RU2709247C1 - Method of simulating retinal pigment epithelium atrophy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to ophthalmology, and can be used in experimental ophthalmology for simulation of retinal pigment epithelium atrophy. Bevacizumab is introduced into the subretinal space at 0.5 pd below the optic disk in amount of 10 mcl containing 0.025 mg of the preparation to form a subretinal bladder.

EFFECT: method enables to obtain a simple, adequate clinical-functional and morphological model of retinal atrophy, reflecting the course of the process closest to its occurrence with the background of anti-VEGF-therapy.

1 cl, 8 dwg

Description

The invention relates to medicine, namely to ophthalmology, and can be used in experimental ophthalmology to model atrophy of retinal pigment epithelium in order to analyze the effectiveness of various methods of treatment and prevention of this pathology.

In the era of the widespread use of anti-VEGF therapy in world practice, ophthalmologists noted the development of atrophic changes against the background of repeated administration of angiogenesis inhibitors and their effect on the functional outcome of treatment.

The effectiveness of anti-VEGF drugs in stopping the exudative reaction is not in doubt. However, there is a need to determine the duration of treatment using angiogenesis inhibitors in an adequate amount. In one of the largest studies of CATT and IVAN, a direct relationship between the development of macular atrophy and the repeated administration of angiogenesis inhibitors was analyzed and revealed. [Ranibizumab and Bevacizumab for Treatment of Neovascular Age-related Macular Degeneration Comparison of Age-related Macular Degeneration Treatments Trials (CATT) Research Group Daniel F. Martin et al. Ophthalmology July 2012 Vol. 119, Issue 7, P. 1388-1398.] However, this issue is still debatable because studies were conducted that did not reveal a significant relationship between the number of injections of an angiogenesis inhibitor and the development of secondary atrophy during their use.

In accordance with this, it seems relevant to study and analyze the mechanisms of development of secondary atrophy of the retinal pigment epithelium, as well as the search for an optimal, safe method for its treatment.

In modern ophthalmology, there is an active search for the most optimal experimental model of RPE atrophy in reproducibility. To date, several models of RPE atrophy have been developed that reveal different mechanisms and pathogenetic features of its occurrence.

A known method of modeling atrophy of RPE, recreated by activation of the innate mechanisms of apoptosis in mice, rats and rabbits. Systemic intravenous administration of Polyinosine-polycytidylic acid (Poly-I) activates the Toll-like 3 (TLR3) receptor, which triggers apoptosis and is accompanied by the formation of drusen, geographical atrophy and retinal detachment. The severity of degenerative changes was in direct correlation with the amount of acid introduced, then the results were evaluated using optical coherence tomography, electroretinography, and subsequent histological examination. [Ni M. et al. Animal Models of Dry Type Retinal Degeneration // Investigative Ophthalmology & Visual Science. - 2009. - T. 50. - No. 13. - C. 776-776.]

A known method of modeling geographical retinal atrophy, in which the retina was coagulated by a diode laser with a wavelength of 810 nm. Signs of geographical atrophy (atrophy of RPE cells, increased lysosomes of RPE, loss of photoreceptors, loss of the outer nuclear layer and thinning of the choroid) were determined in mice by week 12. In this study, the results were evaluated using electroretinography and subsequent histological examination. [Goverdhan S. et al. A Mouse Model of Geographic Atrophy Resembling Human Geographic Age-Related Macular Degeneration // Investigative Ophthalmology & Visual Science. - 2015. - T. 56. - No. 7. - C. 2365-2365].

A known method for modeling atrophy of retinal pigment epithelium by retrobulbar administration of NaIO3 in low doses. In an experiment in mice, first in vitro, then in vivo, preclinical models of geographical retinal atrophy were obtained [Hanus J. et al. Retinal pigment epithelial cell necroptosis in response to sodium iodate // Cell death discovery. - 2016. - T. 2. - C. 16054.] The introduction of NaIO3 induces oxidative stress, which provokes the processes of apoptosis and necroptosis: there are changes in the form of primary lesion of pigment epithelial cells, and secondary damage to the photoreceptor layer in the form of severe geographical atrophy, and then followed by atrophy of the choriocapillary layer.

The closest analogue of the proposed invention is a method for modeling atrophy of retinal pigment epithelium by introducing Mitomycin C and 0.25% ethylenediaminetetraacetic acid (EDTA) through transscleral access to the subretinal space in pigs. Changes in the form of atrophy of the pigment epithelium, accompanied by secondary choroidal atrophy, were recorded 1 week after drug administration. [DEL PRIORE L. V. et al. Retinal pigment epithelial debridement as a model for the pathogenesis and treatment of macular degeneration // American journal of ophthalmology. - 1996. - T. 122. - No. 5. - C. 629-643].

The above simulation methods were obtained as a result of chemical, biological or physical exposure. These induced models are oriented towards histological proximity to existing degenerative diseases of the retina, some of which are insufficiently supported by the functional and clinical data necessary for an adequate assessment of the modeling of RPE atrophy. A model is needed, supported by full-fledged clinical-functional and morphological data and reflecting the course of the process closest to its occurrence against the background of repeated use of angiogenesis inhibitors in the treatment of degenerative diseases of the retina, which motivates the search for new ways to reproduce it.

The objective of the invention is to develop a simple, easy-to-reproduce clinical-functional and morphological model of retinal atrophy.

The technical result of the invention is to obtain a simple, adequate clinical, functional and morphological model of retinal atrophy, reflecting the course of the process closest to its occurrence against the background of anti-VEGF therapy.

The technical result is achieved by introducing into the subretinal space with the formation of a subretinal bladder of bevacizumab in a volume of 10 μl containing 0.025 mg of the drug, which is administered at a distance of 0.5 pd below the optic disc.

As a preparation for modeling RPE atrophy, we chose Bevacizumab (Avastin), as the cheapest and most affordable angiogenesis inhibitor in medicine.

The volume and amount of bevacizumab administered were determined in studies on albino rabbits. During the experiment, it was shown that subretinal administration of bevacizumab in a volume of 10 μl containing 0.025 mg of the drug with the formation of a subretinal bladder leads to irreversible changes in the form of degeneration of the photoreceptor layer of the retina and atrophy of the retinal pigment epithelium of the retina.

The solution is injected at a distance of 0.5 pd below the optic nerve disk for the best reproducibility and repeatability of the model, visualization using optical coherence tomography, the use of a constant reference point (optic nerve disk) and accurate standardized atrophy area topography.

The method is as follows

Bevacizumab is injected into the subretinal space at a distance of 0.5 pd below the optic disc in a volume of 10 μl containing 0.025 mg of the drug, with the formation of a subretinal bladder.

To simulate animals, they were anesthetized with intramuscular injection of Zoletil and Xylazine. Mydriasis was achieved by instillation of Appamide. The operation was performed on the right eye, leaving a pair of eyes for control. Before surgery, Vigamox and Alcain were instilled into the conjunctival cavity. The eye muscles were isolated and taken on the suture-holders. In the projection of the flat part of the ciliary body, sclerotomy was performed with the installation of 23 Ga ports at 2, 10, and 5 hours, respectively. An irrigation cannula was installed at 5 o’clock. Partial vitrectomy was performed. Bevacizumab was administered using a 1 ml syringe connected to an extension tube (guided by an assistant) and a 41G DORK cannula (guided by a surgeon), previously cut at an angle of 45 °. A cannula was inserted through the upper temporal port into the subretinal space at a distance of 0.5 pd below the optic disc. After correctly positioning the tip, which was visualized by blanching of the retina, the preparation in a volume of 10 μl containing 0.025 mg of bevacizumab was administered subretinally until a bladder formed, which was well visualized by the surgeon using an operating microscope. After surgery, all rabbits were subconjunctively injected with a solution of Gentamicin and Dexamethasone at a dose of 3 mg / kg and 0.2 mg / kg, respectively. In the next 7 days, a solution of Dexamethasone 0.1% 3p / d was instilled into the conjunctival cavity.

Experimental studies were conducted on 10 rabbits (10 eyes) of albino breed, weighing 2.0-2.5 kg. The duration of the experiment was one month. A month later, the animals were withdrawn from the experiment by air embolism after the rabbit was anesthetized (according to the order of the USSR Ministry of Higher Education No. 724 of 11/13/184). Eyeballs were enucleated whole, after which they were fixed in a neutral 10% formalin solution for 1 day. After fixation, the eyeballs were cut into 3 blocks so that the zone of induced atrophy was in the central block. After standard histological wiring, the central pads were embedded in paraffin. Serial sections were prepared from paraffin blocks, which were stained with hematoxylin and eosin according to standard methods. A survey microscopic study was performed on a microscope equipped with a digital camera at a magnification of × 200-400. (Fig. 1. Model of RPE atrophy. Atrophic changes of the choroid, total disappearance of pigment epithelial cells, atrophic spongi of the photoreceptor layer, rarefaction of cellular elements of the outer and inner nuclear layers, absence of ganglion layer cells. Stained with hematoxylin and eosin. Increase × 200; Fig. 2 The control group. The retina of normal structure. Under the retina is determined by the intact uniform layer of pigment epithelium (indicated by arrows). Stained with hematoxylin and eosin. Magnification × 400).

Typical morphological signs observed in experimental modeling of atrophy of the retina are pronounced atrophic changes in the retina, the absence of pigment epithelial cells on Bruch's membrane, dystrophic changes in all layers of the retina with a violation of its histoarchitectonics (degenerative changes in the layer of photoreceptors, foci of cell rarefaction in the external and internal nuclei layers, the disappearance of ganglion cells).

An optical coherence tomography scan was performed on both eyes during the observation period on day 2, 2 weeks, 1 month, 3 and 6 months after surgery.

2 days after surgery, an detachment of the neurosensory retina with a gap corresponding to the injection site and disorganization of the layers of the retina were visualized on an OCT image. At autofluorescence, uneven hyperfluorescence was determined - FIG. 3.

After 2 weeks, there was an uneven fit of the neuroepithelium, spontaneous closure of the gap, increased hyperreflectivity in the study area. On autofluorescence, the hypofluorescence zone corresponding to the atrophy zone surrounded by the rim of speckled hyperfluorescence was determined - FIG. 4, 5.

One month after surgery, the atrophy zone of the retinal pigment epithelium was visualized, leading to increased penetration of the laser beam into the underlying tissue. In the atrophy zone, thinning of the pigment epithelium and choroid, the loss of choriocapillaries was determined. On the autofluorescence of the fundus, the hypofluorescence zone corresponding to the atrophy zone was determined - FIG. 6. These changes were saved to 3 - FIG. 7 and 6 months of observation - FIG. 8.

Before and after surgery, the state of blood flow in the vessels of the eye was assessed using the color duplex scanning method in the modes of color Doppler mapping (CDL) and pulsed Doppler ultrasound. More pronounced changes are observed in the CAC (the average maximum systolic velocity Vsyst in the CAC is 8.2 ± 0.4 cm / s) compared with the paired intact eye (12.2 ± 0.6 cm / s; p <0.001). Hemodynamic disturbance and blood flow deficiency in the system of retinal and choroidal vessels in the eyes with atrophy of retinal pigment epithelium (RPE) are recorded.

The functional activity of the retina was evaluated

electroretinographic methods according to ISCEV standards using the Roland Consult RETIport / scan21 diagnostic system (Germany). We recorded scotopic, maximal, and cone-shaped ERGs for single flashes and rhythmic ERGs (RERGs) for light flickering at a frequency of 30 Hz, as well as a steady state ERG pattern (PERGs) on black and white alternating chess cells with an angular size of 0.8 degrees in healthy rabbits ( before atrophy) and 3 months after modeling. We analyzed the amplitude and time indices of biopotentials and the cone glial index Kg, calculated from the ratio of the amplitude of the b-wave cone ERG to single stimuli to the amplitude of the rhythmic response to light flickers of 30 Hz.

The functional activity of RPE was assessed by the activity of retinal photoreceptors, since their normal functioning depends on the state of RPE and the adequacy of choroid circulation. The change in the bioelectric activity of photoreceptors according to the ERG is an indirect marker of the pathology of RPE cells and allows you to indirectly judge the nature of their violation.

In the eyes with atrophy, ERG was recorded with subnormal amplitudes of a- and b-waves, which were 20-40% lower than the initial values in each animal, which indicated inhibition of the functional activity of photoreceptors, ON-bipolar cells and Mueller cells, as well as ganglion cells retina (on the dynamics of PERG). The most pronounced and characteristic for this model of atrophy was a decrease in the amplitude of the a-wave of the maximum and cone ERG, compared with a more moderate inhibition of the amplitude of the b-wave, RERG and PEG, which indicates significant changes, primarily at the level of photoreceptors and RPE of the retina. These changes also indicated a violation of the photoreceptor / RPE interface. An increase in the b / a index of maximum ERG reflects a violation of the transmission of visual signals from photoreceptors to bipolar cells of the postreceptor retina (external / internal retina interface). The cone glial index in the eyes with atrophy was reduced on average to 0.6-0.7 rel. units with norm values of 1-1.2. Such dynamics of the glial index 4 months after the creation of the experimental model indicates a significant violation of the glio-neuronal relationships and inhibition of the function of the glomerular Mueller cells in the retina when a solution of bevacizumab, which suppresses the activity of VEGF factors, is introduced into the subretinal space in a volume of 0.01 ml.

Thus, modeling of RPE atrophy with a single subretinal injection of bevacizumab in a volume of 10 μl containing 0.025 mg allows one to obtain pronounced changes in the functional activity of the retina, primarily in the photoreceptor layer, along with deficiency of retinal and choroidal circulation, which is typical for exacerbating the degenerative process under the influence of antiangiogenic therapy. The relative simplicity of reproducing the model of atrophy allows it to be widely used in experimental studies.

Claims (1)

A method for modeling atrophy of retinal pigment epithelium, including the introduction of the drug into the subretinal space, characterized in that bevacizumab is used as a preparation in a volume of 10 μl containing 0.025 mg of the drug, which is administered to albino rabbits at a distance of 0.5 pd below the optic disc with the formation of subretinal bladder.

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