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.

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.

X-ray micro-computed tomography in the assessment of penile cavernous fibrosis in a rabbit castration model.

BACKGROUND: Current assessment methods of penile cavernous fibrosis in animal models have limitations due to the inability to provide complex and volume analysis of fibrotic alterations. OBJECTIVE: The aim was to evaluate micro-computed tomography for assessment of cavernous fibrosis and compare it with histological, histochemical, immunohistochemical, and RT-PCR analysis. MATERIALS AND METHODS: A controlled trial was performed involving 25 New Zealand male rabbits with induced testosterone deficiency by orchidectomy. Penile samples were obtained before and after 7, 14, 21, and 84 days from orchidectomy. We consistently performed (a) gray value analysis of corpora cavernosa 3D models reconstructed after micro-computed tomography, (b) morphometry of smooth muscles/connective tissue ratio, collagen type I/III ratio, and area of TGF-beta-1 expression in corpora cavernosa, and (c) RT-PCR of TGF-beta-1 expression. RESULTS: Micro-computed tomography allowed visualization of penile structures at a resolution comparable to light microscopy. Gray values of corpora cavernosa decreased from 1673 (1512-1773) on the initial day to 1184 (1089-1232) on the 21st day (p < 0.005). However, on the 84th day, it increased to 1610 (1551-1768). On 21st and 84th days, there was observed a significant decrease in smooth muscle/connective tissue ratio and a significant increase in collagen type I/III ratio (p < 0.05). TGF-beta1 expression increased on the 84th day according to immunohistochemistry (p < 0.005). RT-PCR was impossible to conduct due to the absence of RNA in obtained samples after micro-CT. DISCUSSION AND CONCLUSIONS: Micro-computed tomography provided 3D visualization of entire corpora cavernosa and assessment of radiodensity alterations by gray value analysis in fibrosis progression. We speculate that gray value changes at early and late fibrosis stages could be related to tissue reorganization. RT-PCR is impossible to conduct on tissue samples studied by micro-CT due to RNA destruction. We also suggest that micro-computed tomography could negatively affect the immunohistochemical outcome, as a significant increase of TGF-beta-1 expression occurs later than histological fibrotic signs.

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.