Critical role of photoreceptor apoptosis in functional damage after retinal detachment.

PURPOSE: Although apoptosis is assumed to play a pivotal role in retinal function loss, its mechanism and real influence on retinal function are still unclear. To investigate the relation between retinal function and apoptosis, we studied photoreceptor apoptosis in experimental retinal detachment (RD). METHODS: We induced RD by subretinal injection of sodium hyaluronate in Brown Norway rats. Apoptotic photoreceptors were detected by TdT-dUTP Terminal Nick-End Labeling (TUNEL). To evaluate the function of the detached retina, electroretinograms (ERGs) were taken on day 1, 3 with corneal electrodes and full-field stimulation. RESULTS: Apoptotic DNA fragmentation appeared 12 hours after RD, was most prominent on day 3, and decreased thereafter. The ERGs showed that the amplitudes of dark-adapted a-waves and light adapted 2 Hz b-waves decreased immediately after RD and continued to decrease over time. The administration of Fas/Fc chimera recombinant protein or a caspase inhibitor, Z-VAD.fmk, failed to prevent either photoreceptor apoptosis or retinal functional damage. In contrast, brain derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) significantly impeded both apoptosis and dysfunction. The ERGs recognized the functional changes sensitively, and these ERG changes correlated well to the amount of photoreceptor apoptosis. Immunohistochemical study showed that apoptosis-inducing factor (AIF), a novel caspase-independent apoptotic factor, was relocalized from mitochondria to the nucleus in this process. CONCLUSIONS: The present results showed that apoptosis was a key phenomenon in the retinal dysfunction in RD and that this process was transmitted mainly by mitochondria-dependent pathways rather than Fas/Fas-L or downstream caspase dependent pathways.

A case of Vogt-Koyanagi-Harada disease with good visual acuity in spite of subfoveal fold.

BACKGROUND: Vogt-Koyanagi-Harada (VKH) disease patients with the complication of subretinal pigmented proliferative tissue tend to have a poor visual prognosis. CASE: We herein report a case of VKH with good visual acuity despite a prominent subretinal fold. OBSERVATIONS: A 24-year-old woman, who experienced several recurrent episodes of VKH disease, had bilateral serous retinal detachment with poor vision (RE 20/40 and LE 20/25). After the administration of high doses of systemic corticosteroids and D-mannitol, the subretinal fluid disappeared and the sensory retinas gradually became reattached. During the course of therapy, prominent pigmented subretinal strands were formed in both eyes. Optical coherence tomography disclosed that the strands existed at the retinal pigment epithelium level. Amazingly, we observed a change in the location of the fold in the posterior retina during the course of the disease. The patient finally showed the "sunset glow" fundi and a subretinal fold that was located almost directly beneath both fovea. Fortunately, this patient was able to recover and finally achieve a good visual acuity (RE 20/17 and LE 20/17). CONCLUSION: We reported a VKH disease patient with a good visual acuity despite a remarkable subfoveal fold, which changed its location during the course of the disease.

Optic disk vasculitis associated with chronic active Epstein-Barr virus infection.

PURPOSE: To report two cases of optic disk vasculitis associated with chronic active Epstein-Barr virus infection (CAEBV). METHOD: We examined the eyes of two patients with CAEBV. RESULTS: In the first case, a 6-year-old boy, visual acuity was 20/20 in both eyes. Papillary and peripapillary exudates were observed in the right eye. Fluorescein angiography showed hyperfluorescence of the optic disk and a leakage from the peripapillary retinal vessels in the right eye. Two months later, the exudates increased and preretinal hemorrhages appeared in the right eye. Visual acuity decreased to 20/60. He was treated with systemic administration of corticosteroid, globulin and acyclovir. Visual acuity returned to 20/20, but peripapillary exudates remained in the right eye. In the second case, a 16-year-old girl, visual acuity was 20/20 in both eyes. The right eye showed optic disk swelling and dilated retinal veins. Fluorescein angiography showed hyperfluorescence of the optic disk but no leakage from the retinal vessels. Visual field examination revealed an enlarged blind spot in the right eye in both cases. These ocular manifestations are compatible with those of optic disk vasculitis, which shows swelling of the optic disk and dye leakage on and around the optic disk in fluorescein angiography, with an almost normal visual acuity and an enlargement of the blind spot. CONCLUSION: Persistent Epstein-Barr virus infection may cause optic disk vasculitis.

Phacoanaphylaxis in Behçet's disease: a clinicopathologic and immunohistochemical study.

PURPOSE: To describe the occurrence of phacoanaphylaxis in enucleated eyes of patients with Behçet's disease. DESIGN: Retrospective, observational, case series and human tissue study. PARTICIPANTS: Twenty-six patients with Behçet's disease who underwent enucleation. METHODS: Histopathologic analysis was performed on 28 enucleated eyes of 26 patients with Behçet's disease. The eyes were divided into two groups, based on the absence or presence of tractional retinal detachment associated with cyclitic membrane formation. Selected eyes with tractional retinal detachment were stained for immunohistochemical examination. MAIN OUTCOME MEASURES: Histopathologic examination of enucleated eyes, including routine histopathologic and immunohistochemical studies. RESULTS: None of the five eyes without retinal detachment showed phacoanaphylaxis. Nine of the 23 eyes with detachment exhibited phacoanaphylaxis, 10 showed no inflammation of the lens, and four were aphakic. There was marked inflammatory cell infiltration in the cyclitic membrane of all nine eyes with phacoanaphylaxis. Immunohistochemical examination demonstrated positive staining for the macrophage markers in the epithelioid and giant cells. The average interval between onset of the ocular manifestations of Behçet's disease and enucleation was 63 months for eyes with phacoanaphylaxis and 35 months for eyes without phacoanaphylaxis (P<0.005). CONCLUSIONS: In Behçet's disease, eyes with long-standing intraocular inflammation complicated by cyclitic membrane formation may develop phacoanaphylaxis. Such patients may benefit from surgical removal of the cyclitic membrane along with the lens in eyes with significant visual function.

Targeted gene transfer to corneal stroma in vivo by electric pulses.

This study was conducted to develop a method of targeted gene transfer by electric pulses to a selected area of corneal stroma in vivo. Plasmid DNA with a green fluorescent protein (GFP) gene under a cytomegalovirus promoter was injected through the corneal pocket into the corneal stroma of the adult Brown Norway rat, and various intensities of electric pulses ranging from 10 to 30 V were delivered to the corneal epithelial side with an electric probe. Direct stereomicroscopy of the fluorescent using real-time imaging was used to determine in vivo gene expression on days 1, 2, 4, 6, 8, 10, 15, and 20 after gene transfer. Transgene expression was detected in the corneal stroma as early as day 1 and until day 15. The most intense expression was noted on days 4 and 6. Gene transfer was most effective using eight electric pulses of 20 V for 50 msec. Histologic study disclosed GFP expression in keratocytes within the targeted area. There was no apparent cell damage in the gene transferred cells. No apparent inflammation was found in the anterior chamber or trabecular cells when electric pulses less than 30 V were used. In summary, the present technique transferred the gene of interest to a highly selected area of corneal stroma with no apparent damage. This method will likely be useful not only for developing gene therapy for corneal diseases but also for corneal research in general.

Re-worsening factor after successful vitrectomy for diabetic retinopathy: optic disc fibrovascular proliferation and macular disease.

PURPOSE: To investigate the factors that influence the visual-changing pattern in proliferative diabetic retinopathy even after successful vitrectomy. METHODS: One hundred and forty-seven consecutive eyes were retrospectively reviewed for 6-48 (average 20) months, and were divided into the following 4 groups based on their changing pattern of vision: group A, the visual acuity improved postoperatively and maintained the maximal corrected vision throughout the observation period (n = 49); group B, the visual acuity improved postoperatively but deteriorated thereafter (n = 68); group C, the visual acuity remained the same as before operation (n = 17), and group D, the visual acuity deteriorated immediately after vitrectomy (n = 13). Various issues including systemic conditions, blood tests, preoperative ocular findings, the operative procedures and postoperative complications were reviewed based on the patient records. These issues were analyzed by Spearman's rank correlation, chi(2) test, an analysis of variance (ANOVA) and the Kruskal-Wallis test. Finally, the discriminate factors between groups A and B were examined by a stepwise logistic regression analysis. RESULTS: The following tendencies were observed in all 4 groups: younger patients tended to show a better visual-changing pattern (p = 0.02); patients with younger age at diabetes onset had a better visual-changing pattern after vitrectomy (p = 0.001), and a lower hemoglobin (Hb) A1c level is associated with a better visual changing pattern (p = 0.017). Preoperative rubeosis and macular detachment were frequently found in groups C and D, as well as postoperative rubeosis, vitreous bleeding and retinal detachment. Finally, a stepwise logistic regression analysis showed both fibrovascular proliferation (p = 0.016) from the optic disc and postoperative macular disease (p = 0.0009) to be significant factors for differentiating group A from group B. CONCLUSIONS: In addition to the factors which have already been indicated to affect the visual outcome of a vitrectomy, preoperative findings such as optic disc fibrovascular proliferation and postoperative macular disease were found to affect the visual-changing pattern after a successful vitrectomy. The optimal timing of surgery is very important not only in order to obtain good visual acuity but also to maintain good vision even after a successful vitrectomy.

Neovascularization in the anterior segment of the rabbit eye by experimental anterior ischemia.

PURPOSE: To investigate the effects of anterior ischemia accompanied by neither retinal nor choroidal ischemia on the anterior segment of the eye. METHODS: Both long posterior ciliary arteries in the right eye of 14 rabbits were directly cauterized with an electric coagulator. The eyes were enucleated 1, 2, 4, 7, 9 or 14 days after cauterization, then fixed with 4% paraformaldehyde. Semi-thin sections were studied by light microscopy. Several sections were stained with Griffonia simplicifolia lectin, which bound specifically to mammalian vascular endothelium. Other specimens were examined immunohistochemically for vascular endothelial growth factor (VEGF) protein. The tissue specimens of the first postoperative day were studied for expression of VEGF mRNA by in situ hybridization. RESULTS: Atrophy of the iris and ciliary body was seen after the second postoperative day. Corneal neovascularization appeared after 7 days. Neovascularization on the anterior surface of the iris and in the trabecular meshwork was detected after the ninth postoperative day. The proliferative tissues with newly formed vessels obstructed the iridocorneal angle 14 days after the treatment. There was no histological change in either the retina or choroid. Immunohistochemically, VEGF protein was detected in the epithelial and vascular cells of the iris on the first and fourth postoperative day. Expression of VEGF mRNA was detected in the epithelial cells of the ciliary body on the day following the treatment. CONCLUSIONS: Anterior segment ischemia, when unaccompanied by retinal ischemia, causes neovascularization in the cornea, iris and trabecular tissue.

Expression of matrix metalloproteinases and their inhibitors in experimental retinal ischemia-reperfusion injury in rats.

Matrix metalloproteinases (MMPs) are endopeptidases that degrade the extracellular matrix (ECM) and are involved in the pathogenesis of retinal degeneration along with tissue inhibitors of metalloproteinases (TIMPs). The present study examined the expression and activation of two specific members of MMPs (MMP-2 and MMP-9) and their related inhibitors (TIMP-1 and TIMP-2) in an experimental retinal ischemia-reperfusion injury. Retinal ischemia-reperfusion injury (RIRI) was induced in adult rats with a ligation method. After one hour of ischemia and a varied reperfusion time (0, 3, 6, 12, 24, 48 and 76 hr), the rat eyes were enucleated. Retinal extracts underwent zymographic analysis to measure the activity of MMP-2/9. The activity of TIMP-1 and TIMP-2 was measured by reverse zymography. The protein level was examined by Western blot. Immunohistochemistry analysis was undertaken to assess the anatomical distribution of MMP-9 in the retina after RIRI. The gelatinolytic activity of ProMMP-2 (72 kDa) was increased markedly at 6 hr after RIRI. ProMMP-9 (92 kDa) was not detected in the control specimens, while it appeared at 3 hr, increased markedly at 6 hr, and reached maximal levels at 24 hr after RIRI. The gelatinolytic activity found ian retinal extracts was shown to be inhibited by 10 m M EDTA and activated in vitro by a known metalloproteinase activator (4-aminophenylmercuric acetate (APMA)), indicating that these enzymes were of the metalloproteinase class. By western blot, MMP-2/9 levels increased parallel to protein activity level in zymography. No corresponding increase in TIMP-1 and TIMP-2 protein activity and protein level was detected by reverse zymography and western blot. Elevated levels of MMP-9 and its distribution in retina were confirmed by immunohistochemistry. Expression of MMP-9 was detected in the inner and outer segments of rat retina, and the level becomes stronger at 24 hr after RIRI. In this study, ProMMP-2 and ProMMP-9 were expressed and increased significantly, but their inhibitors (TIMP-1 and TIMP-2) remained relatively unaltered in ischemic retina after RIRI in rats. These results suggest that MMP-2 and MMP-9 may play an important role in the pathomechanism of retinal ischemic injury.

Recombinant Sendai virus-mediated gene transfer into adult rat retinal tissue: efficient gene transfer by brief exposure.

To determine the usefulness of recombinant Sendai virus (SeV) for ocular gene transfer, the authors characterized SeV-mediated gene transfer to the retinal tissue of adult rats via subretinal injection. Recombinant SeV encoding the lacZ gene achieved frequent transgene expression in the retinal pigment epithelium (RPE) (mean=38.76%), while gene transfer to other retinal cells was rare. These findings are similar to those of previous reports using adenoviruses. Peak reporter gene expression of SeV in cultured RPE cells was similar to that of adenovirus at the same titer; however, SeV achieved high levels of expression after a brief vector-cell contact time, while adenovirus required over 3hr for efficient gene transfer. This finding was also observed in vivo following a brief SeV filling in the subretinal space, and may therefore provide a clinical advantage in avoiding retinal damage due to prolonged detachment. The observed SeV-mediated gene expression in the rat retina was transient. The initial phase of the decrease in luciferase activity could be prevented by daily eye drops of dexamethasone, suggesting that the corticosteroid-sensitive host reaction may affect early clearance of the virus. The late decline of transgene expression (2 weeks) was inhibited by the immunosuppressant, cyclosporin A, in a dose-dependent manner, suggesting that the cytotoxic T-lymphocyte response may be important in this phase. This work represents the first report of SeV-mediated gene transfer to ocular tissue, and identifies recombinant SeV as a new tool for studies of retinal gene transfer and gene therapy.