RU2563368C1 - Method for simulating proliferative vitreoretinopathy in rats - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: normal saline 2 mcl containing concanavalin A 0.5 mcg - 0.5 mg is administered into a rat's vitreous cavity.

EFFECT: method enables producing an adequate model of proliferative vitreoretinopathy 8 weeks later, which is necessary to develop methods for preventing and treating the above pathology.

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Description

Technical field

The invention relates to medicine, in particular to ophthalmology, and can be used in the development of methods for the prevention and treatment of proliferative vitreoretinopathy (PVR).

State of the art

PVR is a pathological condition resulting from trauma, diabetic retinopathy, and regmatogenous retinal detachment. PVR is characterized by the proliferation of pigment epithelial cells, glial cells, macrophages and fibroblasts on the surface of the retina, which leads to the formation of membranes, which subsequently acquire contractile ability. The process is accompanied by the formation of folding of the exfoliated retina and its pronounced shortening. PVR is the most common cause of relapse of retinal detachment after surgery.

Prevention and conservative treatment of this pathology have not been developed. Very promising is the development of treatment on experimental models.

There are various approaches to the modeling of PVR: the introduction of different types of cells into the vitreous body (I. Zapuskalov et al. Patent RU 2182369. IP Khoroshilova et al. Patent RU 2161335. Sugita G. et al. Intravitreal autotransplantation of fibroblasts. // Am J Ophthalmol. 1980. Vol. 89, No. 1. P. 121-130. Fastenberg D. et al. A comparison of different cellular inocula in an experimental model of massive periretinal proliferation. // Am. J. Ophthalmol. 1982. Vol. 93, No. 5. P. 559-564. Hui YN, Sorgente N., Ryan S. Posterior vitreous separation and retinal detachment induced by macrophages // Graefe's Arch. Clin. Exp. Ophthalmol. 1987. Vol. 225. P. 279-284. García-Layana A. et al. Porcine model of proliferative vitreoretinopathy with platelets // Curr Eye Res. 1997. Vol. 16, No. 6. P. 556-563) eye populations (Chinn C. et al. Strain-dependent gene expression in a lens extraction PVR model // Invest Ophthalmol Vis Sci. 2005. Vol.46. P. E-Abstract 5528. Cleary P., Ryan S. .. Experimental posterior penetrating eye injury in the rabbit. II. Histology of wound, vitreous, and retina. // Br J Ophthalmol. 1979. Vol. 63, No. 5. P. 312-321), enzyme administration (Frenzel EM et al. A new model of proliferative vitreoretinopathy. // Invest. Ophthalmol. Vis. Sci. 1998. Vol. 39, No. 11. P. 2157-2164. Valeria Canto Soler M. et al. A Mouse Model of Proliferative Vitreoretinopathy Induced by Dispase. // Experimental Eye Research 2002. 75 (5), 491-504. Tan J. et al. Ocular pathogenesis and immune reaction after intravitreal dispase injection in mice. / / Mol Vis. 2012; 18: 887-900).

All of the above methods for simulating TACs have disadvantages.

In models with intravitreal injection of cells, as a rule, only one type is introduced, and the initiation of pathology begins from the stage of the process in which these cells participate. The proliferative process in the body affects various types of cells, and their proliferation in the formation of membranes is not the first link in the pathological process, i.e. it is impossible to study the initiation mechanisms on such models, it is impossible to investigate the mechanisms and therapeutic approaches of postoperative complications. The method of modeling PVR using various surgical procedures (removal of the lens and vitreous body, cryopexy, detachment of the retina or its removal, penetrating eye injury) leads to severe damage to the eye and a violation of its structure and integrity. These models can be used to study the risks of developing PVR only with specific eye pathologies. The introduction of enzymes inside the eye leads to the destruction of the intercellular matrix and the violation of the blood-ophthalmic barrier, and hemophthalmus, as an additional risk factor, increases the aggressiveness of the model and the likelihood of developing PVR. However, inflammation is a key element in the development of PVR in all of these models, and the inflammatory response may be the trigger for the development of PVR. Concanavalin will lead to the activation of inflammatory mediators with minimal impairment of the permeability of the blood-ophthalmic barrier (with a minimum likelihood of developing hemophthalms) and with a minimal destructive component.

Closest to the claimed method is a method for modeling proliferative vitreoretinopathy in rats by intravitreal administration of a suspension of cellular elements in physiological saline (I. Zapuskalov et al. Patent RU 2182369). There are no closer methods for modeling the TAC to the declared one.

The main disadvantage of the prototype is that the initiation of the formation of epiretinal membranes begins with the introduction of cells into the vitreous body, while in the real pathogenetic picture in humans, cell migration is not an early stage of the process. In addition, when cells of a certain type are introduced into the vitreous body, at the initial stages the set of secreted factors by these cells is limited, since these are cells of the same type. Thus, this model does not reflect the detailed dynamics of the full-fledged pathogenetic mechanism of PVR.

The concanavalin model is most relevant because it reflects the inflammatory pathogenesis of PVR, providing selectivity for the development of the inflammatory response by activating lymphocytes of various groups (Dwyer JM, Johnson C. The use of concanavalin A to study the immunoregulation of human T cells. // Clin Exp Immunol. 1981 Nov; 46 (2): 237-49. Lei HY, Chang CP. Lectin of Concanavalin A as an anti-hepatoma therapeutic agent. // J Biomed Sci. 2009 Jan 19; 16:10. Toshiaki Nakano et al. Immunological aspects and therapeutic significance of an autoantibody against histone HI in a rat model of concanavalin A-induced hepatitis. // Immunology. 2010 April; 129 (4): 547-555). Concanavalin A was used in the cataractogenesis model to form the posterior subcapsular cataract in albino rabbits; however, all the eyes into which concanavalin was injected contained fibrin in the vitreous. Epiretinal hemorrhage has been observed in some cases (Gwon A. et al. Concanavalin A-induced posterior subcapsular cataract: a new model of cataractogenesis. // Invest Ophthalmol Vis Sci. 1993 Dec; 34 (13): 3483-8). There are currently no analogues of the concanavalin model of TAC.

Disclosure of invention

The objective of the invention is to provide a new method for modeling proliferative vitreoretinopathy in rats with a minimal destructive component, which will be caused by endogenous cells and factors; to achieve high performance and stability of the result, namely, growth on the surface of the retina of the membranes. The result is achieved by introducing a solution of concanavalin A into the vitreous cavity of the eye of the rat, which leads to the development of inflammation in the eye with almost no violation of the blood-brain barrier.

The technical result of the invention is to obtain PVR in the eye of rats, reflecting the pathogenesis of the process, close to changes in humans, and ensuring the stability of the result. In this case, a relatively “pure” inflammatory reaction with a minimal destructive component develops. The technical result is achieved through the use of concanavalin A - mannose-binding lectin of plant origin.

According to the present invention, a method for simulating proliferative vitreoretinopathy in rats is characterized in that a solution of concanavalin A is injected into the vitreous cavity of the rat eye in a dose range of 0.5 μg to 0.5 mg in a volume of 2 μl, while membrane formation on the retina surface is observed after 8 weeks after administration of the pro-inflammatory agent. The initial stages of the development of proliferative vitreoretinopathy are observed 1-2 weeks after the administration of concanavalin A.

8 weeks after the introduction of lectin, enucleation of the eye can be performed for subsequent histological analysis of the membranes formed on the surface of the retina.

Concanavalin A has T-mitogenic activity and activates mast cells, causing a pseudo-allergic reaction. This leads to the activation of the inflammatory process and proliferation of glial cells, fibroblasts and immune cells with minimal disturbance of the blood-phthalmic barrier. The formation of pro-inflammatory factors stimulates the development of a proliferative response, which is one of the key points in the pathogenesis of proliferative vitreoretinopathy.

Brief Description of the Drawings

The invention is illustrated by figures 1-5.

In FIG. 1 shows the proliferation of a layer of glial retinal cells and the development of wavy changes, especially affecting the outer layer. In FIG. 2 - compaction of the structures of the cortical layers of the vitreous body, the formation of rosette structures. In FIG. 3 depicts an epiretinal membrane formed on the surface of the retina in response to the introduction of concanavalin into the vitreous cavity; FIG. 4 shows pronounced wavy changes in the retina and the beginning of the formation of rosette structures, FIG. 5 - retinal detachment.

Position 1 denotes the retina, position 2 - the epiretinal membrane, position 3 - traction of the retina, position 4 - the vitreous body, position 5 - wavy retinal changes, the beginning of the formation of rosette structures, position 6 - retinal detachment, position 7 - the lens, position 8 - proliferation layer of glial cells.

Examples

Rat No. 20. Alkain 0.5%, tropicamide 1.0% and vitabact 0.05% were instilled into the right eye under general anesthesia. Concanavalin diluted in isotonic solution, a dose of 0.5 mg in a volume of 2 μl was injected into the vitreous cavity through a puncture in a flat part of the ciliary body with a 30G needle. In the postoperative period, in the early days, there was a slight mydriasis, floating opacities in the vitreous. After 5 days, wavy changes in the layers of the retina formed; in the vitreous body, the initial compaction of the structures of the cortical layers. The cornea is not changed. Postcapsular cataract.

Histologically. In the vitreous body near the inner surface of the retina, densification of the structures of the cortical layers, in the retina, the proliferation of the layer of glial cells. There is a development of wavy changes, especially affecting the outer layer (Fig. 1-2).

Pathological diagnosis: the initial stage of PVR.

Rat No. 12. Alkain 0.5%, tropicamide 1.0% and vitabact 0.05% were instilled into the right eye under general anesthesia. Concanavalin diluted in isotonic solution, a dose of 0.5 μg in a volume of 2 μl was injected into the vitreous cavity through a puncture in the flat part of the ciliary body with a 30G needle. In the postoperative period, in the early days, there was a slight mydriasis, floating opacities in the vitreous. After 8 weeks, cords, retinal folding and the formation of rosette structures, and local detachment formed in the vitreous body. The cornea is not changed. Postcapsular cataract. Histologically. In the vitreous body near the inner surface of the retina - compaction of the structures of the cortical layers. There is a violation of the integrity of some layers, including local degradation of the layer of rods and cones; the development of wavy changes, especially affecting the outer nuclear and inner nuclear layers. The reciprocal penetration of retinal layers and the formation of rosette structures are visible. Retinal detachment is visible on the cut. Epiretinal membranes that organize the “pulling” traction component are revealed above the retina (Fig. 4).

The contractile properties in the newly formed epiretinal membrane were manifested in the formation of folds and foci of the exfoliated retina (Figs. 4, 5).

Pathological diagnosis: proliferative vitreoretinopathy, retinal detachment.

The implementation of the invention

The method is as follows.

1. All manipulations were performed on Wistar rats anesthetized with chloral hydrate in male rats (400 mg / kg ip).

2. A local anesthetic alkaine 0.5% (Alcon) was instilled into the right eye of the rat, the pupil was dilated using a 1.0% solution of tropicamide (Rompharm company), Vitabact 0.05% (Novartis) was used as an antiseptic.

3. Concanavalin diluted in isotonic solution in an amount of 0.5 μg - 0.5 mg in a volume of 2 μl was injected into the vitreous cavity through a puncture in a flat part of the ciliary body with a 30G needle.

The proposed method was used in an experiment on 35 rats in the case of an administered dose of 0.5 mg to assess the initial stage of PVR development (1-7 days) and in 15 rats in the case of an administered dose of 0.5 μg to assess the long-term effects of administration of the pro-inflammatory agent (14 28 and 56 days). In all experimental animals, an identical result was obtained on days 1–7 — the initial stages of compaction of the structures of the cortical layers in the vitreous body and wavy retinal changes, which is confirmed by histological examination. On day 56, the development of PVR is observed in all experimental animals, which is also confirmed by histological examination.

After the experiment, clinical observation was carried out for 1-7 days (dose of 0.5 mg) and 1, 2, 4 and 8 weeks (dose of 0.5 μg). Then, enucleation of the eye and decapitation of the animal were performed. The eyes were fixed in Davidson's solution and subjected to histological examination. After fixation, the eyes were dehydrated in ascending alcohol and embedded in paraffin. Paraffin sections were stained with hematoxylin-eosin. Clinically, after the introduction of concanavalin A into the eyes of experimental animals, mild mydriasis was observed in the first three days, and cataracts were observed in 100% of the animals. The maximum recorded number of hemophthalmus (20% of the eyes) occurs on days 1 and 7 after the administration of the pro-inflammatory agent. After 8 weeks, the formation of pronounced epiretinal membranes was observed in 100% of the rats, hemophthalmus was in 20% of the eyes, and cataract by the 8th week of the experiment was observed in 100%.

As a control, we used rats (N = 20), which received an intravitreal injection of an isotonic sodium chloride solution (0.9%). In these rats, the development of PVR was not observed throughout the experiment.

Claims (1)

A method for modeling proliferative vitreoretinopathy in rats, characterized in that 2 μl of an isotonic solution containing 0.5 μg - 0.5 mg of concanavalin A is injected into the cavity of the vitreous body of the rat eye.

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