Method for experimental in vivo simulation of cortical cataract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to ophthalmology, and concerns simulation in vivo of cortical cataract. That is ensured by a double-side surgical sympathectomy by excising a superior cervical sympathetic ganglion.

EFFECT: by simplicity and effectiveness of modulation, the method provides forming the cortical lens-form opacity, which is morphologically and immunohistochemically identical to lens cell changes in age-related lens opacity in a human.

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The invention relates to medicine, in particular to ophthalmology, and is intended to be used in research practice, ophthalmology, gerontology, histological, biological and pharmacological laboratories is available effortless, and highly specific method for experimental modeling of cortical type of cataract in vivo, thereby greatly enhancing the effectiveness of ongoing studies on the mechanisms of formation and prevention of age-related cortical cataract in humans.

According to the world health organization, among the causes of blindness in the proportion of cataract accounts for 43%. It is important to note the extremely high incidence (over 97%) among persons older than eighty years.

Research cataractogenic factors, biochemical and biophysical aspects of cataractogenesis behind the rate of improvement of technology of surgical treatment of age-related cataract. Differences in prevalence of cataract among the population of different regions, the timing and localization of turbidity, the dynamics of progression, as well as maintaining the transparency of the lens in a certain part of elderly people do not allow one to link the issue of the treatment of age-related cataract only with the possibilities of surgery and confirms the necessity of finding new ways to�servative treatment of this disease.

There is no doubt that the development of age-related cataract occurs as a result of changes in the body. Evidence of this is noted by many authors, the relationship between the incidence of certain types of age-related cataract and General somatic pathology, and the development of age-related cataract in all cases, the observations on both eyes.

It is proved that in patients with age-related cortical cataract prevailing tone of the sympathetic division of the autonomic nervous system, which identifies specific pathogenetic features of systemic degenerative changes in tissues and organs of patients of this group (Korsakov N. In. "The role of the autonomic nervous system in the formation of age-related cataract in humans" // Materials of scientific-practical conference dedicated to the 75th anniversary of Professor V. N. Saparova. - Cheboksary, 2006. - P. 35-37).

The question of trophic function of the nervous system is the most important question in a relevant biological problem about the factors maintaining the stability of tissue differentiation and tissue metabolism of living organisms. Universal mechanism of pathology A. D. Speransky seen in the neuro-degenerative processes that are associated with the starting stage disease processes.

Fundamental differences neurotransmitter security of processes fo�formation of cortical and nuclear cataract (Korsakov N. In., Sergeeva V. E. "Features boninovo status of the lens in the conditions of formation of different types of age-related cataract in humans" // Ophthalmosurgery. - M., 2007. - No. 3. - P. 44-47) prove the existence of different pathogenic mechanisms leading to the formation of different types of neurodystrophic opacities in the crystalline lens of the eye.

Thus, the type of age-related cataract reflects the pathological process of aging of the various divisions of the autonomic nervous system of the patient.

However, the high financial costs of modern microsurgical techniques for the treatment of cataract lead to a significant restriction of their use. For example, in some developing countries are operated only 10% of cataract patients; according to S. K. West, H. A. Quigley (1998), in Africa there are more than 3 million blind cataract patients that do not have the financial capabilities of its surgical treatment; in India undergo surgical treatment only 50% of those patients.

Obviously, drug prevention and therapeutic treatment of the early stages of cataract as alternative to surgical methods is now a special value. It is estimated that the restraint of the development of conservative methods of cataract by 10 years could reduce the number of expensive operations by 50%, and late late diag�of astika age-related cataract in many countries now regarded as an undue burden on the state budget, as a socio-economic problem of national importance.

Objective: develop an affordable, effortless, and highly specific method for experimental modeling of cortical type of cataract in vivo.

The essence of the proposed method is to conduct experimental sympathectomy animal.

Positive effect: pathogenetically based modeling of pathological process in vivo, ease of application, reduction of working time of the experiment, a significant increase in efficiency studies on the mechanisms of formation and prevention of cortical form of age-related cataract.

The closest to the proposed method is a model of cataractogenesis vertebrates in vitro (Krasnov M. S., Gurmit E. P., Gundorova R. A., Yamskova V. P., Y. A. Kapitonov "Model of cataractogenesis vertebrates in vitro // Ophthalmology, 2005. - Volume 2. - No. 2. - P. 43-49) applied the above authors in the development and patenting of a method for producing medicines (AMCOW I. A., Yamskova V. P., A. V. Nagovitsyn, Krasnov M. S. "a method of obtaining a medicinal product for the treatment of cataract". Patent RU №2315607, IPC AC 35/44, AK 38/17, AR 27/02). The prototype is as follows: in the experiments were used the lenses of Mature vertebrate vividly�tion (frogs, rats and smilochiropteryx eyes of cattle). The cultivation of the lenses of vertebrates was carried out in a nutrient medium for 3-8 days. For the induction of cataractogenesis was used a solution of hydrogen peroxide, the concentration of which ranged from 0.1 to 100 mm, and the solution of calcium chloride in the concentration range from 1.25 to 60 mm. Inducers of cataractogenesis was added to the nutrient medium at the beginning of cultivation. The degree of cataract was determined spectrophotometrically on the MR device 700 (Dynatech, Germany), and visually, using the lined substrate. In the latter case, the complete opacity of the lens was determined by the disappearance of the lines of the substrate under the lens. Induction of cataractogenesis by hydrogen peroxide in amphibians observed opacity of cortical layers, and the core remained intact. Induction of cataractogenesis by calcium chloride in amphibians observed clouding of the crystalline lens nucleus, cortical layers remain transparent, in some cases, observed the formation of a slight touch in the form of flair. The results reflect the dependence of localization induced cataract from the nature of the studied chemicals in two species of frogs. Induction of cataractogenesis in the lens of the bulls as in the case of calcium chloride, and in the case of the hydrogen peroxide gas� hydrogen arose cortical clouding moderate 3-4 days of cultivation. When cultured lenses of rats with hydrogen peroxide had any clouding of the crystalline lens nucleus and to a greater extent of cortical layers, we have developed a total opacity. Calcium chloride has caused a total clouding of the lenses of rats at the same time of cultivation. The results indicate differences in the regulation of functioning calcium-dependent enzyme systems, and also systems of lipid peroxidation in the membranes of cells and lens fibers in animals species studied.

Famous model of cataractogenesis vertebrates in vitro, mentioned above, may not be widely adopted in research and practice of ophthalmology, gerontology, histological, biological and pharmacological laboratories due to its high cost (environment for cultivation, the cost of electricity for prolonged cultivation), complexity (multistage, multicomponent), high costs of working time (over 8 days) and variability in modeling types of cataract in different experimental animals (specific models).

The following models of experimental cataractogenesis close to the proposed model due to changes in the conditions of life inside a living organism (in vivo), however they currently n� supported by Russian patents.

One of the models of experimental cataractogenesis in vivo based on the use of fotofabrikas effect of ultraviolet radiation with a wavelength shorter than 480 nm against a transparent refracting media of the eye (Linnik L. F., M. A. Ostrovsky et al."Artificial lenses that absorb UV rays: safety, efficacy and prospects for use in ophthalmic surgery (review of literature)" // Ophthalmosurgery. - M., 1991. - No. 4. - P. 3-7). Especially dangerous for the lens is UV light with a wavelength of 280-315 nm (range In). Clinically proven that people exposed to such radiation, regardless of gender and race are more likely to develop cortical cataracts type (Cruickshanks K. J. "Sunlight exposure and risk of lens opacities in an population-based study // Arch. Ophthal. - 1998. - V. 116. - N. 12. - P. 1666).

Another model of experimental cataractogenesis in vivo based on the use of higher concentrations of calcium, fluorine and silicon in drinking water and food experimental animals (Gophers, V. L., Andreev A. N., Stepanov R. V. et al."The role of biogeochemical factors in the pathogenesis of age-related cataract" // Ophthalmological journal. - 1990. - No. 5. Pp. 296-299).

This experimental model of cataract formation in vivo, based on the impact of TGF-β (transforming growth factor-β) induces appearance� epithelial-mesenchymal transition in cells of the lens (De Iongh RU., Wederell, E., Lovicu FJ., McAvoy JW. "Transforming growth factor-beta-induced epithelial-mesenchymal transition in the lens: a model for cataract formation" // Cells. Tissues. Organs. - 2005. - V. 179. - N. 1-2. - P. 43-55).

These methods, having the advantage of the specificity of the model in relation to specific types of cataracts, can not meet the requirements of modern medical science, as they are quite time-consuming, costly and require a significant investment of working time.

Thus, to date, there are no ways to get the most effective simulation of the experimental cortical cataract in vivo with minimal costs.

The proposed method is based on the identified basic differences in neurotrophic control of processes of formation of cortical and nuclear cataract (Korsakov N. In., Sergeeva V. E. "Features boninovo status of the lens in the conditions of formation of different types of age-related cataract in humans" // Ophthalmosurgery. - M., 2007. - No. 3. - S. 44-47; Pastev N. P., Korsakov N. In., Pozdeeva N. And., Sergeev, V. E., "the Frequency and nature of General somatic diseases, related to the formation of different types of age-related cataract in humans" // Ophthalmosurgery. - M., 2011. - No. 1. - P. 45-49), indicating a logical manifestations of age-related involution of the various divisions of the autonomic nervous system of patients.

Object of the invention is to develop DOS�upny, effortless and highly specific method for experimental modeling of cortical type of cataract in vivo, which will increase the efficiency comparison of different anticatarrhal substances, allowing for more efficient (pathogenetically substantiated) therapy cortical type of age-related cataracts and reduce the cost of state funding for remedial measures.

A method of experimental modeling of the cortical type of cataract in vivo, based on the change in tone of the sympathetic division of the autonomic nervous system by conducting bilateral surgical sympathectomy experimental animal.

The proposed method allows high efficiency (80%) to initiate the formation of the cortical type of cataract experimental animal in both eyes and, most importantly, allows you to recreate experimentally histological and phenotypic changes in the cells of the lens with age-related cortical cataract identified in humans.

The invention has novelty, meets the requirements of inventive step and may find wide application in research practice, ophthalmology, gerontology, histological, biological and pharmacological laboratories.

Predlojeny� method is as follows. Under General anesthesia to produce bilateral sympathectomy of the upper cervical sympathetic ganglion (cervical ganglion superius) surgically:

- layers to dissect the skin and subcutaneous tissue on the anterior margin of the m.stemocleidomastoideus;

- to dissect the neurovascular bundle of the neck;

- v.jugularis intema and a.carotis intema slipping medially;

- at the level of 2-3 cervical vertebra to hold the allocation of n.vagus and cervical ganglion superius, located medial to n.vagus;

- make a sympathectomy of the upper cervical sympathetic ganglion surgically;

- to produce hemostasis, to seal the layered sutures in the wound.

As a result of bilateral surgical sympathectomy experimental animal in 5-7 months at biomicroscopy of the anterior segment of both eyes are seen the initial signs of cortical cataract mainly in the lower-nasal quadrant: intensely reflective, specialisee haze of grayish-white color facing the base to the periphery of the lens and located in the region of its bark. 12-14 months from the time of the experiment, the area described above, grayish-white cortical opacities of the lens of both eyes is significantly increased, forming a dazzling wedge-shaped opacities with the base facing the periphery of the lens.

In addition, it is noted by�quench signs of hydration in the area of the cortex of the lens - dissociation of cells called lens fibers, the formation of cracks and water vacuoles (Fig.1, 2).

Described macroscopic changes lenses experimental animal fully consistent with the clinical picture-maturing cortical age-related cataract types in humans can be detected by biomicroscopy of the anterior eye segment during standard ophthalmic examination (Fig.3).

Another important proof of the high specificity of the proposed model is identical histological and immunohistochemical changes of the cells of the lens during formation simulated by the proposed method experimental cortical cataract animal and age-related cortical cataract (according to the results of a morphological study of the postoperative material darkened lenses).

Comparative histological analysis of sections of crystalline lens with age and modeled cortical cataracts after their obesitological stained with hematoxylin-eosin showed identical morphological changes: distinct hydration of cortical lens, dissociation from lenticular cells-fibers, wedge-shaped space filled with detritus and vacuoles. Nuclear division of the lens is squeezed obvodnennye cortical masses, but has no structural otklonenie� from the norm (Fig.4-6).

Comparative immunohistochemical staining with monoclonal antibodies to the neuron-specific enolase (NSE), protein S-100 (S-100), vimentin (Vim), α-smooth muscle actin (α-SMA) and partitocracy (EMA) in all departments of intact crystalline lens of the eye and the experimental animal did not reveal any specific staining, as an intact crystalline lens, as part of the "barrier of the body" (the blood-barrier), with no immunohistochemical label of the basic tissue types of the body. However, when forming in the lens of age or simulated by the proposed method cortical cataract found expressed immuno-positive reaction to certain monoclonal antibody - NSE, Vim and protein S-100, and the area of its existence is limited to the cortical division of the lens (PL. 1), which suggests the formation of identical transformation of the phenotype of cells of the crystalline lens.

The possibility of pathogenetically proved experimental modeling of cortical type of cataract, recreating phenotypic changes in the cells of the lens with age-related cortical cataract in humans, is confirmed by complete coincidence immunohistochemical characteristics presented in table 1.

Table 2 shows the advantages of using the proposed method compared izvestnymi.

Table 1
Comparative immunohistochemical characterization of cells of the crystalline lens of rabbit and human in experimental and age-related cortical cataract
Immunohistochem. methodNeuron-specific enolase (NSE)Protein S-100Vimentin (Vim)α-gedcoms. actin (α-SMA)Partitocracy (EMA)
The object of the observationrabbitpeoplerabbitpeoplerabbitpeoplerabbitpeoplerabbitpeople
Intact lensStaining revealed noStaining revealed noStaining revealed no Staining revealed noStaining revealed no
Cortical type of cataract(+) in the region of the cortex(+) in the region of the cortex(+) in the region of the cortex(+) in the region of the cortex(+) in the region of the cortex(+) in the region of the cortex(-)(-)(-)(-)
Note: (+) - immunopositive reaction;
(-) - mononegative reaction.

Table 2
The advantages of the proposed method for experimental modeling of cortical type of cataract in vivo over other known methods
BenchmarksMethod Krasnov M. S. and co-authorsMethod of Linnik L. F. and co-authorsMethod Suslikov V. L. and co-authorsMethod claimed
Physiology experimental model - (in vitro)+ (in vivo)+ (in vivo)+ (in vivo)
The cost of funds+++ (significant)+++ (significant)+++++ (maximum)+ (minimum)
The complexity (multistage)+++ (significant)++ (moderate)+++++ (maximum)+ (minimum)
Staff time+++ (8 days)+++++ (some years)++++ (1 year)+ (30 minutes)
Morphologically proven pathogenetic validity+ (minimum, at the level of visual control)+ (minimum, at the level of visual control)+ (minimum, at the level of visual control)+++++ (maximum, phenotypic matching)
The expediency of application+++ (significant) + (minimum)++ (minor)+++++ (maximum)

Image CAPTIONS

Fig.1. The intact rabbit lens. Method biomicroscopy of the anterior segment of the eyeball. Slit lamp XG-ZG-06.

1 - absence of opacities in the lens cortex, 2 - iris, 3 - the cornea.

Fig.2. The lens of the rabbit, startled experimental cortical cataract. Method biomicroscopy of the anterior segment of the eyeball. Slit lamp XG-ZG-06.

1 - extensive wedge-shaped grayish-white opacity in the lens cortex, 2 - iris.

Fig.3. Age-related cortical cataract person. Method biomicroscopy of the anterior segment of the eyeball. Slit lamp XG-ZG-06.

1 - wide wedge-shaped grayish-white opacity in the lens cortex, 2 - iris.

Fig.4. Sagittal slice of the intact lens. Coloring with hematoxylin-eosin. SW.: about. 90, at around 15. Biolam 70. The gomal 1,7.

1 - capsule; 2 - nuclei of epithelial cells of the crystalline lens; 3 - nucleus Equatorial cells-fibers. SW.: about. 90, approx. 15.

Fig.5. Sagittal slice of the lens of the rabbit, startled experimental cortical cataract. Coloring with hematoxylin-eosin. SW.: about. 40, at around 15. Biolam 70. The gomal 1,7.

1 - the lens cortex with signs of hydration (dissociation of cell-fibers), 2 - wedge-shaped space�of nsta, filled with detritus and vacuoles, 3 - core of the lens, muffled obvodnennye cortical masses.

Fig.6. Sagittal cut of the crystalline lens of the eye, the affected cortical cataract. Coloring with hematoxylin-eosin. SW.: about. 40, at around 15. Biolam 70. The gomal 1,7.

1 - the lens cortex with signs of hydration (dissociation of cell-fibers), 2 - wedge-shaped space filled with detritus and vacuoles, 3 - core of the lens, muffled obvodnennye cortical masses.

The method of experimental modeling of the cortical type of cataract in vivo, characterized in that the method of surgical bilateral sympathectomy experimental animal by excision of the superior cervical sympathetic ganglion initiate the formation of cortical lens opacities, clinical, morphological and immunohistochemical characteristics are identical to the changes in the cells of the lens with age its opacity in humans.



 

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