inflammation 8.2 ± 0.17 14.5 ± 0.16

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Claims (1)

A method of treating corneal ulcers, including the introduction of antibacterial and anti-inflammatory drugs and the installation in the conjunctival cavity of the eye of a cell suspension of mononuclear cells isolated from the patient’s peripheral blood by density gradient fractionation, characterized in that the cell suspension obtained from 4 ml of blood directly after centrifugation is used for installations which is washed and diluted at least three times with an isotonic sodium chloride solution, and installations are carried out at a frequency 6-8 times a day at intervals of 3-4 days, the course includes 2-3 procedures.