A. A. Varaksin

Features of adult neurogenesis and neurochemical signaling in the Cherry salmon Oncorhynchus masou brain.

We investigated the distribution of gamma aminobutyric acid, tyrosine hydroxylase and nitric oxide-producing elements in a cherry salmon Oncorhynchus masou brain at various stages of postnatal ontogenesis by immunohistochemical staining and histochemical staining. The periventricular region cells exhibited the morphology of neurons and glia including radial glia-like cells and contained several neurochemical substances. Heterogeneous populations of tyrosine hydroxylase-, gamma aminobutyric acid-immunoreactive, as well as nicotinamide adenine dinucleotide phosphate diaphorase-positive cells were observed in proliferating cell nuclear antigen-immunoreactive proliferative zones in periventricular area of diencephalon, central grey layer of dorsomedial tegmentum, medulla and spinal cord. Immunolocalization of Pax6 in the cherry salmon brain revealed a neuromeric construction of the brain at various stages of postnatal ontogenesis, and this was confirmed by tyrosine hydroxylase and gamma aminobutyric acid labeling.

Cystathionine β-synthase in the CNS of masu salmon Oncorhynchus masou (Salmonidae) and carp Cyprinus carpio (Cyprinidae)

Immunohistochemical labeling showed the presence of cystathionine β-synthase in neurons of the ventral spinal column, medulla oblongata, the cells and fibers of the cerebellum, optic tectum, and telencephalon of the masu salmon Oncorhynus masou and the carp Cyprinus carpio. We found considerable interspecies differences in the localization and optical density of immunoreactive structures in brain areas of masu salmon and carp which are presumably related to the specificities of the feeding and behavior of these fishes. In carp, the medulla oblongata and spinal cord had intensely labeled vessels, which were absent in masu salmon. The periventricular area of the medulla oblongata and ventral and lateral areas of the cerebellum of the carp had strongly CBS-positive cells without outgrowths. The sizes of cells and their location in the brain and interrelations with H2S-producing neurons suggest that periventricular area of the carp brain has H2S-producing glia.

Reparative Neurogenesis in the Brain and Changes in the Optic Nerve of Adult Trout Oncorhynchus mykiss after Mechanical Damage of the Eye

Reparative proliferation and neurogenesis in the brain integrative centers after mechanical eye injury in an adult trout Oncorhynchus mykiss have been studied. We have found that proliferation and neurogenesis in proliferative brain regions, the cerebellum, and the optic tectum were significantly enhanced after the eye injury. The cerebellum showed a significant increase in the proliferative activity of the cells of the dorsal proliferative zone and parenchymal cells of the molecular and granular layers. One week after the injury, PCNA-positive radial glia cells have been identified in the tectum. We have found for the first time that the eye trauma resulted in the development of local clusters of undifferentiated cells forming so called neurogenic niches in the tectum and cerebellum. The differentiation of neuronal cells detected by labeling cells with antibodies against the protein HuC/D occurred in the proliferative zones of the telencephalon, the optic tectum, cerebellum, and medulla of a trout within 2 days after the injury. We have shown that the HuC/D expression is higher in the proliferative brain regions than in the definitive neurons of a trout. In addition, we have examined cell proliferation, migration, and apoptosis caused by the eye injury in the contra- and ipsilateral optic nerves and adjacent muscle fibers 2 days after the trauma. The qualitative and quantitative assessment of proliferation and apoptosis in the cells of the optic nerve of a trout has been made using antibodies against PCNA and the TUNEL method.

Cell proliferation and apoptosis in optic nerve and brain integration centers of adult trout Oncorhynchus mykiss after optic nerve injury.

Fishes have remarkable ability to effectively rebuild the structure of nerve cells and nerve fibers after central nervous system injury. However, the underlying mechanism is poorly understood. In order to address this issue, we investigated the proliferation and apoptosis of cells in contralateral and ipsilateral optic nerves, after stab wound injury to the eye of an adult trout Oncorhynchus mykiss. Heterogenous population of proliferating cells was investigated at 1 week after injury. TUNEL labeling gave a qualitative and quantitative assessment of apoptosis in the cells of optic nerve of trout 2 days after injury. After optic nerve injury, apoptotic response was investigated, and mass patterns of cell migration were found. The maximal concentration of apoptotic bodies was detected in the areas of mass clumps of cells. It is probably indicative of massive cell death in the area of high phagocytic activity of macrophages/microglia. At 1 week after optic nerve injury, we observed nerve cell proliferation in the trout brain integration centers: the cerebellum and the optic tectum. In the optic tectum, proliferating cell nuclear antigen (PCNA)-immunopositive radial glia-like cells were identified. Proliferative activity of nerve cells was detected in the dorsal proliferative (matrix) area of the cerebellum and in parenchymal cells of the molecular and granular layers whereas local clusters of undifferentiated cells which formed neurogenic niches were observed in both the optic tectum and cerebellum after optic nerve injury. In vitro analysis of brain cells of trout showed that suspension cells compared with monolayer cells retain higher proliferative activity, as evidenced by PCNA immunolabeling. Phase contrast observation showed mitosis in individual cells and the formation of neurospheres which gradually increased during 1-4 days of culture. The present findings suggest that trout can be used as a novel model for studying neuronal regeneration.

Are in vivo and in vitro assessments of comparative and combined toxicity of the same metallic nanoparticles compatible, or contradictory, or both? A juxtaposition of data obtained in respective experiments with NiO and Mn 3 O 4 nanoparticles

Comparative and combined damaging effects of NiO and Mn3O4 nanoparticles were estimated on cultures of several established human cell lines. The cytotoxicity indices used were: (a) reduction in cellular dehydrogenase activity, (b) decrease in the ATP-content, (c) for SH-SY5Y cells also decrease in the tyrosine hydroxylase content. The combined cytotoxicity was modeled using the Response Surface Methodology. When assessing the stability of metal oxide nanoparticles (MeO-NPs) in cultural media used by us, we found that the addition of the fetal bovine serum (FBS) to them renders NiO-NPs and, to even greater extent, Mn3O4-NPs exponentially slow soluble while without FBS their dissolution was virtually undetectable. At the same time, sedimentation of these MeO-NPs noticeably slowed down in the presence of the same FBS. We have found dependence of cell damage on concentrations of MeO-NPs and higher cytotoxicity of Mn3O4-NP compared with NiO-NP. Thus, comparative assessment of the NPs unspecific toxicity obtained in our animal experiments was reproduced by the inxa0vitro tests. However, with respect to manganese-specific brain damage inxa0vivo discovered previously, present experiments on neurons inxa0vitro showed only a certain enhancing effect of Mn3O4-NP on the action of NiO-NP, but the role of NiO-NP in the combination prevailed.

Combined Subchronic Toxicity of Aluminum (III), Titanium (IV) and Silicon (IV) Oxide Nanoparticles and Its Alleviation with a Complex of Bioprotectors

Stable suspensions of metal/metalloid oxide nanoparticles (MeO-NPs) obtained by laser ablation of 99.99% pure elemental aluminum, titanium or silicon under a layer of deionized water were used separately, or in three binary combinations, or in a ternary combination to induce subchronic intoxications in rats. To this end, the MeO-NPs were repeatedly injected intraperitoneally (i.p.) 18 times during 6 weeks before measuring a large number of functional, biochemical, morphological and cytological indices for the organism’s status. In many respects, the Al2O3-NP was found to be the most toxic species alone and the most dangerous component of the combinations studied. Mathematical modeling with the help of the Response Surface Methodology showed that, as well as in the case of any other binary toxic combinations previously investigated by us, the organism’s response to a simultaneous exposure to any two of the MeO-NP species under study was characterized by a complex interaction between all possible types of combined toxicity (additivity, subadditivity or superadditivity of unidirectional action and different variants of opposite effects) depending on which outcome this type was estimated for and on effect and dose levels. With any third MeO-NP species acting in the background, the type of combined toxicity displayed by the other two remained virtually the same or changed significantly, becoming either more or less unfavorable. Various harmful effects produced by the (Al2O3-NP + TiO2-NP + SiO2-NP)-combination, including its genotoxicity, were substantially attenuated by giving the rats per os during the entire exposure period a complex of innocuous bioactive substances expected to increase the organism’s antitoxic resistance.

GFAP and PCNA Marking in the cerebellum of masu salmon’s (Oncorhynchus masou) juvenile after mechanical injury

The objective of this work was to study proliferation processes and the role of glia and neural stem cells in the event of injurious action on cerebellum of masu salmon’s (Oncorhynchus masou) juvenile. Using the immunoperoxidase staining of the glial fibrillary acidic protein (GFAP) and proliferating cells nuclear antigen (PCNA), processes of proliferation and gliogenesis after mechanical trauma of cerebellum of cherry salmon’s (Oncorhynchus masou) juvenile were studied. After the trauma, the intensity of proliferation and migration processes varies in different zones. Proliferation processes decrease after the trauma in lateral and basal zones, and migration increases. In the dorsal zone, on the contrary, migration processes significantly decrease and proliferation increases. In the dorsal matrix zone of a cerebellum, intense cell proliferation was detected. In the dorsal, lateral, and basal zone of the molecular layer of cerebellum after traumatic damage, neurogenic niches containing PCNA and cells, as well as a heterogeneous population of PCNA-cells, were identified. At the location of neurogenic niches, fibers of radial glia and small single intensely or moderately labeled GFAP cells were discovered. As a result of damaging action, GFAP+ fibers of radial glia, which form differently directed radially oriented bundles, appeared in the dorsal matrix zone. Such structural formations have not been discovered in intact animals. We suppose that, after the trauma, structural reconstruction connected with partial spatial reorientation of the radial glia fibers and formation of specific directions for cells formed in this zone occurs in the dorsal matrix zone. As a result of the trauma, in masu salmon’s cerebellum, elements of the radial glia, including both cells possessing typical morphology and cell fragments presented as long radially oriented processes or cell body containing initial fragments of radial fibers, appeared.

Persistent and reparative neurogenesis in the juvenile masu salmon Oncorhynchus masou telencephalon after mechanical injury

A superficially located periventricular proliferative area with PCNA-immunopositive (PCNA+) cells, which corresponds to the pallial periventricular zone (PVZ) of other fish species, including its dorsal, lateral, and medial compartments, is discovered in the telencephalon of the juvenile masu salmon Oncorhynchus masou. The PCNA+ cells are also identified in the parenchyma of the masu salmon intact brain, and their maximum concentration is observed in the medial zone. After a mechanical injury, the zones of induced neurogenesis—neurogenic niches and sites of secondary neurogenesis surrounded by radial glial fibers—appear in the masu salmon telencephalon. The PVZ of the juvenile masu salmon pallium contains clusters of undifferentiated HuCD-immunopositive (HuCD+) neurons. A change in the HuCD+ cell topography is observed in the mechanically injured masu salmon telencephalon, namely, neurogenic niches in the lateral zone and an increase in the cell distribution density and cell migration patterns in the medial zone. A high level of persistent neurogenesis is characteristic of the juvenile masu salmon brain.

Cell proliferation and differentiation in primary cultures of the juvenile brain of the masu salmon Oncorhynchus masou

The central nervous system of fish has a high ability to produce new nerve cells in the adult organism in certain neurogenic areas of the brain throughout life. Analysis of proliferative activity and the ability to neuron differentiation was performed in cultured cells of the brain and spinal cord of the juvenile masu salmon Oncorhynchus masou. Proliferating cell nuclear antigen (PCNA) was used as a proliferative marker, while a marker of neuronal differentiation, the neuron protein HuCD, detected neurons. The results showed that cell proliferation occurs mainly in the suspension cell fraction. In monolayer, only a few cells were found to express PCNA. The results of immunocytochemical analysis allow us to conclude that proliferative activity in primary cultures from the O. masou brain is mainly connected with the suspension fraction of small cells. In contrast, the presence of the cells expressing cystathionine β-synthase, a marker of H2S synthesis, and the cells expressing PCNA in the monolayer, may indicate the possible participation of H2S in proliferative activity of neurons in primary cultures. The data obtained suggest that the hydrogen sulphide can be involved in the process of neuronal differentiation; however, the nature of its participation remains largely unknown.

Localization of NADPH-diaphorase and neuronal NO-synthase in the digestive tract of the Masu salmon, Oncorhynchus masou (Osteichthyes: Salmonidae)

The morphology, localization, and distribution of nitrergic neurons in the digestive tract of juvenile and adult Masu salmon, Oncorhynchus masou, were studied using histochemical and immunohistochemical methods. We revealed general and age-related peculiarities in the distribution and number of NO-ergic enteric neurons. These neurons were detected in juveniles only in the esophagus and the stomach, while in the adult fish, the neurons were present throughout the gastrointestinal tract. The total number of labeled cells was considerably lower in the proximal intestine than in the esophagus and the stomach, but was some-what greater in the distal intestine. Both in juveniles and adults, neurons and nerve fibers were located within the branches of the vagus nerve, in the myenteric nervous plexus and the circular muscle, and, more rarely, in the submucous nervous plexus and in the longitudinal muscle. Unipolar and bipolar neurons and a large number of undifferentiated and subdifferentiated cells were found, with the latter being more numerous in juveniles. Among multipolar cells, the neurons of Dogiel’s morphological types I and II occurred and they were more abundant in adult fish. Moreover, positive nerve fibers were present in the muscle wall of numerous blood vessels and the swim bladder ducts of juvenile and adult fish.