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

Abstract

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.

Keywords: apoptosis; brain; nerve regeneration; neural regeneration; neurogenic niches; neurospheres; optic nerve; proliferation; radial glia cells.

Figure 1

Morphological structure and apoptosis in the optic nerve of trout Oncorhynchus mykiss. (A) General view of the contralateral optic nerve. (B) Cells (black arrows) and TUNEL-labeled granules (red arrow) in the contralateral nerve. (C) Patterns of cell migration (type I cells shown by black arrows, type II cells – red arrows, apoptotic bodies - yellow arrows) in the ipsilateral optic nerve. (D) Accumulation of apoptotic bodies (yellow arrows) in the proximal ipsilateral optic nerve. (E) A large cluster of types III and IV cells (red arrows) in mesaxons of ipsilateral optic nerve, red arrows indicate the different types of TUNEL-labeled elements. (F) General view of the proximal portion of the ipsilateral optic nerve, white arrows show type I migrating cells, and black arrows indicate TUNEL-labeled apoptotic bodies. (G) TUNEL-labeled fibers in the proximal ipsilateral optic nerve (red arrows). Immunoperoxidase TUNEL labeling in combination with methyl green staining. Scale bars: 200 μm (A) and 50 μm (B–G). (H) The number of cells stained with methyl green and TUNEL-labeled cells (mean ± SEM) per visual field in contralateral and ipsilateral nerves (n = 5 in each group; #P < 0.05, ##P < 0.001, vs. ipsilateral and contralateral nerves). (I) Number of TUNEL-labeled elements per visual field in the contralateral and ipsilateral nerves; post hoc Tukey's test was used to determine significant differences in contralateral and ipsilateral nerves.

Figure 2

Localization of proliferative cell nuclear antigen (PCNA) in damaged optic nerve of trout 1 week after optic nerve injury. (A) Clusters of intensely labeled type II and III cells (contoured by oval) in the deep layers of the damaged optic nerve. (B) Accumulation of immunopositive type I cells (contoured by square) in the surface layers of the damaged nerve, a red asterisk here and in figure D shows cells where mitosis was finished. (C) Migratory stream of moderately labeled cells in the superficial layers of optic nerve (shown by black arrows). (D) Stratification of migrating and moderately PCNA-labeled type I cells (indicated by black arrows) and highly immunopositive type IV cells (contoured by square), the area of migration is limited by dotted lines. Scale bars: 50 μm for A–D. (E) Number of TUNEL⁺, PCNA-immunopositive (PCNA⁺) elements and cells stained by methyl green per visual field in damaged nerve (n = 5 in each group; #P < 0.05). (F) Number of PCNA⁺ cells of types I–IV in damaged optic nerve (mean ± SEM). (G) Optical density of PCNA immunolabeling in cells of types I–IV in damaged optic nerve (mean ± SEM). (H) Number of PCNA⁺ cells in contralateral and ipsilateral nerves per 400-fold visual field; post hoc Tukey's test was used to determine significant differences in contralateral and ipsilateral nerves (n = 5 in each group; #P < 0.05).

Figure 3

Localization of proliferative cells nuclear antigen (PCNA) in trout cerebellum 1 week after optic nerve injury. (A) PCNA-immunopositive (PCNA⁺) cells in the cerebellar dorsal matrix zone (DMZ) (delineated by a solid line), the accumulation of PCNA-immunonegative (PCNA^–) cells under the DMZ delineated by the dotted line, yellow arrows show intensively labeled oval cells in the granular layer (GrL), red arrows show rod-shaped migrating cells. (B) Dorsal part of the molecular layer (ML) of the cerebellum. Accumulation of PCNA^– cells is delineated by circle, black arrows indicate the PCNA^– tangentially migrating cells, red arrows point to PCNA^– small round cells, white arrows indicate weakly labeled radially migrating cells, and NN represents neurogenic niche. PCNA⁺ cells in the cerebellar DMZ are delineated by dotted line. (C) Cellular composition of DMZ. Type I cells are shown by red arrows, type II cells by yellow arrows, type III cells by blue arrows, and type IV cells by white arrows. (D) Ventral part of cerebellar body. Black arrows show PCNA⁺ cells in molecular (ML), ganglionic (GL) and granular layers. (E) Infraganglionic plexus (IFGP) in dorsal part of the cerebellum. Blue arrows show the PCNA⁺ cells in IFGS, red arrows point to PCNA^– cells in GrL, ovals delineate a cluster of PCNA⁺ cells in the granular layer. (F) Fragment of ganglionic layer containing neurogenic niche (contoured by square). Peroxidase PCNA immunolabeling on transversal brain sections in situ. Scale bars: 100 μm (A, B, D) and 50 μm (C, E, F). (G) Optical density of PCNA labeling in types I–IV cells in cerebellar DMZ (mean ± SEM). (H) Number of PCNA⁺ types I–IV cells in cerebellar DMZ; Student's t-test was used to determine significant differences in control animals and animals after injury (n = 5 in each group; *P < 0.05, **P < 0.001, vs. control group). UOD: Units of optical density.

Figure 4

Localization of proliferative cell nuclear antigen (PCNA) in the potic tectum of trout 1 week after optic nerve injury. (A) General view of optic tectum. Black arrows show PCNA-immunopositive (PCNA⁺) cells of radial glia, blue arrows show PCNA⁺ cells in deep layers, red arrows point to PCNA-immunonegative (PCNA^–) cells. (B) Radial glia (contoured by square) in stratum marginale (SM) of optic tectum. SO: Stratum opticum. (C) PCNA⁺ radial glia (white arrows) and PCNA^– cells (red arrows) at high magnification. (D) PCNA⁺ cells (red arrows) in stratum griseum et album superficiale (SGAS), stratum griseum centrale (SGC), stratum album centrale (SAC), PCNA^– cells (black arrows); cell cluster forming the neurogenic niche is contoured by oval. (E) Number of PCNA⁺ elements: radial glial cells and immunopositive cells in the deep layers of optic tectum. Tukey's post-hoc test was used to determine significant differences in radial glia cells and immunopositive cells in deep layers. Error bars represent SEM (n = 5; #P < 0.05). (F) Optical density of PCNA-labeling in radial glia and cells in deep layers of optic tectum (mean ± SEM). UOD: Units of optical density.

Figure 5

Phase contrast monitoring in primary culture of trout brain cells. (A) Cells in monolayer; colored arrows indicate the different types of cells: big cell (yellow arrow), cells with outgrowth (red arrow) and cell without outgrowth (black arrow). (B) Suspension fraction of brain cells after 1 day of culture. Red arrow shows single cells. (C) Suspension fraction of cells on the 2^nd day of culture. Ovals contour cell conglomerates, and red arrows indicate single cells. (D) On the 4^th day of culture. (E) General view of neurospheres. (F) Heterogeneous conglomerates of cells in suspension on the 4th day of culture. Scale bars: 50 μm (A–D, F), 10 μm (E). (G) Number of types II–V cells in suspension after 1–2 days of culture. Tukey's post-hoc test was used to determine significant differences in number of cells (n = 7 in each group; #P < 0.05, ##P < 0.001). (H) Size of cell conglomerates after 1, 2 and 4 days of culture (mean ± SEM).

Figure 6

Proliferative cell nuclear antigen (PCNA) labeling of trout brain cells after 4 days in primary culture. (A) Highly active PCNA-immunopositive (PCNA⁺) cells (black arrows), moderately active PCNA⁺ cells (red arrows) and PCNA-immunonegative cells (blue arrows); (B) Conglomerate of highly and moderately labeled PCNA⁺ cells (contoured by oval); arrows represent the same cells as in A. Scale bars: 50 μm (A, B). (C) Tukey's post-hoc test was used to determine significant differences in number of PCNA+ cells. Error bars represent (mean ± SEM, #P < 0.05, ##P < 0.001). (D) Optical density of PCNA⁺ cells (mean ± SEM).