Retinal function and PKC alpha expression after focal laser photocoagulation

Abstract

Purpose: To examine the effects of focal laser photocoagulation on general and local retinal function and to relate electrophysiological findings with changes in protein kinase C (PKC) alpha expression.

Methods: Twelve rabbits were treated with 70 spots of laser photocoagulation in the central cone-rich retina. The operated eyes were investigated with electroretinography (full-field ERG and multifocal electroretinography, mfERG) preoperatively and at 1, 3, and 5 weeks after surgery. The expression of PKC alpha was examined at all three time points using immunohistochemistry, and PKC alpha mRNA levels were quantified using real-time polymerase chain reaction (PCR). Immunohistochemistry for glial fibrillary acidic protein (GFAP) and hematoxylin and eosin staining was employed to monitor the extent and dynamics of the morphological response.

Results: The full-field ERG revealed a significant increase in b-wave amplitudes derived from the isolated rod response (blue light) at all three time points after surgery (p < 0.05). Supernormal b-wave amplitudes were also found for the combined rod-cone response at 3 weeks (white light), and for the isolated cone response (light-adapted 30-Hz flicker) at 5 weeks after treatment. In the mfERG, amplitudes derived from the central retina did not change postoperatively, while the implicit time was significantly increased at all time points. Immunohistochemistry for PKC alpha revealed a reduced expression of the enzyme in rod bipolar cells 1 and 3 weeks after laser treatment compared with untreated controls. Five weeks postoperatively, no PKC alpha labeling in rod bipolar cells was found in any part of the retina. Real-time PCR 1 and 3 weeks after treatment displayed a decreased level of PKC alpha mRNA compared to the controls. Immunolabeled tissue sections from laser-treated eyes displayed GFAP expression in Müller cells in the treated as well as untreated retina 1 week postoperatively. At 3 and 5 weeks, GFAP labeling was less pronounced and was concentrated around the laser-treated spots.

Conclusions: Focal laser treatment in the rabbit eye induces local and wide-spread alterations in both rod- and cone-mediated retinal function in the form of supernormal b-wave amplitudes in the full-field ERG and increased latency in the mfERG. The electrophysiological abnormalities are accompanied by a progressive down-regulation of the PKC alpha isoenzyme in rod bipolar cells, reaching far beyond the treated area. PKC alpha is down-regulated directly by impaired protein synthesis, and also possibly indirectly by protein consumption related to GFAP up-regulation. The results indicate that focal laser photocoagulation interferes with PKC-alpha-mediated inhibitory regulation of inner retinal signal transmission.