Immunocytochemical localization of basic fibroblast growth factor and glial fibrillary acidic protein after laser photocoagulation in the Royal College of Surgeons rat.
OBJECTIVE
Argon laser photocoagulation slows photoreceptor degeneration in the Royal College of Surgeons (RCS) rat, as does intravitreal injection of basic fibroblast growth factor (bFGF). We hypothesize that up-regulation of retinal bFGF is a consequence of laser lesioning in RCS rats. Therefore, we examined the localization of bFGF after laser and correlated this with Mailer cell glial fibrillary acidic protein (GFAP) expression, which is known to increase after injury.


METHODS
A total of 34 RCS rats at postnatal day 23 were anaesthetized (ketamine 40 mg/kg) and their retinas were irradiated with a grid pattern of 40 non-overlapping argon green lesions with a power of 120 mW for 0.2 s using a 50 microm spot size. At 0, 6, 12, 24 and 48 h and 7, 14 and 21 days post-lesion, rats were anaesthetized and their eyes were enucleated and cryostat sectioned and the sections were processed using either an antibody to bFGF or GFAP using the standard avidin-biotinylated peroxidase complex method. Five age-matched RCS rats without laser lesions served as controls.


RESULTS
Basic fibroblast growth factor immunoreactivity (IR) was normally located within cells in the ganglion cell layer inner nuclear layer and in retinal pigment epithelium cells and in the extracellular matrix/cell membranes of the outer nuclear layer (ONL). In lasered retinas, there was elevated bFGF-IR in the coagulated outer segments for the first 24 h. Retinal blood vessels/Müller cells/astrocytes were moderately labelled in and near each lesion immediately after lesion and became more intense after 48 h and persisted for at least 21 days. There was an elevation of bFGF-IR in the ONL on the lesion flanks at 14 days. Muller cell GFAP-IR was first detected at 6 h post-lesion and spread for a considerable distance beyond the lesion site. At 7 and 14 days, Müller cells at the lesion site had sprouted, while those on the flanks were still GFAP-IR.


CONCLUSIONS
Following laser lesion there was an increase in bFGF at the lesion core only for the first 24 h. However, elevated levels of bFGF were observed in the ONL at 14 days, which extended into the lesion flanks for a similar distance to that over which increased photoreceptor survival is found. These results provide support for the hypothesis that laser lesions induce bFGF and this may be the mechanism whereby photoreceptors are spared. Müller cell activation is consistent with growth factor stimulation, but was more widespread than the bFGF changes in ONL. However, blood vessel labelling was similarly widespread and so the responses may be linked between Müller cell GFAP reaction and blood vessel bFGF localization after laser lesions.
Chu
Y
Y
Centre for Ophthamology and Visual Science, Lions Eye Institute, University of Western Australia, Nedlands, Australia. ychu@cyllene.uwa.edu.au
Humphrey
M F
MF
Alder
V V
VV
Constable
I J
IJ
D000818:Animals; D005109:Extracellular Matrix / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D017075:Laser Coagulation; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D051381:Rats; D011922:Rats, Mutant Strains; D012160:Retina / Q000378:metabolism* / Q000601:surgery; D012162:Retinal Degeneration / Q000378:metabolism* / Q000601:surgery; D012165:Retinal Ganglion Cells / Q000378:metabolism; D015854:Up-Regulation
D005904:Glial Fibrillary Acidic Protein; D016222:Fibroblast Growth Factor 2