Nestin contributes to laser choroidal and retinal neovascularization.

Purpose: Choroidal and retinal neovascularization plays an essential role in various ocular diseases. In this study, we examined the role of nestin in this process. Nestin is an intermediate filament protein known to play several roles, including as a marker of neural progenitor and proliferating endothelial cells. Methods: We used Brown Norway rats, in which choroidal and retinal neovascularization was induced using intraocular laser impacts. The role of nestin was examined using angiography, western blot from the second to the 14th day after laser impacts, and intraocular injection of nestin siRNA. The localization of the protein was specified by co-immunoreactivity with glial fibrillary protein (GFAP), glutamine synthetase (GS), and von Willebrand factor (vWF). Results: In the control retina, nestin was found principally in glial structures in the ganglion cell layer, as confirmed by nestin/GFAP immunolabeling. Two days after the laser impacts, the nestin expression extended to numerous radial processes at the site of the impacts. With Bruch's membrane ruptured, these processes penetrated into the choroid. Nestin immunolabeling remained high from the third to the seventh day but appeared reduced on the 14th day. The nature of these processes was not clearly defined, but co-immunolabeling with GFAP suggested that they were principally in activated Müller cells from the third day after the laser impacts. However, the co-immunoreactivity of nestin and GS, a marker of mature functional Müller cells, could be observable only from the seventh day. Nestin was also observed in some vascular cells, as demonstrated by the co-immunoreactivity of the protein with vWF in the choroid and retina. As observed on angiography, the numbers of choroidal and retinal blood vessels were significantly increased (principally on the seventh day) after the laser impacts. An intraocular injection of nestin siRNAs led to a significant decrease in the number of blood vessels. Conclusions: Our results confirmed the presence of nestin in glial (e.g., astrocytes), reactive Müller, and endothelial cells. They demonstrated their critical involvement in a rat model of retinal and choroidal neovascularization experimentally induced using ocular laser impacts.

Nestin expression in the retina of rats with inherited retinal degeneration.

Nestin is found in radial glia and neuronal/glial progenitor cells during retinal development, and is re-expressed after acute damage in the retina of adult mammals. We have investigated nestin expression in the retina of the Royal College of Surgeons (RCS) rat model of human inherited blindness, Retinitis pigmentosa (RP). During the first postnatal week, nestin immunoreactivity was located in elongated processes resembling radial glia in both control and dystrophic animals. During the second postnatal week, the density of nestin immunoreactive radial processes decreased progressively starting in the outer retina. At postnatal day 20 (PNd20), Nestin immunoreactive radial processes were no longer visible, with immunoreactivity restricted to structures resembling Müller end-feet and/or astrocytes located in the ganglion cell layer (GCL) in both control and dystrophic rats. These morphological results were confirmed by Western blotting and qPCR analysis. The level of nestin remained low in control animals at different time points up to 1 year, but we observed a re-expression of this protein from PNd30 in the dystrophic animals. The morphology of cells re-expressing nestin resembled that of radial glia and/or Muller cells, but co-localization of nestin and glutamine synthetase (GS: a marker of mature Müller cells) was only partial. Interestingly, whereas Western blot analysis confirmed the increase in protein levels from PNd30 onwards, mRNA levels remained low in dystrophic rats. Additional studies demonstrated that the discrepancy between protein and mRNA contents could be due to a dysfunction in proteasome activity as often observed in neurodegenerative pathologies. In conclusion, because of its localization in astrocytes and in radial processes resembling radial glia in the pathologic adult retina, nestin may be involved in mechanisms such as cell migration, generation of new neurons or glial cells and/or in retinal (re)modeling in dystrophic adult animals. The lack of concomitant up-regulation of mRNAs in adult dystrophic animals suggests that the pathology could lead to transcriptional and/or metabolic changes involving the stabilization of the half-life and/or dysregulation of degradation processes of nestin protein.