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.

Hydrogen Sulfide in Proliferating and Differentiated Cells in Primary Cultures of Juvenile Brain of Masu Salmon <i>Oncorhynchus masou</i>

Analysis of proliferative activity and the ability to neuron differentiation was performed in cultured cells of the brain and spinal cord of juvenile masu salmon Oncorhynchus masou. Proliferating cell nuclear antigen (PCNA) was used as a proliferative marker, while the markers of neuronal differentiation—a neuron protein HuCD, and a neuron-specific transcriptional factor with two DNA- binding sites Pax6—detected neurons. The results showed that cell proliferation occurred mainly in the suspension cell fraction. In monolayer, a few cells were only found to express PCNA. The results of morphological and immunohistochemical 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, a positive correlation between the cells expressing cystathionine β-synthase (CBS), a marker of H2S synthesis, and the cells expressing PCNA in the monolayer, indicates the participation of H2S in proliferative activity of neurons in primary cultures. The data obtained suggest that the hydrogen sulphide is also involved in the process of differentiation.