Abstract
Abstract: :
Purpose: Endothelins are powerful vasoconstrictor peptides, that also act as astrocytic growth factors, regulators of gap junctions conductance and blood–retinal barrier permeability. Since they could be involved in the glial response to retinal injury, we have studied the expression of endothelins and their receptors in mouse retinas submitted to constant–light–induced photoreceptor degeneration. Methods:BALB–c mice were bred under cyclic low level illumination. Experimental animals were kept under 1,500 lux for periods of 3, 6, 9 and 18 days. Retinas obtained from perfusion–fixed mice were incubated with antibodies against glial fibrillary acidic protein (GFAP), endothelins 1 and 3 (ET–1 and ET–3) or their receptors ET–A, ET–B. Areas of astrocyte immunoreactivity (ir) were measured in retinal wholemounts using Kontron 400. For RT–PCR, RNA was extracted from non–fixed retinas. Results:Almost half of the outer nuclear layer disappeared after 6 days of constant illumination, whereas only a single layer of cell nuclei remained after 18 days. Gliosis, as evidenced by the area of GFAP immunoreactivity, increased after 3 days of illumination. In normal retinas, ET–1–ir was only detected in astrocytes, as shown by co–localization with GFAP. ET–3–ir could not be observed. ET–A–ir and ET–B–ir appeared on endothelial cells. ET–A–ir was also present in ganglion cells and outer plexiform layer (OPL). Astrocytes exhibited very low levels of ET–B–ir. In light–injured retinas, astrocytic ET–1–ir area steadily increased during the experimental period. Significant changes, however, were only present at the 18 day stage. The ratio ET–1/actin mRNA increased in a similar fashion. By contrast, area of astrocytic ET–B–ir showed an early increase, significant differences being detected by the 6 day stage. No changes in endothelial and ganglion cell immunoreactivities could be detected, but ET–A–ir disappeared from OPL. Conclusions:Gliosis, affecting both Müller cells and astrocytes, was an early event in our experimental model. The endothelinergic system seemed to be specifically associated to astrocytes, since neither the ligands nor the receptors could be detected in Müller cells. The early increase in astrocytic ET–B immunoreactivity suggests that this receptor might probably be involved in the gliotic response.
Keywords: pathology: experimental • retinal glia • immunohistochemistry