RU2568368C1 - Method for simulating proliferative vitreoretinopathy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: Interleukin 1-b 2000 U in an amount of 0.1 ml is introduced intravitreally into both rabbit's eyes. That is followed by sequential intravitreal administration of platelet-derived growth factor PDGF AA of osteoblastic sarcoma in an amount of 0.1 ml in the concentration of 2000 pcg/ml.

EFFECT: producing a clinically and cytopathogenetically adequate model of PVR applicable to study the efficacy of medicinal treatment and prevention of PVR experimentally.

2 ex, 6 dwg

Description

The present invention relates to ophthalmology and is intended to model proliferative vitreoretinopathy (PVR).

Proliferative vitreoretinopathy (PVR) is a complex heterogeneous disease that is characterized by the formation of cell membrane formations on the inner surface of the retina. A feature of these membranes is their traction properties, leading to traction retinal detachment with loss of visual functions. PVR, as a multifactorial pathological process, most often occurs as a complication in such clinical conditions as regmatogenous retinal detachment, traumatic retinal detachment. The development of PVR after surgical treatment of retinal detachment causes relapse of the disease - retinal non-adherence.

The formation of PVR is based on the proliferation of retinal pigment epithelial cells (RPE) and glial retinal cells. Treatment of PVR presents great difficulties associated with the localization of the process mainly in the posterior part of the eye. Therefore, for an objective judgment on the effectiveness of drug exposure during PVR, it is necessary to create an adequate experimental model.

Over the past 25 years, numerous studies have been carried out to simulate TAC.

A well-known conventional method for simulating PVR is known, in which the main model material is fibroblasts from tissue culture, which are administered intravitreal. However, this model is not a pathogenetically oriented model that reflects the clinical course of PVR. The model demonstrates the formation of fibrous membranes on the surface of the retina, but does not reflect the leading role in the PVR proliferation of RPE cells that are not identified in the formed membranes (Sugita G, Tano Y, Machemer R, Abrams G, Claflin A, Florentino G. Intravitreal autotransplantation of Fibroblasts Am J Ophthalmol 1980; 89: 121-30). (Hitchins C.A., Grierson J. Intravitreal injection of fibroblasts. The pathological effects on the ocular tissue of the rabbit following an intravitreal injection of analogus skin fibroblasts. Br J Ophth. 1998. vol. 72, p. 488). Currently, it is known that as a result of the violation of the hematoretinal barrier, a specific inflammatory process is launched involving: platelet-derived growth factor PDGF, tumor necrosis factor beta-TGF-b, epiretinal growth factor - EGF, tumor necrosis factor alpha-TGA-a,and others; pro and anti-inflammatory cytokines: interleukins - IL-1, IL-6, IL-8, IL-10 and interferon gamma - INF-v. The interaction of these factors leads to processes of migration, proliferation and differentiation of cells involved in the development of PVR (Lei H, Rheaume MA Kazlauskaa A. Recent developments in our understanding of how platelet-derived growth factor (PDGF) and its receptors contribute to proliferative vitreoretinopathy. Exp Eye Res. 2010; 98 (3): 376-381.)

Models that are based on the participation in the pathogenesis of PVR of inflammatory factors are called inflammatory.

Known inflammatory - cytokine model for inducing PVR in the experiment, which was first used by S. Baudouin et al. when modeling PVR in rats by introducing a xanthine-xanthine oxidase complex into ST. Baudouin C, Ettaiche H., Imbert F .: Inhibition of preretinal proliferation by free radical scavengers in an experimental model of tractional retinal detachment.// Exp. Eye Res., 1994, V. 59 (6), P. 697-706.

It is known to use the natural homologous cytokine complex of the drug Superlim ImBio (Nizhny Novgorod, registration certificate No. 000516 / 01-2001) obtained from pig blood with the activity of IL-1, IL-6, TNF-a, TGF-b, MIF (Khoroshilova-Maslova I.P., Babizhaev Μ.Α., Ilatovskaya L.V., Kiseleva O.Α., Gankovskaya L.V. Protocol of the method for the prevention of proliferative vitreoretinopathy. Patent for the invention No. 2175223. Moscow, 2001).

With the introduction of this complex to rabbits, the inflammatory process in the uveal tract and the accumulation of macrophages and lymphocytes on the surface of the inner boundary membrane of the retina with its damage took place intravitreal in the vitreous body. A study to simulate PVR using the Superlymph drug was continued to optimize the dose of the cytokine complex. A model of PVR with 90% reproducibility was obtained by administering Superlimf at a dose of 0.15 ml 1000 μm through the flat part of the ciliary body to rabbits (Ph.D. dissertation, N.L. Leparskaya, Role of proliferative vitreoretinopathy in pathogenesis, clinic and treatment of traumatic retinal detachment (clinical experimental study) Moscow. 2005. pp. 114-121).

It is known to simulate PVR that intravitreal administration to rabbits of one pro-inflammatory cytokine - human interleukin IL-1b in 250 units in 0.1 ml simultaneously with 10 mechanical injuries of the retina by an insulin needle. At the same time, the importance of inflammation and trauma as triggering factors for the development of PVR (HGF regulation of RPE proliferation in an IL-1b / retinal hole-induced rabbit model of PVR. Gregory I. Liou, Vytautas A. Pakalnis, Suraporn Matragoon and ets. Mol Vis. 2002 Dec 20; 8: 494-501.

There is a known method of simulating PVR, which made it possible to study the role of platelets as the main component of the blood coagulating system in the formation of PVR, in which the platelet mass serves as a model material (Baudouin Ch, Utache M., Imbert F., Droy-Letaix M., Gustaud D., Lapalus Ph. Inhibition of preretinal proliferation by free radical scavengers in an experimental model of fractional retinal detachment. Experimental Eye Research. 1994. 59. 697-706).

There is a known method for simulating PVR, in which activated ADP platelets are injected into the eye cavity, characterized by pronounced expression of fibronectin, which facilitated platelet aggregation and adhesion on the inner boundary membrane of the retina with its destruction and subsequent development of PVR (Babizhayev MA, Seguin MS, Gueyne J., Evstigneeva RP, Agryeva EA, Zheltukhina. L Camosine (b-alanyl-L-histidine) and carcinine (b-alanylhistidine) act as natural antioxidants with hydroxyl radical scavenging and lipid peroxidase activities.// Biochemical journ., 1994, 304, p. 509-516).

There is a known method for simulating PVR in rabbits, in which heterogeneous platelets were used to standardize the simulation with the activation of the platelet mass of the donor by the introduction of thrombin, followed by stimulation in a centrifuge. With intravitreal administration of activated platelets, PVR developed in 91.7% of cases. Depending on the injected platelet mass dose, either the glial type of proliferation (administration of 0.5 ml) with complete retinal lysis or the epithelial type of proliferation with partial retinal lysis and the formation of epiretinal membranes, 0.3 ml, were observed. This model revealed the role of proliferation of individual retinal structures, but was of little use for the study of various drugs in order to prevent the proliferative process. (Candidate dissertation NL Leparskaya. Role of proliferative vitreoretinopathy in the pathogenesis, clinic and treatment of traumatic retinal detachment (clinical and experimental study) Moscow. 2005. Pages 105-114).

The high activity of autologous cells of the retinal pigment epithelium in the process of development of proliferative changes in the retina made it possible to use them as inducers of PVR in the pigment epithelial cell model. In rabbits, retinal pigment epithelial cells were obtained using trypsin from enucleated eyes, mixed with Eagle's medium, and then a suspension containing 3.5–9.6 × 102 cells was introduced into the remaining eye. After 1-3 months, histological examination revealed the formation of cell aggregates on the inner border membrane (Khoroshilova-Maslova I.P., Ilatovskaya L.V., et al. Patent method for modeling proliferative vitreoretinopathy, patent RU 2161335, 2000). The three-component model according to the authors gave 100% reproduction of the TAC in a period of 1 month.

Three components were introduced intravitreal to rabbits: a homologous retinal pigment epithelium, a natural complex of cytokines, and plasma fibronectin (an adhesive protein that enhances cell aggregation, which plays an informational role in fibrillogenesis).

However, the described models differ significantly from the natural clinical course of PVR, since there is an inadequate severity of the proliferative and inflammatory response, there is no uniformity of morphological signs of the pathological process.

The closest analogue of the present invention is a method for simulating PVR, based on a single administration to rabbits of an intravitreal worked-out dose of the pro-inflammatory cytokine inerleukin IL-1b. The introduction of a dose of 2000 Ε is accompanied by the formation of epiretinal membranes, the model has 100% reproducibility. The disadvantage of this modeling method is the lengthy process of PVR formation with insufficient severity of the clinical picture. (Development of a pathogenetically substantiated standard experimental model of proliferative vitreoretinopathy induced by cytokines. IP Khoroshchilova-Maslova, NL Leparskaya, MK Nabieva, LD Andreeva. ROSH. Vol. 6, No. 1, 2013. Pages 78-80).

In our opinion, the PVR model should reflect the staged process of migration and proliferation of cells on the inner border membrane of the retina with its damage and the formation by proliferating cells of newly formed membranes with contractile properties. A similar model will reflect the course of the process in the human eye.

Thus, it remains relevant to create a standard model of PVR, reflecting the pathogenesis of PVR, corresponding to the natural course of the process under clinical conditions, which was the object of the invention.

The objective of the invention was the creation of a model of PVR, reflecting the pathogenetic features of cytopathogenesis of PVR.

The technical result of the proposed method is to obtain a clinically and cytopathogetically adequate model of PVR with the severity of the proliferative and inflammatory response, the same morphological signs of the pathological process, the staged process of migration and proliferation of cells on the inner border membrane of the retina with its damage and the formation of proliferating cells of newly formed membranes with contractile properties.

The technical result is achieved through the use of a model corresponding to the natural course of the process under clinical conditions using intravitreal single sequential administration of Interleukin 1-b 2000 Ε and platelet-derived growth factor PDGF AA human osteosarcoma at a concentration of 2000 pcg / ml.

One of the most important factors currently known that regulate the process of membrane formation on the surface of the retina and the activity of the PVR process is PDGF (platelet-derived growth factor), a platelet-derived growth factor. The role of PDGF in the regulation of growth and proliferation of retinal pigment epithelium is known. PDGF is expressed by retinal pigment epithelial cells, muller cells, glial cells, PDGF was detected in epiretinal membranes (Campochiaro et al. Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media. (1989) Exp. Eye Res. 49, 217-227; Mudhar et al., (1993) PDGF and its receptors in the developing rodent retina and optic nerve. Development 118, 539-552; Yoshida et al, (1992) Platelet-derived Growth Factor gene expression in cultured human retinal pigment epithelial cells. Biochem. Biophys. Res. Comm. 189: 66-71).

PDGF isoforms are known: PDGF-AA, PDGF - AB, PDGF - BB. Studies have shown that the activity of PVR development is dependent on the quantitative level of PDGF-A and PDGF-B. Of greatest importance in membrane formation is the isoform of PDGF - A. (Vinores SA, Henderer JD, and ets. Isoforms of Platelet-derived Growth Factor and its Receptors in Epiretinal membranes: immunolocalization to Retinal Pigmented Epithelial cells. Experimental Eye Res. 1995. 60, 607-619).

On clinical material (a study of subretinal fluid), it was found that with an increase in PDGF - AA of more than 400 pcg / ml, diffuse PVR developed in 100% of cases. (Ph.D. Kuznetsova IS. Prediction and early diagnosis of progression of PVR after successful surgical treatment of regmatogenous retinal detachment. Moscow. 2012. Pp. 152-153)

Thus, to optimize the interleukin model of PVR induced by IL-1b as an activator, we decided to use PDGF.

In the first series of experimental morphological studies, the effect of various doses of PDGF on eye tissue was studied. In the experiment, 6 rabbits weighing 2.5-3.0 kg of Chinchilla breed were used. The animals were kept in the vivarium of the Federal State Institution Scientific Research Institute GB named after Helmholtz in terms of relevant regulatory documents of the Rosselkhoznadzor No. 1045-73.

Platelet growth factor PDGF AA human osteosarcoma was administered once in various doses once intravitreal in a volume of 0.1 ml: group 1 - at a dose of 2000 pkg / ml, 2 rabbits (4 eyes), group 2 - at a dose of 5000 pcg / ml, 2 rabbits (4 eyes ), Group 3 - at a dose of 20,000 pkg / ml 2 rabbits (4 eyes). The injection was performed with an insulin needle under local anesthesia with a 0.5% Alcain solution (Alcon). The duration of the experiment was 1 month. After 1 month animals were removed from the experiment by air embolism.

The results of the first series of experimental morphological studies showed that the clinical analysis in all cases, conducted by methods of direct and reverse ophthalmoscopy on the dates 7, 14, 21 and the day of slaughter, did not reveal the uveitis clinic, changes in the anterior section of the eye. On the fundus there was a clinic of “baldness” of the pigment epithelium, depigmentation in the central parts of the fundus. In group 3, the depigmentation zones were more extensive.

Morphological studies were carried out according to the following method: enucleated eyes were fixed in 10% buffered formalin, then opened by dissection into 3 parts. The central part was embedded in paraffin. Serial paraffin sections were stained with hematoxylin and eosin, as well as picrofuxin according to Van Gieson. For microscopic examination, a Leica microscopic system was used.

Microscopic examination of the eyes of group 1 rabbits showed that the anterior segment of all 4 eyeballs was unchanged. The main changes were localized in the cells of the retinal pigment epithelium (RPE). The dissociation of RPE cells associated with their active migration was noted. The cells that emerged from the reservoir had a rounded shape, with the nucleus moving to the periphery. The disappearance of pigment and the appearance of processes in migrating cells were noted. Along with active migration, sections of exposure in the reservoir appeared. In the outer layers of the choroidal zone - the accumulation of migrating RPE cells. The same cells settled on the inner surface of the retina and on the fibrils of the vitreous. The destruction of the outer segments of the rods and cones with retinal detachment from RPE, the decrease in the number of nuclear layers in the outer granular layer of the retina with a sharp thinning of the outer plexiform layer were revealed.

Microscopic examination of the eyes of rabbits of group 2 showed that the same changes were found as in group 1, localized in RPE cells. Dissociation of RPE cells, their migration, foci of destruction of RPE cells with the development of exposure of the RPE layer and limited retinal detachments were noted. Due to the pigment deposits in the outer layers of the choroid, a pigment layer has formed. At the same time, in the eyes of group 2, atrophic changes in the retina were more pronounced: the number of cell rows in the nuclear layers decreased to 3-5. The plexiform layer was not visualized. Retinal gliosis was revealed, which was characterized by hyperplasia of Mueller cells, which replaces zones of atrophic ganglion cells. Newly formed vessels appeared in this group, which were localized on the surface of the optic disc.

Microscopic examination of the eyes of rabbits of group 3, revealed that the anterior segment is not changed. The main changes are concentrated in the posterior part: extensive RPE dissociation, its exit from the formation with the formation of exposure sites, migrating cells in small quantities were determined on the inner surface of the retina, pronounced destruction of the layer of rods and cones with the formation of a flat retinal detachment. In the retina there is atrophy, a sharp reduction in the number of layers in the granular layers (external and internal). Thinning of the inner plexiform layer and the absence of the outer, atrophy of the layer of ganglion cells with compensatory hyperplasia of Mueller cells.

Thus, in the first series of experimental work with the introduction of intravitreal platelet-derived growth factor (PDGF), the dose-dependent nature of the changes is shown. The main target of action was RPE cells, i.e., the stimulation of the formation of active migrating RPE cells obtained in the experiment can characterize the development of PVR with the introduction of intravitreal PDGF in the amount of 2000 pg / ml. At higher doses of PDGF administration (5000 and 20,000 pg / ml), a progressive change in RPE cells and a violation of the metabolism of the outer layers of the retina, where photoreceptors are localized, were noted. The death of the photoreceptor layer at high doses leads to retinal atrophy, where the layer of rods and cones disappears, the outer granular layer and the outer plexiform layer sharply thin, and the layer of ganglion cells atrophies. As a result of atrophic processes, compensatory hyperplasia of the retinal supporting cells — Mueller cells — occurs. That is, as a model mass, it is reasonable to choose a dose of 2000 pkg / ml, due to which activation of RPE cell migration is triggered.

Thus, the dose of the activator of the interleukin model was worked out - the dose of PDGF AA 2000 human osteosarcoma PCG / ml.

In the second series of experimental work, we used the worked-up doses, introducing interleukin once successively Interleukin 1-b at a dose of 2000 Ε in a volume of 0.1 ml and platelet growth factor PDGF AA human osteosarcoma at a dose of 2000 pcg / ml in a volume of 0.1 ml.

8 rabbits (16 eyes) were used. The injection was performed with an insulin needle under local anesthesia with a 0.5% Alcain solution (Alcon). The duration of the experiment was 1 month. After 1 month Animals were removed from the experiment by air embolism.

Clinical analysis in all cases, carried out by methods of direct and reverse ophthalmoscopy on the dates 7, 14, 21 and the day of slaughter, did not reveal the clinic of uveitis, changes in the anterior section of the eye. On the fundus there was a clinic of “baldness” of the pigment epithelium, depigmentation in the central parts of the fundus, a slight “dullness” of the surface of the retina was noted on the retina.

The main evidence-based research method was the morphological identification of TAC. Morphological studies were carried out according to the method described above.

Research results

Morphological studies in all cases (16 eyes) revealed the development of PVR in the form of a thin-fiber epiretinal membrane (ERM) on the inner surface of the retina. The ERM was focal in nature, in places it was soldered to the internal boundary membrane (VPM), and in some places it was far from it. The retina in the foci of PVR was folded, retaining its usual layered structure. Attention was drawn to the very weak severity of the inflammatory process: single lymphocytes are scattered in the vitreous, around the ciliary processes. Changes in RPE were natural: RPE cells settled on the vitreous membranes. As a result of such migration, foci of “baldness” with exposure of the Brukhov membrane were formed in the pigment epithelium. RPE cells settling on the inner boundary membrane began to actively proliferate. RPE cells underwent transformation in proliferates: they lost pigment, changed their shape, and actively synthesized the intercellular matrix, including collagen (Fig. 3). As a result of such a transformation, a delicate fibrous membrane formation was formed on the inner surface of the retina, which in places was intimately soldered to the retina, and in some places it was freely separated from it.

The regularity of the changes made allows us to recommend this model for use in studying the effectiveness of drug treatment and prevention of PVR in the experiment.

The method is as follows. The rabbit is given local anesthesia, for example, using a 3-fold installation with an interval of 3 minutes of 0.5% Alkain solution. A puncture is performed through the flat part of the ciliary body at a distance of 2.5 mm from the limb with the 32G insulin needle with the introduction of intravitreal Interleukin 1-b at a dose of 2000 Ε in a volume of 0.1 ml, then platelet-derived growth factor PDGF AA human osteosarcoma at a dose of 2000 pkg / ml in a volume of 0.1 ml

Example 1. Rabbit No. 1. Modeling: Alkain solution 0.5% (Alcon) was installed in both eyes 3 times with an interval of 3 minutes. Intravitreal injection was performed in each eye once sequentially. A puncture was performed through the flat part of the ciliary body at a distance of 2.5 mm from the limb with a 32G insulin needle with the introduction of intravitreal Interleukin 1-b at a dose of 2000 Ε in a volume of 0.1 ml, then platelet growth factor PDGF AA human osteosarcoma at a dose of 2000 pcg / ml in a volume of 0.1 ml

The duration of the experiment was 1 month. After 1 month, the rabbit was slaughtered by air embolism. The eyes were enucleated, subjected to histological examination: fixation in 10% buffered formalin, dehydration in alcohols of an ascending strength. The eyes were opened by dissection into 3 parts. The central part was embedded in paraffin. Serial paraffin sections were stained with hematoxylin and eosin, as well as picrofuxin according to Van Gieson. Histological examination showed rounding of RPE cells, exposing Bruch's membrane, their dissociation and active migration into the vitreous body (Fig. 1), as well as proliferation of RPE cells on the inner border membrane (Fig. 2) with the formation of newly formed epiretinal membranes (Fig. 3) .

Example 2. Rabbit No. 5. Modeling: Alkain 0.5% solution (Alcon) was installed 3 times in both eyes with an interval of 3 minutes. Intravitreal injection was performed in both eyes once sequentially. A puncture was performed through the flat part of the ciliary body at a distance of 2.5 mm from the limb with a 32G insulin needle with the introduction of intravitreal Interleukin 1-b at a dose of 2000 Ε in a volume of 0.1 ml, then platelet growth factor PDGF AA human osteosarcoma at a dose of 2000 pcg / ml in a volume of 0.1 ml

The duration of the experiment was 1 month. After 1 month, the rabbit was slaughtered by air embolism. The eyes were enucleated, subjected to histological examination: fixation in 10% buffered formalin, dehydration in alcohols of an ascending strength. The eyes were opened by dissection into 3 parts. The central part was embedded in paraffin. Serial paraffin sections were stained with hematoxylin and eosin, as well as picrofuxin according to Van Gieson. Histological examination showed active dissociation of RE cells into the vitreous body (Fig. 4). Metaplasia and proliferation of RPE cells on the inner border membrane (Fig. 5), the formation of an epiretinal membrane on the surface of the retina (Fig. 6).

Thus, the presented examples demonstrate the changes that occur in the RPE and on the surface of the retina with the onset of the formation of epiretinal membranes, i.e. the obtained PVR model is clinically and cytopathogenetically adequate and can be used to determine the effectiveness of the antiproliferative effect of drugs.

Claims (1)

A method for modeling proliferative vitreoretinopathy in rabbits, including a single intravitreal administration of 2000 Ε Interleukin 1-b in a volume of 0.1 ml, characterized in that platelet growth factor PDGF AA osteosarcoma 0.1 ml with a concentration of 2000 pg / ml is sequentially intravitreal once administered.

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