Comparative study of experimental choroidal neovascularization by optical coherence tomography and histopathology.

PURPOSE: To study the development, progression, and regression of experimental choroidal neovascularization (CNV) by correlating the cross-sectional images from sequential optical coherence tomography (OCT) with histopathologic sections of the same retinal regions. METHODS: Laser photocoagulation was performed in the posterior pole of the eye of 4 rhesus monkeys to induce CNV. Funduscopy, fluorescein angiography (FAG), and OCT were performed on day 1 and weekly for 13 weeks. Histological serial sections of CNV tissue were compared to corresponding OCT images. RESULTS: In the developmental stage of CNV, the CNV was observed by OCT as a nodular high reflex area continuing from the highly reflective retinal pigment epithelium (RPE). Histopathological studies showed that the CNV was composed of tightly packed proliferated RPE and immature vascular endothelial cells. In the active stage, OCT revealed a thick multi-layered high reflex area under the sensory retina. This high reflex area corresponded with the CNV membrane that consisted of newly formed blood vessels with wide vascular lumens and proliferated spindle-shaped RPE cells. In the regressive stage, OCT revealed a dome-like, white-colored highly reflective layer continuing from the RPE layer with moderate reflection beneath the layer. Histopathologically, the neovascular tissue was enveloped by mono-layered, cuboidal-shaped RPE cells with melanin granules. CONCLUSION: Optical coherence tomography images clearly demonstrated the positional relationship between the CNV and the RPE. Optical coherence tomography imaging provides information on the CNV which complements conventional examinations by funduscopy and FAG.

Ciliochoroidal Detachment Following Scleral Buckling Surgery for Rhegmatogenous Retinal Detachment.

Purpose and Methods: We observed the peripheral choroid, ciliary body, and depth of the anterior chamber by ultrasound biomicroscopy (UBM) in 31 eyes with rhegmatogenous retinal detachment before and after scleral buckling surgery. Scleral encircling was performed in 11 eyes and segmental scleral buckling in 20 eyes.Results: With UBM, ciliochoroidal detachment was detected in all eyes (100%) following scleral encircling and in 8 eyes (40.0%) following segmental scleral buckling. After scleral encircling procedure, the eyes with preoperatively bullous and wide retinal detachment showed a severe ciliochoroidal detachment and edema of the ciliary body. Shallowing of the anterior camber occurred in all 11 eyes (100%) after scleral encircling and in 12 of 20 eyes (60.0%) after segmental scleral buckling. Marked shallowing with closure of the angle and elevated intraocular pressure occurred in 2 eyes.Conclusion: The results showed that careful postoperative examinations for the anterior segments, chamber angle, and intraocular pressure are necessary with slit-lamp examination and applanation tonometry after scleral buckling surgery.

Enhancement of dedifferentiation and myoid differentiation of retinal pigment epithelial cells by platelet derived growth factor.

AIMS: To clarify factor(s) involved in morphological dedifferentiation of retinal pigment epithelial (RPE) cells in vitro from mitotically quiescent hexagonal cells to flattened cells that lack epithelial characteristics and concurrent myoid differentiation. METHODS: RPE cells which retained their differentiated hexagonal morphology were isolated from bovine eyes by mechanical pipetting. Dedifferentiation and myoid differentiation of RPE cells were examined by microscopic observation and immunohistochemical analysis using antibodies against cytokeratin, an epithelial marker, and alpha smooth muscle actin, a marker of myoid differentiation. The contractile ability of RPE cells was evaluated by collagen gel contraction assay. RESULTS: Platelet derived growth factor (PDGF) enhanced morphological changes in the RPE from hexagonal-shaped cells to flattened cells. Coincident with this morphological alteration, the expression of cytokeratin in RPE cells decreased and expression of alpha smooth muscle actin began and was increased in a time dependent manner. These alterations were completely blocked by collagen synthesis inhibitors. Interleukin 1beta, transforming growth factor beta1, insulin-like growth factor I, and basic fibroblast growth factor had little or no effect on the dedifferentiation. PDGF also potentiated the RPE induced collagen gel contraction. CONCLUSIONS: These results demonstrate that PDGF enhanced the dedifferentiation of RPE cells, the initial step of proliferative vitreoretinopathy (PVR), as well as myoid differentiation and collagen gel contraction. PDGF may have a versatile role in the pathogenesis of PVR involving collagen synthesis.

[Ciliochoroidal detachment following scleral buckling surgery for rhegmatogenous retinal detachment].

PURPOSE AND METHODS: We observed the peripheral choroid; ciliary body, and depth of the anterior chamber by ultrasound biomicroscopy (UBM) in 31 eyes with rhegmatogenous retinal detachment before and after scleral buckling surgery. Scleral encircling was performed in 11 eyes and segmental scleral buckling in 20 eyes. RESULTS: With UBM, ciliochoroidal detachment was detected in all eyes (100%) following scleral encircling and in 8 eyes (40.0%) following segmental scleral buckling. After scleral encircling procedure, the eyes with preoperatively bullous and wide retinal detachment showed a severe ciliochoroidal detachment and edema of the ciliary body. Shallowing of the anterior camber occurred in all 11 eyes (100%) after scleral encircling and in 12 of 20 eyes (60.0%) after segmental scleral buckling. Marked shallowing with closure of the angle and elevated intraocular pressure occurred in 2 eyes. CONCLUSION: The results showed that careful postoperative examinations for the anterior segments, chamber angle, and intraocular pressure are necessary with slit-lamp examination and applanation tonometry after scleral buckling surgery.

Optical coherence tomographic findings of idiopathic polypoidal choroidal vasculopathy.

BACKGROUND AND OBJECTIVE: To identify the histological level of abnormal vessels associated with idiopathic polypoidal choroidal vasculopathy (IPCV), we examined IPCV with Optical Coherence Tomography (OCT). PATIENTS AND METHODS: Fourteen patients diagnosed with IPCV were examined with Indocyanine green (ICG) angiography and OCT. RESULTS: ICG angiography demonstrated branching vascular networks with polypoidal dilatations at the terminals beneath the retinal pigment epithelium (RPE). OCT showed dome-like elevation of the RPE, and moderate reflex or nodular appearance were seen beneath the RPE. CONCLUSION: The abnormal vessel associated with IPCV is supposed to be choroidal neovascularization with polypoidal dilatations at the terminals between Bruch's membrane and RPE. We consider that this disease is a peculiar form of age-related macular degeneration.

Occult retinal pigment epithelial detachment in hyperviscosity syndrome.

We document and evaluate a serous retinal detachment in a patient with hyperviscocity syndrome. Optical coherence tomographic images of the serous retinal detachment in a patient with hyperviscocity syndrome were correlated with slit-lamp biomicroscopic findings, fundus photographs, fluorescein angiograms, and indocyanine green angiograms. Fluorescein angiography demonstrated venous and capillary bed abnormalities but no leakage or pooling of fluorescein corresponding to the retinal pigment epithelial detachment (PED) beneath the serous retinal detachment. Indocyanine green angiogram disclosed a delay of intrachoroidal circulation. Optical coherence tomography (OCT) revealed a large retinal pigment epithelial detachment beneath the serous retinal detachment. The occult retinal PED beneath the neurosensory retinal detachment was detected only by OCT in a patient with hyperviscosity syndrome. We suggest that gamma globulin, which is the hyperviscosity material, accumulated in the subretinal pigment epithelial space and blocked the leakage or pooling of fluorescein corresponding to the retinal pigment epithelial detachment.

Histopathological findings of X-linked retinoschisis with neovascular glaucoma.

BACKGROUND: X-linked retinoschisis (XLRS) is rarely complicated by neovascular glaucoma. Only a few reports of XLRS histopathological findings with neovascular glaucoma have been published. METHODS: A 41-year-old man with XLRS complicated by neovascular glaucoma in his left eye was examined with electroretinography, B-scan, ultrasound biomicroscopy and computed tomography. He was examined by ophthalmoscopy and fluorescein angiography in the other eye. An enucleation was performed in his left eye due to uncontrollable high intraocular pressure and persistent ocular pain. We examined the enucleated eye histopathologically. RESULTS: Examination of the enucleated eye showed nuclear sclerosis of the lens, pigmented retrolental membrane and retinoschisis which separated the inner layer of the retina and made a large space in the vitreous cavity without any apparent detachment of the outer layers of the retina. Sclerotic vessels were present histopathologically in both the inner and outer layers of the retina. There was a peripheral anterior synechia, ectropion uveae and a fibrovascular membrane, which contained many lumina of neovascularization, indicating marked rubeosis iridis. Small cystic spaces were observed in both the schitic retina in the peripheral region and the foveal schisis at the outer layer of the retina. The photoreceptor cells had become markedly atrophied and multiple regions of calcification were observed. The optic nerve showed severe atrophy with gliosis, but the central retinal artery and vein were still open within the nerve. CONCLUSIONS: These histopathological findings suggest that rubeosis iridis may have developed secondarily to retinal ischemia due to occlusion of the retinal blood vessels.

Idiopathic polypoidal choroidal vasculopathy in Japanese patients.

OBJECTIVE: To describe the vascular nature and clinical features of idiopathic polypoidal choroidal vasculopathy in Japanese patients. METHODS: Patients thought to have idiopathic polypoidal choroidal vasculopathy were examined with binocular ophthalmoscopy, slitlamp biomicroscopy with a contact lens, fluorescein angiography, and indocyanine green angiography. RESULTS: From January 1993 to December 1997, 35 eyes in 32 patients were diagnosed as having idiopathic polypoidal choroidal vasculopathy. Men were predominantly affected (22 patients [69%]). Most patients were unilaterally involved (29 patients [91%]) and elderly, with a mean age of 65.7 years (range, 44-82 years). Ocular manifestations were relatively mild, with serous or hemorrhagic detachments of the retinal pigment epithelium and neurosensory retina in the posterior pole. Most patients had a favorable course, although some experienced recurrence, and a few eyes developed disciform scarring. In all patients, indocyanine green angiograms demonstrated branching vascular networks with polypoidal dilations at terminals of the network beneath the retinal pigment epithelium. These lesions were mostly in the macula (33 eyes [94%]), with a few in the peripapillary area. CONCLUSIONS: Idiopathic polypoidal choroidal vasculopathy in Japanese patients differs from that in American patients. It seems that this disorder occurs in elderly Japanese patients and should be treated as a distinct clinical entity. It is probably a peculiar form of choroidal neovascularization beneath the retinal pigment epithelium. We propose the term "polypoidal choroidal neovascularization" for this disorder.

[Vascular endothelial growth factor promotes experimental choroidal neovascularization in monkey eyes].

PURPOSE: To evaluate the effect of vascular endothelial growth factor (VEGF) on experimental choroidal neovascularization (CNV) in monkey eyes through clinical, morphometric, and histological observations. METHOD: CNV was induced in both eyes of 6 rhesus monkeys by intense photocoagulation by red krypton laser. Immediately after photocoagulation, 2.5 micrograms of exogenous human VEGF was injected into the vitreous of the left eye in each animal. The right eyes served as controls. The eyes were enucleated 3 days to 12 weeks after photocoagulation and were examined by light and electron microscopy. RESULTS: VEGF-treated eyes developed remarkable serous retinal detachment around the sites of photocoagulation with manifest CNV one week after photocoagulation. Although there was no difference in the incidence of CNV between the treated and control eyes, the treated eyes showed more intense leakage of fluorescein from the CNVs for up to 4 weeks after treatment. Morphometrically, the CNVs were significantly larger and continued to grow longer in the treated than in the control eyes after one week of photocoagulation. Histologically, newly formed vessels with a distinct lumen were present in the treated eyes after 3 days of photocoagulation. CONCLUSION: Intravitreal injection of human VEGF promotes experimental choroidal neovascularization in monkey eyes.

Phosphorothioate oligonucleotides induction into experimental choroidal neovascularization by HVJ-liposome system.

PURPOSE: The purpose of this study was to determine whether the inactivated hemagglutinating virus of Japan (HVJ)-liposome method can induce phosphorothioate oligonucleotides effectively into an experimentally-induced choroidal neovascularization of rats. We also examined whether antisense phosphorothioate oligonucleotides against VEGF could be induced into choroidal neovascularization as a therapeutic agent by the HVJ-liposome method. METHODS: The experiments were conducted on a rat model of choroidal neovascularization. FITC-labeled phosphorothioate oligonucleotides were coencapsulated in liposomes. The liposomes were coated with the envelope of inactivated HVJ and injected into the vitreous cavity following photocoagulation of pigmented rat eyes. The eyes were removed following injection, fixed, frozen and cut into thin sections. Induction of oligonucleotides was observed under a laser confocal scanning microscope for fluorescence and the development of choroidal neovascularization was evaluated histopathologically. RESULTS: Phosphorothioate oligonucleotides were effectively induced into ganglion cells and into the cells of the choroidal neovascularization induced by laser photocoagulation. Highly effective induction of oligos was observed 3 to 14 days after intravitreal injection of HVJ-liposomes after which the level decreased. Antisense oligonucleotides against VEGF were induced specifically into cells in the choroidal neovascularization, however neovascularization was still observed. CONCLUSIONS: Phosphorothioate oligonucleotides can be effectively induced into ganglion cells, and specifically into cells in choroidal neovascularization. Although antisense oligonucleotides against VEGF failed to prevent choroidal neovascularization, the HVJ-liposome method provided a highly effective means of inducing antisense oligos for in vivo antisense therapy.

Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization.

PURPOSE: Vascular endothelial growth factor (VEGF) is an angiogenic peptide that has been suggested to be important in the pathogenesis of choroidal neovascularization. We investigated the transcription of VEGF and its receptor KDR/flk-1 genes during the development of experimentally induced choroidal neovascularization. METHODS: Rat VEGF or KDR cDNA was inserted in PGEM or pBluescript to prepare antisense or sense riboprobes. Multiple krypton laser burns were applied to the posterior pole of pigmented rat eyes according to a previously described protocol which produces choroidal neovascularization. At intervals of up to 4 weeks after photocoagulation, the eyes were removed and cut into thin sections. The sections were subjected to in situ hybridization with digoxigenin (DIG)-labeled single-strand rat VEGF and KDR cDNA riboprobes. RESULTS: In normal adult rat retinas, VEGF and KDR mRNA expression was mainly observed in the ganglion cell and the inner nuclear layers. During the development of neovascularization, VEGF and KDR mRNAs were detected in retinal pigment epithelial-like cells, fibroblast-like cells and endothelial cells in neovascular lesions. The level of expression was strongest at 1 week after photocoagulation in lasered lesions, and decreased by 4 weeks after photocoagulation. CONCLUSIONS: Our findings demonstrate that expression of VEGF and its receptor KDR may play a role in the formation of experimentally induced choroidal neovascularization. In this study, VEGF and its receptor were co-localized, suggesting that an autocrine and/or paracrine mechanism may be operative.

Surgical outcome on combined procedures of lens extraction, intraocular lens implantation, and vitrectomy during removal of the epiretinal membrane.

BACKGROUND AND OBJECTIVE: The authors evaluated the outcome of combined procedures of lens extraction, intraocular lens implantation, and vitrectomy during procedures for the removal of the epiretinal membrane, and attempt to clarify which factors affected the outcome of surgery. PATIENTS AND METHODS: Surgery for the removal of the epiretinal membrane was performed and a 2-year follow-up program was maintained for 32 eyes at the authors' clinic during the past 5 years. The combined procedure was undertaken in 15 eyes, whereas a simple vitrectomy was performed in 17 eyes. Results were analyzed by postoperative ophthalmoscopic and slit-lamp examinations for visual acuity and complications. RESULTS: Six months after surgery, visual acuity improved similarly in both groups. But, 2 years after surgery, 11 eyes (65%) that underwent a simple vitrectomy showed decreased visual acuity due to the advancement of cataracts. The postoperative visual acuity at 6 months correlated to preoperative visual acuity, and the advancement of cataracts tended to correlate inversely to the age of the patients. Major complications did not occur in any of the cases. CONCLUSION: Earlier surgical management is better for treatment of the epiretinal membrane, and a combined procedure has advantages in cost reduction and is less troublesome, especially for elderly patients.

Expression of vascular endothelial growth factor and its receptor, KDR, following retinal ischemia-reperfusion injury in the rat.

PURPOSE: There is considerable evidence that vascular endothelial growth factor (VEGF) mediates ocular neovascularization in retinal vascular diseases. We investigated the time-dependent changes in the expression of VEGF and its receptor KDR/ Flk in a transient retinal ischemia-reperfusion injury model. METHODS: Transient retinal ischemia was induced by increasing the intraocular pressure in albino rats eyes for 45 min. In situ hybridization was used to identify the retinal cells synthesizing VEGF mRNA and KDR mRNA at various times following reperfusion. Immunohistochemical analysis was also carried out to detect VEGF immunoreactivity. RESULTS: In the control, non-ischemic retinas, signals for VEGF mRNA and KDR mRNA were observed in the cells of the ganglion cell layer. Immunoreactivity to VEGF was also found in the nerve fiber layer, the ganglion cell layer, and the retinal pigment epithelial (RPE) cell layer. Immediately and 6 h after reperfusion, VEGF and KDR mRNA expression was markedly decreased, but recovered by 24 h to the levels observed in normal retinas. Immunoreactivity for VEGF was also decreased immediately and 6 h after reperfusion, and was detected in the endothelial cells of the retinal vessels after 24 h. Immunoreactivity to VEGF recovered by 48 h after reperfusion. CONCLUSIONS: The hybridization pattern of VEGF and KDR mRNA in the ganglion cell layer strongly suggests that the ganglion cells are the major source of this growth factor. The decrease of VEGF mRNA, KDR/Flk mRNA and VEGF protein levels after ischemia and recovery after reperfusion suggest that transient hypoxia might mediate short-term down-regulation of VEGF and KDR mRNA.

Granulocyte-macrophage colony-stimulating factor expressed in T cells mediates immunity against herpes simplex virus type 1 encephalitis.

A model of herpes simplex virus type 1 (HSV-1) infection was developed in rats to study systemic immune responses elicited by intravitreous inoculation of the virus. HSV-1 inoculation led to distinct granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing memory T cells, which did not develop in rats inoculated with either HSV-1 intraperitoneally or inactivated HSV-1 intravitreously. On subsequent intraperitoneal viral boosting, systemic GM-CSF production was elicited as a secondary immune response that caused neutroeosinophilia. To examine the role of GM-CSF in anti-herpetic immunity, cytokine-producing and -nonproducing rats were intravitreously challenged with HSV-1, which causes lethal encephalitis. Only intravitreously primed rats were protected upon production of GM-CSF. Furthermore, pretreatment with recombinant GM-CSF protected unimmunized rats against the encephalitis. It is thus strongly suggested that the production of GM-CSF leads to anti-HSV-1 immunity against the transneuronal spread of challenged HSV-1 within the visual system.

Prevention of ornithine cytotoxicity by proline in human retinal pigment epithelial cells.

PURPOSE: To investigate the relationship between ornithine-delta-aminotransferase (OAT) deficiency and ornithine accumulation and the specific degeneration of retinal pigment epithelial (RPE) cells in gyrate atrophy. METHODS: Human RPE cells, human hepatoma cells, and human fibroblast cells were treated with 5-fluoromethylornithine (5-FMOrn), a specific irreversible inhibitor of OAT. Ornithine cytotoxicity was determined by using a [3H]thymidine incorporation assay and immunohistochemical staining for cytokeratin. The effects of various metabolites of ornithine and arginine, such as creatine, creatine phosphate, I-delta 1-pyrroline-5-carboxylic acid (L-P5C), and proline, which may be deficient in gyrate atrophy on RPE cell damage by ornithine, were determined by the same procedures. RESULTS: When the human RPE cells, HepG2 hepatoma cells, and WI-38 fibroblast cells were treated with 0.5 mM 5-FMOrn for 30 minutes, which inactivated OAT, ornithine exhibited severe time- and dose-dependent inhibition of DNA synthesis in the human RPE cells but not in the HepG2 hepatoma cells or WI-38 fibroblast cells. The inhibition of DNA synthesis was accompanied by drastic changes in morphologic appearance, disorganization of the cytoskeleton, and cell death. Ornithine or 5-FMOrn alone did not exhibit such cytotoxicity to the RPE cells. Proline prevented the cytotoxicity of ornithine. CONCLUSIONS: These findings suggest that an elevated level of ornithine combined with an increased sensitivity to ornithine as a result of OAT deficiency may be crucial to the specific RPE degeneration in gyrate atrophy. They suggest also that abnormalities of proline metabolism may be involved in the progress of gyrate atrophy.

Time-course expression of vascular endothelial growth factor as related to the development of the retinochoroidal vasculature in rats.

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.

[Results of vitreous surgery for proliferative vitreoretinopathy].

During the past four years, we performed vitreous surgery on 73 eyes with rhegmatogenous retinal detachments complicated with severe proliferative vitreoretinopathy (PVR). We analyzed the surgical outcome of PVR according to the revised classification of PVR Grade C (1991). After a mean follow-up period of 19 months, the retinas were successfully reattached in 62 of 73 eyes (85%). The reattachment rate in the eyes with only posterior proliferation was high (96%), regardless of the extent of posterior proliferation. However, the reattachment rate in the eyes associated with anterior proliferation was markedly low (57%), depending on the extent of anterior proliferation. Among 62 eyes with successfully reattached retinas, 39 eyes (63%) had an improved postoperative visual acuity. These results demonstrated that the eyes with anterior PVR have a worse reattachment rate than the eyes with only posterior PVR. Using the revised classification of PVR, we were able to analyze the surgical outcome of PVR which could not be classified by the old classification.

Involvement of prostaglandin E2 in rabbit corneal injury by anterior segment ischaemia.

The involvement of prostaglandins (PGs) in the development of anterior segment ischaemia after occlusion of the bilateral long posterior ciliary arteries was investigated in rabbit eyes. In this experimental ischaemia, the tissue weight and protein content in the peripheral cornea and the protein content in the aqueous humour increased on the first postoperative day. Topically applied cyclooxygenase inhibitor diclofenac (0.1%) reduced corneal inflammation and further suppressed the elevation in the tissue weight and protein content in the peripheral cornea on day 1 after ischaemia, but did not affect the changes in the aqueous humour. Subconjunctivally administered PGE1 and PGE2 induced corneal oedema and increased corneal protein content in diclofenac-treated and ischaemia-induced eyes, but PGD2, PGF2alpha, and the stable PGI2 analogue cicaprost did not evoke any change. In fact, PGE2 content was markedly increased in the aqueous humour on day 1 after ischaemia, and diclofenac suppressed the increase. In addition, CPT-cAMP increased the corneal tissue weight and protein content in organ culture. These observations suggest that PGE2 may play an important role in developing corneal oedema at the initial stage of ischaemic damage, possibly through the cAMP-mediated pathway.

Vascular endothelial growth factor expression in choroidal neovascularization in rats.

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.

Expression of transforming growth factor-beta mRNA in experimental choroidal neovascularization.

PURPOSE: Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that modulates biological events as diverse as wound healing and angiogenesis and which may be important in the pathogenesis of choroidal neovascularization. We investigated the mRNA expression of TGF-beta isoforms in a model of experimental choroidal neovascularization induced by krypton-laser photocoagulation. METHODS: Rat TGF-beta 1, mouse TGF-beta 2 or TGF-beta 3 cDNAs was inserted into the pBluescript vector to prepare antisense and sense riboprobes. Intense laser burns were applied to the posterior poles of the eyes of pigmented rats according to a protocol described for producing choroidal neovascularization in these animals. At intervals up to 4 weeks after photocoagulation, the eyes were obtained and cut into thin sections. The sections were subjected to in situ hybridization with digoxigenin (DIG)-labeled single-strand riboprobes synthesized from each TGF-beta cDNA. RESULTS: In normal adult rat retinas and choroids, TGF-beta 1 mRNA was found only in cells of the ganglion cell layer, TGF-beta 2 mRNA was found in cells of the ganglion cell layer and choriocapillaris endothelium, whereas TGF-beta 3 mRNA was not detected at all. During the process of neovascularization, TGF-beta 1 and TGF-beta 2 mRNAs (the latter being expressed more prominently) were detected in retinal pigment epithelial cells, fibroblast-like cells and the endothelium of the neovascular region. TGF-beta 2 was the predominant isoform of TGF-beta, and its expression was especially strong in the endothelium of the choroidal neovascularization at 2 weeks. However, TGF-beta mRNAs was decreased in cells 4 weeks after photocoagulation. CONCLUSIONS: Our findings suggest that TGF-beta may act in the retina as a neurotrophic agent, since TGF-beta 1 is normally transcribed in ganglion cells and TGF-beta 2 is also transcribed in ganglion cells and choriocapillaris endothelium. TGF-beta 1 and TGF-beta 2 mRNA expression were increased in photocoagulated lesions from 3 days to 2 weeks after laser treatment. Therefore, it is likely that TGF-beta acts as a mediator of the neovascularization process.