[Competence factors of retinal pigment epithelium cells for reprogramming in the neuronal direction during retinal regeneration in newts].

Retinal pigment epithelium (RPE) cells that have the unique ability to reprogram retinal cells @in vivo@ were analyzed in the adult newt. Our own data and that available in the literature on the peculiarities of the biology of these cells (from morphology to molecular profile, which can be associated with the capability of phenotype change) were summarized: It was established that the molecular traits of specialized and poorly differentiated cells are combined in RPE of the adult newt. It was registered that persistent (at a low level) proliferation and rapid change of specific cytoskeleton proteins can contribute to the success of RPE cell reprogramming in the neuronal direction. Each of the considered factors of competence for reprogramming can be found for animal RPE, whose cells are not able @in vivo@ to change the phenotype to a neuronal one; however, their totality (supported by the epigenetic state permissive for conversion) is probably an internal property of only newt RPE.

[FGF2 signaling pathway components in tissues of the posterior eye sector in the adult newt Pleurodeles waltl].

The FGF2 signaling pathway components in tissues of the posterior wall in the normal and regenerating eye of the adult Pleurodeles waltl newt were detected for the first time. The fgf2 gene expression was found in the retina, retinal pigment epithelium, and choroid using polymerase chain reaction (PCR). A high homology of the mRNA nucleotide sequence of the most conservative fgf2 gene region in the P. waltl with the fgf2 orthologs in other vertebrates was proved. The Fgf2 protein aminoacid sequence of the P. waltl newt demonstrates even more homology with this growth factor in other vertebrates. The Fgf2 protein with a molecular weight 35 kDa was found in the studied eye tissues using Western blot hybridization. Localization of the Fgf2 protein and its Fgfr receptors was immunohistochemically studied in the pigment epithelium, choroid, central and growth retina regions of the newt native eye, and in the connective cilium of photoreceptors. Using real-time PCR and immunohistochemistry methods, it was found that the fgf2 gene down-regulation and a decrease in the intensity of the immunochemical reaction of its protein product (Fgf2) occur in the early period after the retina removal (in 4-8 days) (as compared with those in the same department of the unoperated eye).

[Morphogenetic changes during newt tail regeneration under changed gravity conditions].

Gravity-dependent shape alterations in newt tail regenerates are described, which were previously noticed in experiments onboard satellites Foton M2, M3 and in corresponding laboratory controls. Laboratory conditions were developed that allow reproducing this phenomenon persistently in the adult newts Pleurodeles waltl (Michahelles, 1830). The newts kept in an aquarium (in partial weightlessness) after 1/3 tail amputation developed normal lanceolate regenerates, while those that stayed on a moist mat (exposed to greater gravity than in aquarium) developed curved tail regenerates. Dynamics of the shape alterations were described using computer morphometric analysis. The curve was shown to develop at stage III of regeneration and to be caused by bending of the developing axial structures: the ependymal tube and the cartilage rode. Cellular processes were described that accompany the tail shape changes, such as cell migration and formation of dense aggregates. Unequal proliferation throughout the wound epidermis and blastema was revealed using BrdU assay. Proliferation increased within dorsal and apical regions of the regenerates in the newts kept on the mat cell compared with the aquarian animals.

[In vitro organotypic cultivation of adult newt and rat retinas].

Adult rat and newt retinas were studied during long organotypic 3D cultivation. A high proliferation level was discovered in the region of growth by applying DNA synthesis markers and in vitro mitosis registration in newt retina. Aggregates were formed in the retina spheroid cavity because dedifferentiated cells migrated into this region. Small cell populations in nuclear layers also had dividing and migration capacity. Rosette formation has been shown in newt retina. It is a characteristic of fetal retinal development under pathological conditions. The antiG FAP antibody dye demonstrated an increase in the parent M@uller cell population and generation of a small cell pool with short GFAP-extensions de novo. Recoverin expression studies detected its translocation from photoreceptor extensions to the cell bodies. Moreover, protein was presented in some cells inside the spheroid. It has been shown for the first time that cell proliferation occurred in the developing adult rat retinal spheroid in vitro; BrdU-positive cells and multiple mitoses were revealed in this zone. However, the source of proliferation was not in the peripheral retina, and stable macrophages and glial cells located among neurons of the inner nuclear layer had the ability to divide. The antiGFAP antibody showed an increase in GFAP fibers in the rat retina as well as in the newt retina. Recoverin translocated into photoreceptor perikaryons and the outer plexiform layer in cultivated rat retina. Interestingly, some cells with probably de novo expression of recoverin were discovered in rat and newt retinas.

[The retinal pigment epithelial cells of the adult newt and rat under conditions of in vitro organotypic culture].

To understand why the retinal pigment epithelium (RPE) has different potentials for neural differentiation in lower and higher vertebrates, the RPEs of adult newts and rats were compared under similar in vitro cultivation conditions. The RPEs of both animal species were organotypically cultivated within the posterior eye wall under constant rotation in the serum medium free of growth factors. Comparison of the cell morphology, proliferation, and expression of pan-neural markers demonstrated that the RPE cells of adult newts and rats under similar in vitro conditions displayed both similarities and differemces. They were able to synthesize DNA but rarely divided mitotically. In addition, part of the RPE cells of both the newt and the rat were dislodged from the layer, migrated, and acquired a macrophage phenotype. However, the majority of the cells retained the initial morphology and remained within the layer. In several cases, these cells displayed the initial characteristics of neural differentiation, namely, expression of pan-neural proteins. The difference between the newt and rat RPE cells was in the ability of the former to generate in vitro an additional row of dedifferentiated NF-200-positive cells, characteristic of in vivo newt retinal regeneration. These data demonstrate that the RPE cells of the adult newt and rat retain the potential of manifesting neural cell traits; however, more advanced changes towards differentiation are characteristic of only the newt RPE.

[Transcriptional factor Pitx2: localization during triton retina regeneration].

For the first time immune-chemical analysis of transcriptional factor Pitx2 localization during triton retina regeneration after its removal and also in tissues of a nonoperated eye of an adult triton has been carried out. Protein Pitx2 has been found in the nucleus of the earliest neuroblasts that form the germ of the retina. At a later stage of retina regeneration, Pitx2 was found in the nucleus of differentiating cells of ganglionic layers that correspond to Pitx2 protein localization in the native retina. Protein Ptix2 has also been found in the nucleus of less differentiated cells of the peripheral region of regenerative and native retina. It was demonstrated that protein Pitx2 is expressed not only in retina but also in other tissues of the posterior sector of the eye (pigment epithelium, choroid) using immune-histochemical and Western blot hybridization. It is supposed that transcriptional factor Pitx2 has been involved in the control of subsequent stages of retina regeneration from pigment epithelium cells.

[A study of the localization and accumulation of S-phase cells in the retina of newt Pleurodeles waltl after experimental pigment epithelial detachment].

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15-20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals.

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age-induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 microM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

[Studies on the effect of the adhesion protein isolated from bovine eye on cell proliferation in the newt cornea].

The effect of the adhesion protein isolated from the bovine cornea was studied on the model of mechanical injury (cross cutting of the cornea). In the concentration of 10(-12) mg/ml, the protein influenced the proliferation of corneal epithelial cells in newt Pleurodeles waltl in vivo. Experiments were conducted using autoradiography, and the nuclear labeling index (NLI) was determined at different times after surgery and in different corneal regions. This adhesion protein significantly induced proliferation of corneal epithelial cells relative to control groups with the injured eyes treated with the serum adhesion protein at the same concentration or water. The differences between the experimental and control animals were most pronounced 7 days after surgery. By day 14, they were less pronounced but still significant. On day 28, no significant differences in NLI were observed between the three groups, although these values remained higher than in intact animals. An increased pool of proliferating cells in the corneal epithelium was observed both in the affected and intact areas. The data obtained indicate that the biological activity of this protein is not species specific and that it can be a proliferation factor for corneal epithelial cells.

[Regulatory proteins of vertebrate eye tissues]. / Reguliatornye belki tkanei glaza pozvonochnykh.

In our work the new proteins likely belonged to the microenvironment of pigmented epithelium cells and retinal neurons in mammalian eye were studied. We attempted to understand the role of these proteins in the maintenance of normal morphological and functional state of these eye tissues. Earlier for the first time we identified the adhesion molecules with physico-chemical and biological properties much different from other known cell adhesion molecules of bovine eye. Probably, they represent one family of low molecular weigh, highly glicosylated proteins, that express biological activity in extremely low doses--10(-10) mg/ml. The homogeneity of studying proteins is confirmed by HPLC and SDS-electrophoresis in PAAG. It is shown also that these proteins are N-glycosylated, because they contain mannose and N-acetilglucosamine residues. They demonstrate as well a high calcium-binding activity, with Kd corresponded to 10(-4)-10(-6) mg/ml. For a study of the biological effect of these glycoproteins in extremely low doses, a new experimental model was proposed and developed. It was the cultivation in vitro of the posterior part of the eye obtained from the newt Pleurodeles waltl. In short-time culture system it was demonstrated that the studied glycoproteins could stabilize pigment epithelium cell differentiation and cellular interactions in the neural retina in vitro. In addition, glycoproteins, obtained from the pigmented epithelium of bovine eye could decrease the rate of bipolar cell apoptosis in the neural retina. Therefore, the novel adhesion glycoproteins, expressing their biological activity in extremely low doses, pretend to be the regulatory molecules with vivid gomeostatic effects necessary for the delicate adjustment of cell behavior action and function in sensory tissues.

[An organotypic culture of the newt retina together with other tissues of the posterior eye segment as a model for studying the effects of the cell adhesion glycoproteins]. / Model' organotipicheskogo kul'tivirovaniia setchatki vmeste s tkaniami zadnego sektora glaza tritona dlia izucheniia deistviia adgezivnykh glikoproteinov.

The adult newt retina explanted together with the posterior eye wall and cultivated for a short time in a serum-free medium was tested as an experimental model by several criteria, including the expression of protein markers of the main retinal cell types. Some differences in the expression of specific photoreceptor, interneuron, and glial cell proteins and the localization of acetylcholinesterase activity were found to appear during in vitro cultivation. Using this model, preliminary tests of new cell adhesion glycoproteins from the bovine retina and pigment epithelium were conducted, and the role of pigment epithelial cell proteins in improving cell viability in the cultivated newt retina was revealed. Moreover, the fraction of basic adhesion proteins from the bovine pigment epithelium improved the survival potential of the macroglial (Muller) cell population, compared to that in the control.

[Peculiarities of eye morphogenesis in embryonic Japanese quails developed in microgravity]. / Osobennosti morfogeneza glaza u émbrionov iaponskogo perepela, razvivshikhsia v usloviiakh nevesomosti.

This work is a part of comprehensive research into the effects of space flight on Japanese quail ontogenesis. Analysis of eye morphogenesis in the embryonic Japanese quails developed in microgravity discovered considerable deviations and abnormalities. Ocular abnormalities in the embryonic quail were mainly micro-ophthalmic and associated with disproportional growth of the pigmental epithelium and neural retina which resulted in plication and a broken sandwich structure of the retina.

[Retina of vertebrates: an internal cell reserve for regeneration]. / Setchatka pozvonochnykh: vnutrennii kletochnyi rezerv dlia regeneratsii.

The recent data were summarized concerning the presence in the retina of fish, amphibians and birds of additional sources of growth and regeneration, alternative to the already known sources, such as growth zone of eye, pigment epithelium, and cells--precursors of rods, and which are localized in the inner nuclear layer of retina. These sources are represented by as yet not finally identified oval small cells and cells of Muller glia. Both types of cells are capable of proliferating and producing precursors for various differentiated cells, including photoreceptors or their additional precursors. The current immunochemistry data are provided, which were obtained using markers of proliferation, proneural phenotype, and specific cell differentiation in the growing retina and in the retina after various damages. The regulatory mechanisms and methods of the stimulation of proliferation of the cells, which are sources of increase in the number and restoration of photoreceptors, interneurons, and glial cells of vertebrate retina, are discussed.

[An immunohistochemical study of localization of the calcium-binding protein recoverin in retina of the newt Pleurodeles waltl]. / Immunokhimicheskoe izuchenie lokalizatsii kal'tsii-sviazyvaiushchego belka rekoverina v setchatke tritona Pleurodeles waltl.

The presence and localization of the calcium-binding protein recoverin, initially found in photoreceptors of the bovine eye, were immunochemically studied in retina of the new Pleurodeles waltl. Polyclonal monospecific antibodies against recoverin were raised and the methods of immunoblotting and indirect immunofluorescence were used. A protein with an apparent molecular mass of 26 kDa was found in the retina extract, which was specifically stained by the antibodies against recoverin. Localization of recoverin was studied on the retina sections: an intense reaction was found in the inner segments and a weak reaction was found in the basal part of the outer segments of photoreceptors and in Landolt's clubs of displaced bipolars. The results we obtained suggest for the first time the presence of recoverin in the retina of a representative of the Urodeles and indicate to interspecific conservativeness of this protein and differences of its localization in the retina photoreceptors in different species. The data obtained open a possibility of using recoverin as a marker protein of photoreceptors and displaced bipolars in studied of retina regeneration in newts.

[Changes in the relative number of bipolar-like cells in the retina of Pleurodeles waltl as a function of age and as a result of light exposure]. / Izmeneniia otnositel'nogo chisla smeshchennykh bipoliarov setchatki glaza tritonov Pleurodeles waltl v zavisimosti ot vozrasta i v rezul'tate oblucheniia svetom.

The study of the population of bipolar-like cells (displaced bipolars) was continued in order to establish their role in development and regeneration of the retina in lower vertebrates. The size of the population of these cells was estimated on serial semithin sections in the retina of normal eyes in adult and young newt Pleurodeles waltl, as well as in adult newts subjected to long-term bright illumination. The population of displaced bipolars was significantly increased with reference to all cells of the outer nuclear layer. In young and illuminated animals, their numbers were approximately 1.3 and 1.4 times that in the adult animals not exposed to constant light. The results obtained favor the earlier suggestion of the involvement of the displaced bipolars in growth and restoration of the outer nuclear layer in the retina of newts during development and after trauma.

[Markers of retinal-type cell differentiation in studies of eye development and regeneration in vertebrates]. / Markery differentsirovki kletochnykh tipov setchatki v issledovaniiakh razvitiia i regeneratsii glaza u pozvonochnykh.

Data on the use of various immunochemical markers specifically indicating cell types of the neural retina and pigment epithelium are reviewed. It is demonstrated how this approach can be applied to the analysis of specific features of vertebrate retinal development, including the order and timing of differentiation of the main cell types, their interdependence in the course of this process, and factors controlling the latter. Problems concerning the state of differentiation and its change in the cells of retinal pigment epithelium and glial cells are discussed in respect to their analysis with the aid of specific protein markers. The current state of retina regeneration research involving the use of labelled cell sources and regenerated cells in lower vertebrates is analyzed. Problems in the search for new markers of retinal photoreceptor, macroglial, and microglial cells and their use in experiments are addressed.

[Study of adhesive proteins from neural tissues of the 8-day old chicken embryonic eye]. / Issledovanie adgezivnykh belkov iz neiral'nykh tkanei glaza 8-dnevnykh kurinykh zarodyshei.

Two groups of proteins were isolated from the retina and pigment epithelium of eight-day-old chick embryos. Experiments with suspension cultures of retinal cells demonstrated that only the retinal extracts and the fraction of its acidic proteins can stimulate cell aggregation in vitro. Analysis by the method of high-performance liquid chromatography showed that fractions of acidic and basic retinal proteins, which markedly differ in their electric charge and biological activity, have similar composition. To study the effect of these proteins on the morphological and functional state of pigment epithelium in vitro, a new experimental model is proposed, with the posterior segment of the newt (Pleurodeles waltl) eye used as a test tissue. The fraction of basic proteins isolated from the chick embryonic pigment epithelium stabilized cell differentiation in the newt pigment epithelium. The analyzed proteins proved to be biologically active at extremely low doses, corresponding to 10(-12) M solutions.

[Discovery of internal sources of the neural retinal regeneration after its detachment in Pleurodeles. II. The radioautography study]. / Obnaruzhenie vnutrennikh istochnikov regeneratsii neitral'noi setchatki posle ee otsloiki u tritonov. II. Radioavtograficheskoe issledovanie.

We continued to study internal sources of neural retina regeneration in adult lower vertebrates. A radioautographic study carried out was aimed at the visualization of proliferating cells in different areas of the retina and at different times after its detachment in the newts Pleurodeles waltl. Analysis of serial standards and semithin sections has shown several types of cells capable of incorporating a labeled DNA synthesis precursor. The marker of proliferating cells was incorporated in the cells of Mullerian glia, pigment epithelium, and growth zone and in small neural cells of the vitreal part of the inner nuclear layer. A source of formation of neuroepithelial "insertions" responsible for restoration of the complete cell composition of the retina damaged as a result of detachment should be searched among these cell types. Neither bipolar cells with Landoldt's club in the inner nuclear layer, nor bipolar-like cells in the outer nuclear layer, which are a source of formation of additional photoreceptors, were found among the 3H-thymidine- labeled cells. A conclusion was drawn that the bipolar-like cells of P. waltl are postmitotic cells, that did not reach terminal differentiation.

[Retinal regeneration after dissection of the optic nerve in newts exposed on board the Bion-11 biosatellite]. / Regeneratsiia setchatki posle pererwzki zritel'nogo nerva u tritonov, éksponirovannykh na bortu biosputnika "Bion-11".

The work brought up initial information on the impacts of space flight (SF) on regeneration of nerve tissues in vertebrata. Summarized are data of analysis of the retinal regeneration following section of the ocular nerve and blood vessels in space-flown adult newts (Pleurodeles waltlii). Two weeks in SF were found not to impede the regeneration of retina as its growth was fully dependent on the same cell sources as in the condition of 1 g. In the newts which had been operated 2 wk prior to launch, recovery of retina in SF proceeded more intensively (phases V-VI) compared with the synchronous controls (phase IV). According to the morphometric analysis, differentiation of regenerates' layers in the space animals was also a more rapid process. The proliferative activity of cells in regenerates estimated with the 3H-timidine radioautography turned to be higher, too: the labeled nuclei index in early non-differentiated regenerates was in 1.2 to 1.5 times higher than in the control. Immunohistochemical array with the help of GFAP antibodies performed at the late phases of regeneration revealed an activating effect of SF on the Muller glia cells. These findings indicate that microgravity can stimulate general retinal regeneration and activate regenerate cells, specifically those involved in morphogenesis.