GFAP expression in the optic nerve and increased H(2)S generation in the integration centers of the rainbow trout (Oncorhynchus mykiss) brain after unilateral eye injury

Hydrogen sulfide (H(2)S) is considered as a protective factor against cardiovascular disorders. However, there are few reports on the effects of H(2)S in the central nervous system during stress or injury. Previous studies on goldfish have shown that astrocytic response occurs in the damaged and contralateral optic nerves. Glial fibrillary acidic protein (GFAP) concentration in the optic nerves of rainbow trout has not been measured previously. This study further characterized the astrocytic response in the optic nerve and the brain of a rainbow trout (Oncorhynchus mykiss) after unilateral eye injury and estimated the amount of H(2)S-producing enzyme cystathionine β-synthase (CBS) in the brain of the rainbow trout. Within 1 week after unilateral eye injury, a protein band corresponding to a molecular weight of 50 kDa was identified in the ipsi- and contralateral optic nerves of the rainbow trout. The concentration of GFAP in the injured optic nerve increased compared to the protein concentration on the contralateral side. The results of a quantitative analysis of GFAP(+) cell distribution in the contralateral optic nerve showed the largest number of GFAP(+) cells and fibers in the optic nerve head. In the damaged optic nerve, patterns of GFAP(+) cell migration and large GFAP(+) bipolar activated astrocytes were detected at 1 week after unilateral eye injury. The study of H(2)S-producing system after unilateral eye injury in the rainbow trout was conducted using enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemistry of polyclonal antibodies against CBS in the integrative centers of the brain: telencephalon, optic tectum, and cerebellum. Enzyme-linked immunosorbent assay results showed a 1.7-fold increase in CBS expression in the rainbow trout brain at 1 week after unilateral eye injury compared with that in intact animals. In the ventricular and subventricular regions of the rainbow trout telencephalon, CBS(+) radial glia and neuroepithelial cells were identified. After unilateral eye injury, the number of CBS(+) neuroepithelial cells in the pallial and subpallial periventricular regions of the telencephalon increased. In the optic tectum, unilateral eye injury led to an increase in CBS expression in radial glial cells; simultaneously, the number of CBS(+) neuroepithelial cells decreased in intact animals. In the cerebellum of the rainbow trout, neuroglial interrelationships were revealed, where H(2)S was released, apparently, from astrocyte-like cells. The organization of H(2)S-producing cell complexes suggests that, the amount of glutamate produced in the rainbow trout cerebellum and its reuptake was controlled by astrocyte-like cells, reducing its excitotoxicity. In the dorsal matrix zone and granular eminences of the rainbow trout cerebellum, CBS was expressed in neuroepithelial cells. After unilateral eye injury, the level of CBS activity increased in all parts of the cerebellum. An increase in the number of H(2)S-producing cells was a response to oxidative stress after unilateral eye injury, and the overproduction of H(2)S in the cerebellum occurred to neutralize reactive oxygen species, providing the cells of the rainbow trout cerebellum with a protective effect. A structural reorganization in the dorsal matrix zone, associated with the appearance of an additional CBS(+) apical zone, and a decrease in the enzyme activity in the dorsal matrix zone, was revealed in the zones of constitutive neurogenesis. All experiments were approved by the Commission on Biomedical Ethics, A.V. Zhirmunsky National Scientific Center of Marine Biology (NSCMB), Far Eastern Branch, Russian Academy of Science (FEB RAS) (approval No. 1) on July 31, 2019.