Method of correction of degenerative changes of the retina by pathology of various origins
The invention relates to ophthalmology and is used for correction of degenerative changes of the retina by pathology of various origins. Transplantation of the patient's suspension of cultured neuronal stem cells. The suspension is injected retrobulbarno in the amount of 5 million cells in 1 ml of physiological solution. The method allows to achieve acceleration of reparative regeneration of the retina of the recipient.
The invention relates to ophthalmology and is intended for the treatment of certain types of pathology of the retina dystrophies various origins, post-traumatic changes, lesions of the retina in systemic diseases).
Despite the wide selection of commonly used treatments, restoring the structural organization of the retina and its functional activity remains an actual problem. Numerous medications, physical therapy techniques, laser and surgical interventions are not sufficiently effective (Lysenko Century C. Development of pathogenetically oriented complex methods of treatment of involutional Central chorioretinal dystrophies: Dissert. Kida is to preservati, leading to significant vision impairment. This explains the need for new modern methods of correction of this pathology.
Know the use of peptide bioregulators in ophthalmology. In 1985, at the Military Medical Academy, Saint-Petersburg) C. H. Khavinson and co-authors from the retina of the eye of cattle have identified a complex of peptides that stimulate the function of the retina, called ritalin or medication (EN 2073518, 20.02.97 - the closest equivalent). Treatment with use of ritilin conducted in patients suffering from Central chorioretinal dystrophy of the retina, diabetic retinopathy, pigmentary amyotrophia of the retina, as well as in complex with microsurgical treatment of wounds and injuries of the eye (Trofimova S. C. Application of peptide bioregulators in the treatment of diabetic retinopathy: author. dis. Kida. the honey. Sciences. - SPb., 1999; Maksimov I. B. peptide Complex correction in microsurgical treatment of eye injuries and their consequences: author. dis. Dr. med. Sciences. - M., 1996). The mechanism of action of peptide bioregulators based on the restoration and conservation of regulatory mechanisms of cell-cell interaction, which is manifested, in particular, the recovery is generally effective and efficient in the early stages of the pathological process.
In this regard, our interest in the question of the feasibility of using cultured in vitro neuronal stem cells in some types of disorders of the retina.
Stem cells are the provision of emergency repair in the case of a massive disaster tissue damage. The share of stem cells account for about 0,1-0,01% of the total cell mass of the body. The imbalance in the number and activity of self-renewal of differentiated clones of stem cells is the root cause of many of the lesions at the cellular level. Stem cells have a number of significant advantages: can ensure the regeneration of damaged areas through the production of various growth factors and key metabolites; able to expand the program embryogenesis and differentiation, thereby restoring the lack of active cells. Transplantation of stem hematogenous cells is widely used in the treatment of leukemia. Substitution of cell therapy using neural stem cells tested in the clinic for the treatment of stroke patients. After implantation of the donor cells marked clinical improvement in most patients (Kondziolka D., Wechstler L., Goldstein S, et al. // Am. Soc. Neur. Tr is the technical result of the invention is the acceleration of reparative regeneration of the retina by use of suspension cultured in vitro neuronal stem cells.
The technical result is achieved by transplantation of suspension of neuronal stem cells into the retro-bulbar space.
Source of donor material is the brain tissue of human embryos in the 1st trimester of pregnancy (8-12 weeks). Abortive material obtained from licensed institutions MOH, operating under Zakonodatelstva of the Russian Federation on health protection of citizens and in accordance with the approved list of medical conditions (Order of Minister of health No. 302 from 28.12.93 "On approval of the list of medical indications for abortion" and the Annex to that order No. 3 from 05.04.94). The cultivation is carried out in serum-free medium consisting of DMEM (50%), F12 (50%), the main factor toast fibroblasts (hFGF-b), epidermal growth factor (hEGF) and factor in the survival of neuronal cells (N2-supplement) (C. N. Svendsen, M. G. ter Borg, R. J. E. Armstrong, A. E. Rosser, S. Chandran, T. Ostenfefeld, M. A. Candwell. A new method for rapid and long-term growth of human neural precursor cells // J. Neirosci. Methods. 85 (1998) 141-152).
Immediately prior to transplantation of the cell suspension thoroughly washed from the culture medium, counted the number of viable cells using the iodide of propecia and Trypanosoma blue and suspension in minimal concentration of 5 million cells in 1 ml The dose of injected neural stem cells tested experimentally. Stay unfrozen material with a positive temperature should not exceed 2 hours.
The method is as follows. Method of injection into the retro-bulbar space transplanted cultured neuronal stem cells in suspension at a dose of 5 million cells in 1 ml of physiological solution.
Example 1. As a model of retinal dystrophy we took experimental toxic dystrophy of the retina, caused by intravenous injection of a 4% solution monoiodotyrosine acid (MIUC) at the rate of 10 mg per 1 kg of the rabbit according to the method Noell, W. K. (Noell, W. K. Corneal cheuges durind pilocarpine gel theapy // Ameer. J. Ophthalmolm. - 1951. - Vol.101, No. l. - P. l3-15; Noell, W. K. Monin dected comtal ulcers // Ophthalmology. - 1958. - Vol.14, No. 1. - P. 44-51). The degree of retinal damage was assessed by the indicators of the functional status, namely on the level of bioelectric activity determined by the method of electroretinography. This dose MYUK causes severe damage to the retina. After 20 min after the introduction of the wave amplitude "b" was on average 20% of the original value. The next day after the introduction of MYUK method of retro-bulbar injection prostranstva solution. ERGS were recorded in both groups - control and experimental (10 eyes in each group) for 3, 5, 10, 20 days from the beginning of the experiment. The data obtained show that the use of neuronal stem cells in the experimental group leads to a significant increase of wave "b" ERG compared with control animals eyes. therapeutic effect of neural stem cells were found in average 10 day observation. Amplitude of wave "b" ERG in the experimental animals by 20 days reached 35% from baseline, and almost 2 times higher than that in the control group.
Thus, the results of these studies suggest that toxic damage to the retinal neuronal stem cells have a pronounced therapeutic effect.
Example 2. Study of the effect of transplantation of neuronal stem cells in the reparative processes in the retina on the model of laser damage. Damage to the retina when sverhdorogom laser radiation is characterized, as a rule, a violation of the ultrastructure of cells of the retina up to changing the architectonics chorioretinal region and hemorrhages in the retina. Damage to the retina as a result of the direct thermal effects lazernogo, necrosis of the inner and outer segments of photoreceptors. Laser damage to the retina of the rabbit (10 animals) was performed using an argon laser system. Immediately after laser injury to the right (experienced) eyes of each rabbit by injection into the retro-bulbar space transplantability neuronal stem cells (in suspension in a dose of 5 million cells in 1 ml of physiological solution). In the left (control eye) in the same way were injected with saline. In a clinical study of the anterior segment of the eye for any signs of inflammatory or allergic reactions have been identified, indicating the absence of immune conflict. When ophthalmoscopy noted that in the experimental group the appearance of the pigment in the field of laser damage began with 4-5 days in the control group - 7-8-x days. Pigmentation coagulates on the backdrop of the complex was completed by 18-20-m days, with the introduction of the saline - only 25 days. The histological examination showed that in the control eyes, there was complete destruction of retinal layers in the damaged area, while in the experienced eyes of the 10-th day of observation, the structure of the retina in the area coagulative on strengthening of processes of reparative regeneration of the retina in the transplantation of neuronal stem cells.
Given the opportunity to have a stock of donor material, as well as efficient and simple technique of transplantation, the use of neuronal stem cells seems promising for correction of degenerative changes of the retina by pathology of various origins.
Method of correction of degenerative changes of the retina by pathology of various origins, characterized in that the retro-bulbar space transplanted suspension cultured neuronal stem cells in a dose of 5 million cells in 1 ml of physiological solution.
SUBSTANCE: method involves introducing 0.1-0.3 ml of photosensitizing gel preliminarily activated with laser radiation, after having removed neovascular membrane. The photosensitizing gel is based on a viscoelastic of hyaluronic acid containing khlorin, selected from group containing photolon, radachlorine or photoditazine in the amount of 0.1-2% by mass. The photosensitizing gel is in vitro activated with laser radiation having wavelength of 661-666 nm during 3-10 min with total radiation dose being equal to 100-600 J/cm2. The gel is introduced immediately after being activated. To compress the retina, vitreous cavity is filled with perfluororganic compound or air to be further substituted with silicon oil. The operation is ended with placing sutures on sclerotomy and conjunctiva areas. Compounds like chealon, viscoate or hyatulon are used as viscoelastic based on hyaluronic acid. Perfluormetylcyclohexylperidin, perfluortributylamine or perfluorpolyester or like are used as the perfluororganic compound for filling vitreous cavity.
EFFECT: excluded recurrences of surgically removed neovascular membrane and development of proliferative retinopathy and retina detachment; retained vision function.
3 cl, 5 dwg