Characteristics of cellular proliferation in the developing human retina.

The expression of proliferation-associated proteins Ki67 and PCNA was studied in the retinal rudiments of human embryos at 5-8 weeks of development; studies also addressed the numbers of nucleoli in the nuclei of neuroepithelial cells (with consideration of their distances to the apical surface) and DNA-synthesizing cells after transient (20 min) in vitro incubation in serum-free medium containing BrdU. The retinal rudiment of embryos at five weeks of development had neuroepithelium of the typical structure. BrdU-positive nuclei and nuclei with small numbers of nucleoli were located in the basal part of the ventricular zone. However, this organization was disrupted during the initial period of formation of the inner nuclear layer (six weeks). At this time, DNA-synthesizing cells were found even at the apical surface. Retinal rudiments of embryos at 6-7 weeks of development contained an additional area of cell proliferation in the Chievitz layer and the inner nuclear layer. In eight-week embryos, dividing cells were located in the outer nuclear layer, which again acquired the organization typical of neuroepithelium.

[Characteristics of the cell proliferation in the developing human retina]. / Osobennosti kletochnoi proliferatsii v razvivaiushcheisia kletchatke cheloveka.

The retinal primordia were studied in human embryos at developmental weeks 5-8 to examine the expression of proliferation-associated proteins (Ki67 and PCNA), the number of nucleoli in the nuclei of neuroepithelial cells (in respect to their distance to the apical surface) and the distribution of DNA-synthesizing cells after a short-term (20 min) incubation in vitro in serum-free medium containing BrdU. In 5-week embryos, retinal primordium contains the neuroepithelium with a typical structure. BrdU-positive nuclei and nuclei with small number of nucleoli were found in the basal portion of the ventricular zone. However, this organization was disturbed in the initial period of the formation of inner nuclear layer (week 6). At this stage DNA-synthesizing cells were seen even at the apical surface. In retinal primordia of embryos at weeks 6-7 there appeared an additional area of cell proliferation in the Chievitz layer and in the inner nuclear layer. In embryos at week 8 the dividing cells were concentrated in the outer nuclear layer, which has regained the organization, typical for a neuroepithelium.

[Nucleolar apparatus of neuroepithelial cells and organization of the ventricular zone in human neocortical anlage]. / Iadryshkovyi apparat neiroépitelial'nykh kletok i organizatsiia ventrikuliarnoi zony v neokortikal'noi zakladke cheloveka.

Using silver nitrate impregnation nucleolar apparatus was studied in cells of ventricular zone of human neocortex in embryos at 6-13 wks of development. The number of nucleoli in cells grew gradually in the direction to ventricular surface in all cases studied. These data indicate characteristic localization of cell cycle phases in the neuroepithelium and correspond to the results of experiments performed in animals using DNA-labelled precursors. Specific cell population with scanty large nucleoli was found in inner portion of ventricular zone in 6-8 wks old embryos where mitotic figures are usually localized. Some hypotheses on these cells' origin and role are discussed.

[The reaction of the rat thymus to xenotransplantation and immunosuppression]. / Reaktsiia timusa krysy na ksenotransplantatsiiu i immunosupressiiu.

To the complex action of human embryonal neocortex transplanted into the eye anterior chamber and cyclosporin A injected daily to prevent graft rejection rat thymus reacts by accidental involution, layer inversion and vascular disturbances (without hematoma formation). After 3 wks reparative period sets in and by the end of 5th wk thymus approximates to the control in all parameters.

[Reactive invagination of the neuroepithelium in anlages of the human neocortex, tectum of the midbrain, and the retina at various stages of development]. / Invaginatsionnaia reaktsiia neiroepiteliia v zakladkakh neokorteksa, kryshi srednego mozga i setchatki glaza cheloveka na raznykh stadiiakh razvitiia.

Intensity of invagination of neuroepithelium of neocortex of human embryonal anlages of neocortex, tectum of the midbrain and retina was studied as a response to mechanic damage of ventricular zone induced in the course of preparation of the anlages to explanation. It was shown that no invagination formed in 24 hrs of the explantation. In neocortical anlage intensity of invagination was meximal in 6.5 wks embryo. In tectum invagination intensity of th explants lowered gradually from wk 5 to wk 7.

[Rosette formation in explants of human embryonic neocortex (an electron-microscopic study)]. / Obrazovanie rozetok v éksplantatakh neokorteksa émbrionov cheloveka (élektronno-mikroskopicheskoe issledovanie).

Multiple invaginations and closed cavities (rosettes) were developed in 199 fragments of wall of human anterior cerebral vesicle. Contraction of neuroepithelial cells apexes after the principle of the gathered tobacco pouch was involved into the process. This confirms the previous suggestion of the authors on the similarity between the mechanisms of rosettes forming and neurulation. The participation of radial glia cells and neuroblasts in the reorganization of the neocortex germ was also studied.

[Vascularization of the human embryonic neocortex in the rat anterior chamber under cyclosporine A immunosuppression]. / Vaskuliarizatsiia neokorteksa émbrionov cheloveka v perednei kamere glaza krysy v usloviiakh immunosupressii tsiklosporinom A.

Fragments of 9 weeks hyman embryo neocortex were grafted into the rat eye anterior chamber. To prevent rejection cyclosporin A was introduced to the animals. Vascular network forming in transplants was studied for 5 weeks 3 days endothelial buds from the iris vessels appeared in transplants. After 10 days capillaries with erythrocytes appeared in the graft on the border with iris. 3 weeks later vascular network achieved its maximal development, occupying almost the entire graft thickness. The vessels developed diverged from the norm: they had variceal widenings, and focal distribution with significant non-vascular gaps.

[The reactions of different sections of human embryonic neuroepithelium to damage to the ventricular surface during explantation]. / Reaktsii razlichnykh uchastkov neiroépiteliia émbrionov cheloveka na povrezhdenie ventrikuliarnoi poverkhnosti pri éksplantatsii.

Fragments of different regions of brain, spinal cord and retina obtained from 6 up to 7 wk human embryos were placed into media 199. On 24th hr of cultivation multiple invaginations developed in the explant ventricular surface as a response to its damage while preparing to explantation. Maturity extent of neural and glial elements was not essential to invagination intensity. Fragments of tectum and retina that are characterised by permanent shape of ventricular surface displayed minimal activity. These data suggest that the reaction considered reflects a property of neuroepithelial cells that constitutes an important factor in normal brain morphogenesis.

[The structure of the developing blood vessels of the neocortical anlage of the human embryo]. / Stroenie formiruiushchikhsia krovenosnykh sosudov zakladki neokorteksa émbriona cheloveka.

Using light and electron microscopy the structure of blood vessels of neocortical anlage of human 7-12 embryos was studied. It was shown that at the early stage of formation of intraorgan vascular network the wall of blood vessels of ventricular zone successively differentiate, which is characterized by the appearance of second layer of cells (pericytes), accumulation of basement membrane components, widening of the zone of contacts between endotheliocytes and establishment of the contacts with bipolar cells of neocortex anlage. The morphological data obtained assist in comprehension of physiological aspects of formation of blood brain barrier and regulation of blood flow in human embryonal neocortex.

[Vascularization and development of the cytoarchitecture in the human neocortical rudiment]. / Vaskuliarizatsiia i stanovlenie tsitoarkhitektoniki v zakladke neokorteksa cheloveka.

The development of cytoarchitectonics of the brain rudiments in mammals is accompanied by the formation of an intracerebral vascular network. The relationship between these two processes is insufficiently clear. We studied the development of blood vessels and cytoarchitectonics in the neocortical rudiment of 6- to 13-week old human embryos. The light and electron microscopy methods were used, as well as histochemical visualization of NADPH-diaphorase in the vessel cells. The endothelium proliferation was evaluated using antibodies to proliferating cell nuclear antigen. Starting from week 8 of development, the tangentially oriented vessels formed a intraneural network in the ventricular zone of the rudiment, which appears to restrict the motility of neuroepithelial cells. The basal membrane was initially absent, and the neuroepithelial cells were in direct contact with the endothelial cells. During week 9 of development, the tangentially oriented vessels appeared in the intermediate zone. Formations similar to glial legs with short regions of the basal membrane adjoined the walls of inter- and intraneural vessels (note that, according to the published data, glial fibrillary acidic protein is not yet visualized at this stage). Angioarchitectonics depended little on the cell population density in different zones of the rudiment; specifically, the cortical plate did not contain tangentially oriented vessels until week 12-13 of development. The data we obtained suggest that the blood vessels fulfill a special morphogenetic function in the developing neocortex.

[The development of transplants of human embryonic neocortex in the anterior chamber of the rat eye]. / Razvitie transplantatov neokorteksa émbrionov cheloveka v perednei kamere glaza krysy.

Dorsolateral regions of embryonic human neocortex at 9-11 weeks of development were transplanted into the eye anterior chamber of rat. Significant changes, leading to the complete destruction of the original cytoarchitectonies occured during the first three days. Intensive neuroblast degeneration in cortical and interstitial zones as well as decrease of mitotically deviding cell numder were observed 3-6 hours after the transplantation. Neuroepithelial cells form rosettes in ventricular zone by the end of the first day. Rosettes turned into the centres of proliferation, mitotic activity restored to the initial level. Cells migrated out of the rosettes in radial directions. By the end of the third day almost all the cells in cortical and interstitial zones degenerated, transplant was almost completely represented by the ventricular zone cells. Number of mitotic figures exceeded the initial level almost three times, transplant was enlarged, its blood supply was absent. Since the 4-th day mitotic activity decreased, transplant underwent lymphoid infiltration and its complete destruction occured by the end of the second week.

[Mitotic activity and rosette formation in the neuroepithelium of the embryonic human neocortex in vitro]. / Mitoticheskaia aktivnost' i obrazovanie rozetok v neiroépitelii émbrional'nogo neokorteksa cheloveka in vitro.

Fragments of anterior cerebral vesicle wall were exposed in 6-8 weeks human embryos and cultured for up to 48 hrs in media 199. Two types of neuroepithelial cells dynamics were demonstrated: the first one is associated with invaginative movements of the layer (apical contractions and elongation of neuroepithelial cell processes are seen, the other one-with cell mitotic division (apical processes become shorter and wider and nuclei reach germinal zone). Prevalent cell dynamics type is determined by media composition. Type I predominates in serum-free media, while type II-in media containing embryonal serum. The lack of correlation between the extent of neuroepithelial layer bending and number of mitotically dividing cells in it indicates invagination process independence from cell divisions. Thus, neuroepithelial layer invagination is not stimulated by mitotic activity and does not stimulate it.

[Nucleolar apparatus of proliferating and differentiating cells of human embryonic neocortex during formation of the cortical plate]. / Iadryshkovyi apparat proliferiruiushchikh i differentsiruiushchikhsia kletok neokorteksa émbriona cheloveka v period formirovaniia kortikal'noi plastinki.

Human neocortex was studied using light and electron microscopy in embryos 6-10 wks of development. Vast majority of proliferating cells was established to be concentrated in ventricular zone. Nuclear, nucleolar and cytoplasmic organelle structure indicate various level of synthetic processes activity in cells of different layers of the developing human neocortex. The dynamics in nucleolar number in the cells of ventricular zone and cortical plate was demonstrated.