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Background: The pathogenesis of choroidal neovascularization is largely unknown. We investigated vascular endothelial growth factor (VEGF) expression in laser-induced choroidal neovascularization (CNV) in rats. Methods: Intense krypton laser photocoagulation was applied to the posterior poles of the eyes of pigmented rats to induce CNV, which was confirmed by fluorescein angiography and histopathology. The eyeballs were enucleated 1, 3, 7, 14 and 28 days after laser photocoagulation. Cryostat sections were prepared for immunofluorescence staining using anti-VEGF and macrophage marker (ED1) antibodies. The posterior segments of eyeballs pooled from photocoagulated and control rats were submitted for immunoprecipitation and immunoblotting by the anti-VEGF antibody, and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of VEGF mRNA. Results: Very weak immunoreactivity for anti-VEGF antibody was found in the ganglion cell layer, inner nuclear layer, and retinal pigment epithelium (RPE) in the normal retina. In the development of CNV, strong positive staining for anti-VEGF antibody was found in photocoagulated areas in the subretinal space and choroid. Double immunofluorescence staining showed that many cells in lasered lesions were positive both for anti-VEGF and macrophage marker ED1 antibody staining in the early stage of this model. Immunoblots showed a positive band for the VEGF molecule in treated but not control animals. RT-PCR results demonstrated upregulation of VEGF transcripts in the CNV model compared with normal animals. Conclusions: Our findings showed the upregulation of VEGF expression in experimentally induced CNV, where it may be involved in promoting choroidal angiogenesis. Macrophages may be one of the main sources of VEGF in the early stage of the disease.

Insulin receptor substrates (Irs-proteins) integrate signals from the insulin and insulin-like growth factor-1 (IGF1) receptors with other processes to control cellular growth, function, and survival. Here, we show that Irs2 promoted the maturation and survival of photoreceptors in the murine retina immediately after birth. Irs2 was mainly localized to the outer plexiform layer as well as to photoreceptor inner segments. It was also seen in ganglion cells and inner plexiform layer but in smaller amounts. Compared with control littermates, Irs2 knock-out mice lose ∼10% of their photoreceptors 1 week after birth and up to 50% by 2 weeks of age as a result of increased apoptosis. The surviving photoreceptor cells developed short organized segments, which displayed proportionally diminished but otherwise normal electrical function. However, IGF1-stimulated Akt phosphorylation was barely detected, and cleaved/activated caspase-3 was significantly elevated in isolated retinas ofIrs2-/-mice. When diabetes was prevented, which allowed theIrs2-/-mice to survive for 2 years, most photoreceptor cells were lost by 16 months of age. Because apoptosis is the final common pathway in photoreceptor degeneration, pharmacological strategies that increase Irs2 expression or function in photoreceptor cells could be a general treatment for blinding diseases such as retinitis pigmentosa.

Growth factors involved in angiogenesis are critical to both the normal and pathological vascular development in the retina and choroid. In the present experiment, the relationship between the vascular endothelial growth factor (VEGF) expression and the retinochoroidal vasculogenesis in Sprague-Dawley rats was investigated using in situ hybridization and immunohistochemistry. It was found that VEGF was produced mainly by astrocytes and Muller cells in the neural retina, and this was correlated temporally and spatially with the retinal vasculogenesis. In addition, it was observed that, although the VEGF expression in the retinal pigment epithelium (RPE) decreased with increasing age, it persisted from the embryonic stage to adulthood. These findings indicate that the VEGF expression in RPE may play a role in the development of the choroidal vessels as well as in the maintenance of the normal structure and permeability of the choriocapillaris in adults.

• Background: Scatter photocoagulation induces regression of retinal neovascularization, but the mechanism of its therapeutic effect is incompletely understood. To elucidate the mechanism of therapeutic effect of photocoagulation is the main focus of our research. We have already demonstrated basic fibroblast growth factor (bFGF) immunolocalization during retinal wound repair following laser photocoagulation. Transforming growth factor beta (TGF β) reportedly inhibits endothelial cell growth and bFGF-induced cell proliferation in vitro. In the present study, we evaluated the immunohistochemical localization of TGF-β1 and -β2 during wound repair in the rat retina following laser photocoagulation. • Methods: Krypton laser photocoagulation was performed on the eyes of pigmented rats. The eyes were then enucleated on day 1, 3, 7, 14, 28 or 56 following the photocoagulation and enrolled into the analysis of immunohistochemical localization of TGF-β1 and -β2. • Results: Immunoreactivity for TGF-β1 and -β2 was present in the ganglion cell layer and photoreceptor outer segments of the normal adult rat retina. The cytoplasm of RPE cells at the photocoagulated lesion showed intense TGF-β1 and -β2 immunoreactivity on day 3 after laser photocoagulation. Macrophages that migrated into the lesion lacked positive staining for TGF-β1 and -β2. TGF-β immunoreactivity in RPE cells continued to be upregulated for more than 1 month compared with that in normal RPE cells. Controls did not exhibit any positive staining. • Conclusion: An elevated expression of TGF-β immunoreactivity for a longer period of time than bFGF was observed in RPE cells at the photocoagulated lesion in vivo. In the late phase of retinal wound repair, TGF-β may inhibit cell proliferation induced by mitogens, introduce an end stage of cellular events, and induce extracellular matrix induction.

The retinal pigment epithelium (RPE) has a major role in maintaining the homeostasis and visual activity of the retina. Various pharmacological agents are known to affect the pigment epithelial cells primarily1,2 or to affect the neurosensory retina and damage the RPE secondarily3–5. When the physiological functions of the pigment epithelium are altered the overlying retina is also affected, resulting in a decrease of visual function.

Basic fibroblast growth factor (bFGF) is an angiogenic peptide that may be important in the pathogenesis of choroidal neovascularization. We attempted to determine the transcription of the bFGF gene during the development of experimentally induced choroidal neovascularization.Rat bFGF cDNA was inserted in the pBluescript to prepare antisense and sense riboprobes. Multiple krypton laser burns were applied to the posterior poles of the eyes of pigmented rats according to a protocol described for producing subretinal neovascularization in these animals. At intervals of up to 4 weeks after photocoagulation, the eyes were removed and cut into thin sections. The sections were subjected to histopathological analysis, cell proliferation study, or in situ hybridization with digoxigenin (DIG)-labeled single-strand riboprobes synthesized from rat bFGF cDNA.In normal adult rat retinas, bFGF mRNA expression was mainly observed in the ganglion cell layer and the inner nuclear layer. After laser photocoagulation, proliferation of RPE cells, fibroblast-like cells and cells in the choroid in the lesions were observed. Expression of bFGF mRNA was observed in the lesions 3 days to 2 weeks after laser treatment. Signals of bFGF mRNA were detected in the proliferating RPE-like cells, choroidal vascular endothelial cells and fibroblast-like cells, all of which are essential for neovascularization. However, bFGF mRNA expression was no longer detectable in these cells 4 weeks after photocoagulation.Our findings indicate that bFGF is normally transcribed in ganglion cells and the inner nuclear cell layer. During the neovascularization that followed laser photocoagulation, bFGF mRNA expression was detected within the laser lesions. It is thus probable that bFGF acts as a mediator in the neovascularization process.

• Background: The intravitreal injection of ornithine produces selective damage to the retinal pigment epithelium (RPE) and results in a loss of RPE, choriocapillaris and photoreceptor cells. To elucidate the mechanism of secondary retinal atrophy, we investigated the presence of apoptotic cells in a rat model of ornithine-induced retinopathy. • Methods: At 6 and 12 h and 1, 2, 4, 7, 14 and 28 days after an intravitreal injection of L-ornithine hydrochloride in rat eyes, we removed the eyes and subjected them to histopathological examination. We detected apoptotic cells by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end labeling (TUNEL) assay, which stains the 3′-OH ends of fragmented DNA. We used electron microscopy to detect the apoptotic cells morphologically. • Results: RPE cells were selectively damaged immediately after ornithine administration. TUNEL-positive photoreceptor cells appeared exclusively in the photoreceptor cell layer 12 h after ornithine administration. The number of TUNEL-positive cells increased throughout the 2 days following the injection, then decreased markedly. TUNEL-positive cells remained until 28 days, when the photoreceptor cells had disappeared. The ganglion cell layer, inner nuclear layer and damaged RPE cells were negative for TUNEL staining during all stages. The electron microscopic study also revealed the pyknotic nuclei of apoptotic photoreceptor cells. • Conclusion: An intravitreal injection of ornithine caused primary damage to the RPE, and subsequently some of the photoreceptor cells revealed apoptosis by TUNEL assay. These findings suggest the dysfunction of the RPE causes photoreceptor cell death according to the intrinsic program of an apoptotic mechanism.