Abstract
Purpose: :
Laser–induced injury of the retinal pigment epithelium (RPE) and Bruch’s membrane (BM) has been used extensively to experimentally–induce choroidal neovascularization (CNV) in rodents and primates. c–fos, a component of AP1 transcription factor, is known to initiate stress responses such as wound healing by modulating transcription of its target genes through AP1 binding sequences. Here we characterize the expression pattern of c–fos in RPE cells immediately after laser injury.
Methods: :
A ND: YAG 532 nm laser was used to produce 1 laser lesion/quadrant in the eyes of adult Brown Norway rats. Three categories of laser lesions were produced; lesions without rupture of BM, lesions with rupture of BM but no evidence of choroidal bleeding and lesions with rupture of BM and visible choroidal bleeding. Rupture of BM was identified by bubble formation during laser application and confirmed by confocal microscopy. Rats were euthanized at 5, 15, 30, 45, 60 minutes and 3, 4, 6 and 12 hours after laser injury. Eyes were fixed in 4% formaldehyde in PBS. Retinas were removed and the RPE/choroid labeled with fluorescent markers for nuclei (DAPI), RPE actin cytoskeleton (phalloidin) and c–fos, an immediate early gene. Specimens were flat–mounted and multi–plane Z series were collected on a Leica SP2 confocal microscope to evaluate the spatial and temporal patterns of c–fos expression in RPE cells within and surrounding the three categories of laser lesions.
Results: :
Eyes from non–lasered controls and 5 minutes post–laser exhibited detectable c–fos signal in the RPE cytoplasm, but not in RPE nuclei. At 15 minutes and 30 minutes post–laser, scattered c–fos–positive RPE nuclei were visible at the perimeter of the lesions. At these time points, c–fos labeling was greater in lesions with rupture of BM and bleeding, compared to lesions without bleeding. At 45 minutes post–laser, intense c–fos labeling was observed in the nuclei of RPE cells encircling the lesion. Lower but detectable c–fos nuclear labeling was also visible in RPE cells up to 700um from the center of the lesion. Sixty minutes post–laser, c–fos labeling at the perimeter of the lesion reached a peak, while labeling outside this zone decreased. The intensity of c–fos labeling decreased significantly by 3 hrs post–laser. At 6 and 12 hr time points, c–fos labeling was limited to a few RPE nuclei.
Conclusions: :
c–fos protein expression is substantially increased in RPE cells surrounding the lesion 45 minutes after laser injury in this CNV model. The transient elevation of c–fos expression may reflect the earliest events in the cellular cascade preceding CNV formation.
Keywords: retinal pigment epithelium • laser • transcription factors