Abstract
Purpose: :
Unlike humans, teleost fish have the ability to regenerate their retinas after damage. In a prior study, we demonstrated the recovery of visual function in zebrafish subjected to widespread retinal damage inflicted by intraocular injection of ouabain (Sherpa et al., 2008; Dev Neurobiol 68:166-81). Functional recovery required 98 days and corresponded to the time of optic nerve head (ONH) restoration. However, at later recovery times, we observed overproduction of cells in the retinal ganglion cell (RGC) layer and hypertrophy of the ONH. In addition, while the spatiotemporal pattern of RCG gene expression largely recapitulated that seen in embryonic development, the genes encoding fgf8, shh, and brn3b, were not detected during regeneration. The current study has two goals: 1) determine whether a more selective lesion to inner retinal layers will result in fewer histological errors and faster visual recovery; and 2) investigate temporal patterns of RGC gene expression using quantitative as well as qualitative methods after each type of lesion.
Methods: :
Zebrafish were injected intraocularly with 0.5 ul of 200 uM ouabain, creating widespread retinal damage, or with 0.5ul of 40 uM ouabain, creating selective retinal damage to the inner retinal layers (as in Fimbel et al., 2007; J Neurosci 27:1712-24). Radial sections of the retina containing the optic nerve head (ONH) were stained with methylene blue and images of these sections were imported into Scionimage for measurement of ONH diameter. Whole mount Immunohistochemistry was performed using ganglion cell marker islet-1, labeled cells were counted and an estimate of ganglion cell density was calculated. In-situ hybridization, RT-PCR, and QRT-PCR were performed to study the expression of selected genes in regenerating retinas in both broad and selective lesion models. Visual function was monitored by evaluation of the dorsal light reflex.
Results: :
Following selective retinal damage, the ONH returned to normal size at 60 days (Fimbel et al., 2007; J Neurosci 27:1712-24). In addition, eyes evaluated at later time points (up to 210days) did not show ONH hypertrophy and did not contain supernumerary cells in the GCL. Studies of visual function are in progress. Preliminary studies of gene expression using RT-PCR showed that shh was indeed expressed after broad as well as selective retinal damage, at 8 days following the lesion. Further quantitative studies using QRT-PCR are in progress.
Conclusions: :
Regeneration of RGCs may be optimized by the presence of other, undamaged retinal cells. The overproduction of RGCs following widespread retinal damage is probably not related to errors in expression of shh, as we had previously hypothesized.
Keywords: regeneration • ganglion cells • optic nerve