Confocal Laser and Electron Microscopic Investigation of Gap Junctions in Anaplastic Astrocytomas.

Connexin 43 (Cx43) is a multifunction protein that forms gap junction channels and hemichannels and is suggested to play an essential role in oxygen-glucose deprivation, induced via neuroinflammation during astrocytoma expansion into healthy tissue. To prove this assumption we studied connexin 43 localisation and ultrastructure of gap junctions in samples of malignant brain tumour (anaplastic astrocytomas grade III). For confocal laser microscopy, vibratome sections of tumour fragments were incubated in a mixture of primary antibodies to connexin 43 and glial fibrillary acidic protein (GFAP), then in a mixture of secondary antibodies conjugated with a fluorescent label. After the immunofluorescence study, sections were washed in phosphate buffer, additionally postfixed with 1% OsO4 solution, dehydrated and embedded in epoxy resin by a plane-parallel method. Ultra-thin sections obtained from these samples were contrasted with uranyl acetate and lead citrate and viewed under a Jem 1011 electron microscope. Confocal laser examination detected a positive reaction to Cx43 in the form of point fluorescence. These points were of various sizes. Most of them were localised around or at the intersection of small processes containing GFAP. Electron microscopy of the tumour samples containing the most significant number of Cx43 revealed single and closely spaced gap junctions with a typical ultrastructure on the processes and bodies of tumour cells. Sequential analysis in the fields of view revealed 62 gap junctions in the area of 100 µm2. Numerous gap junctions in anaplastic astrocytomas revealed in our study may indicate electrotonic and metabolic transmission between glioma cells, possibly promoting its progression.

Evaluation of Age-Dependent Changes in the Coloration of Male Killifish Nothobranchius Guentheri Using New Photoprocessing Methods.

Fish as model objects have found wide applications in biology and fundamental medicine and allow studies of behavioral and physiological responses to various environmental factors. Representatives of the genus Nothobranchius are one of the most convenient objects for such studies. Male fish belonging to the family Nothobranchiidae are characterized by extremely diverse coloration, which constantly changes, depending on the age of the fish, environmental factors, and social hierarchical status. These fish species are characterized by a short life cycle, which allows changes in coloration, an indicator of the ontogenesis stage, to be estimated. Existing methods of fish color assessments do not allow the intensity of coloration of particular body zones to be clearly differentiated. In the present study, we suggest a method of two-factor assessment of specific fish body zones using modified methods of photofixation and image processing software. We describe the protocol of the method and the results of its application to different-aged groups of male Nothobranchius guentheri. The coloration of selected areas (i.e., red spot on the gill cover (RSGC), black border on the caudal fin (BBCF), and white border on the dorsal fin (WBDF)) differed significantly according to the size and age of the fish (p < 0.05). The data obtained suggest that N. guentheri can be a model for studying aging by the intensity of body coloration in males.

Ultrastructural evidence for presenсe of gap junctions in rare case of pleomorphic xanthoastrocytoma.

The phenomenon of unstable expression of gap junction's proteins connexins remains a "visiting card" of astrocytic tumors with various degrees of malignancy. At the same time, it stays unclear what is detected by the positive expression of connexins in astrocytic tumors: gap junctions, hemi-channels, or connexin proteins in cytosol. In the present work, for the first time, we demonstrate an ultrastructural evidence of gap junctions in pleomorphic xanthoastrocytoma, a rare primary brain tumor, the intercellular characteristics of which are poorly studied and remain very discursive and controversial. The primary tumor mass was resected during craniotomy from a 57-old patient diagnosed with pleomorphic xanthoastrocytoma Grade II based on the histopathological analysis. The immunohistochemical study was conducted with primary antibodies: Neurofilament, Myelin basic protein, Glial fibrillary acidic protein, and Synaptophysin. For electron microscopic examination fragments of tumor tissue were fixed in a glutaraldehyde, postfixed in a 1% OsO4, dehydrated and embedded into resin. After the detailed clinical, histological, and immunohistochemical study we revealed some ultrastructural characteristics of the tumor, as well as the first evidence of direct intercellular connection between the tumor cells via gap junctions. Regularly arranged gap junctions connected the somas of xanthastrocytes with dark cytoplasm containing lipid drops. Besides the localization between the cell bodies, from one to several gap junctions were found between the branches of xanthoastrocytoma in tumor intercellular space in close proximity to tumor cell. Our results may indicate gap junctions as a possible structure for intercellular communication between pleomorphic xanthoastrocytoma cells.

Laminar distribution of gap-junctions in rat somatic cortical columns.

Results about the spindle-like activity peculiarities and the dendro-dendritic gap-junctions numbers in the infragranular and supragranular layers (modules) of the rat somatic cortex columns are presented. During simultaneous recording of extracellular field potentials from the cortical columns, the following was revealed: (i) the spindle-like activity was more synchronized inside one cortical column than in different columns; (ii) in most of the experiments (9 out of 10) an amplitude of the spindle waves was two times more in the supragranular layers than in the infragranular layers. During electron microscopy investigation of the experimental objects used in the electrophysiological study, it was shown that the number of the gap-junctions was more in the supragranular columnar layers as compared with the infragranular layers (4.15% and 2.84% out of the total number of chemical synapses and gap-junctions identified in each module, respectively). The results obtained are discussed as possible structural base of the local electrotonic synchronization of the neuronal activity inside a cortical column.