Phenotypic Features of Oguchi Disease and Retinitis Pigmentosa in Patients with S-Antigen Mutations: A Long-Term Follow-up Study.

PURPOSE: To present phenotypic features of 22 patients with S-antigen (SAG) mutations. DESIGN: Retrospective cohort study. PARTICIPANTS: Twenty-one Japanese patients from 16 families with a homozygous c.924delA mutation and 1 patient with a homozygous c.636delT mutation in the SAG gene. METHODS: Clinical records on symptoms; best-corrected visual acuity; and Goldmann perimetry, fundus photography, fundus autofluorescence (FAF), OCT, and electroretinography results were reviewed. MAIN OUTCOME MEASURES: Best-corrected visual acuity, Goldmann perimetry results, imaging findings, and electroretinography results. RESULTS: Ten patients had Oguchi disease and 12 had retinitis pigmentosa (RP) with mean follow-up periods of 13.8 and 10.2 years, respectively. Retinitis pigmentosa patients were older (mean age, 56.0 years) than those with Oguchi disease (mean age, 22.1 years; P < 0.001) at the initial visit. Night blindness noted in childhood was the most common initial symptom for both Oguchi disease (80.0%) and RP (91.7%) patients. Best-corrected visual acuity in the logarithm of the minimum angle of resolution (logMAR) was well preserved in Oguchi disease patients (mean, 0.02 logMAR in both eyes) but reduced in most RP patients (mean, 1.32 logMAR [right eye] and 1.35 logMAR [left eye]). Similarly, the visual field in the retinal area was preserved in Oguchi disease patients (mean, 677 mm2 right eye and 667 mm2 left eye) and reduced in RP patients (mean, 369 mm2 right eye and 294 mm2 left eye). Fundus images revealed a characteristic golden sheen with no retinal degeneration in Oguchi disease patients, excluding 2 with macular degeneration detected by FAF, OCT, or both and 1 with mild retinal degeneration confirmed by OCT and fluorescein angiography. Pigmentary retinal degeneration most evident posteriorly was observed in RP patients, accompanied by a characteristic golden sheen in 12 of 14 patients undergoing ultra-widefield fundus imaging. OCT showed disrupted macular structure, and FAF revealed variable hypofluorescence. Electroretinography identified absent rod responses in both diseases, along with relative preservation of cone responses in Oguchi disease patients. Three patients showed progressive loss of the golden sheen based on fundus images, including 1 who demonstrated RP 26 years after the initial diagnosis of Oguchi disease. CONCLUSIONS: Retinitis pigmentosa with SAG mutations often shows a characteristic golden sheen surrounding posterior pigmentary retinal degeneration. Oguchi disease can show progressive degeneration in adulthood, rarely resulting in RP.

Establishment of a recessive mutant small-eye rat with lens involution and retinal detachment associated with partial deletion and rearrangement of the Cryba1 gene.

From our stock of SDRs (Sprague-Dawley rats), we established a mutant strain having small opaque eyes and named it HiSER (Hirosaki small-eye rat). The HiSER phenotype is progressive and autosomal recessive. In HiSER eyes, disruption and involution of the lens, thickening of the inner nuclear layer, detachment and aggregation of the retina, rudimentary muscle in the ciliary body and cell infiltration in the vitreous humour were observed. Genetic linkage analysis using crossing with Brown Norway rat suggested that the causative gene(s) is located on chromosome 10. Microarray analysis showed that the expression level of the Cryba1 gene encoding ßA3/A1-crystallin on chromosome 10 was markedly decreased in HiSER eyes. Genomic PCR revealed deletion of a 3.6-kb DNA region encompassing exons 4-6 of the gene in HiSERs. In HiSER eyes, a chimaeric transcript of the gene containing exons 1-3 and an approximately 250-bp sequence originating from the 3'-UTR of the Nufip2 gene, located downstream of the breakpoint in the opposite direction, was present. Whereas the chimaeric transcript was expressed in HiSER eyes, neither normal nor chimaeric ßA3/A1-crystallin proteins were detected by Western blot analysis. Real-time RT (reverse transcription)-PCR analysis revealed that expression level of the Nufip2 gene in the HiSER eye was 40% of that in the SDR eye. These results suggest that the disappearance of the ßA3/A1-crystallin protein and, in addition, down-regulation of the Nufip2 gene as a consequence of gene rearrangement causes the HiSER phenotype.

Level of Vascular Endothelial Growth Factor in the Vitreous Fluid of Proliferative Diabetic Retinopathy Patients and Prognosis after Vitrectomy.

PURPOSE: The aim of this study was to evaluate the relationship between the intravitreal vascular endothelial growth factor (VEGF) level and prognosis of proliferative diabetic retinopathy (PDR). METHODS: The study involved 136 eyes of 114 PDR patients who underwent an initial vitrectomy between 2006 and 2008. Intravitreal VEGF levels were determined using Bio-Plex® (Bio-Rad), with levels of 5,000 pg/mL or more classified as high-VEGF (45 eyes) and levels lower than 5,000 pg/mL as low-VEGF (91 eyes). Diabetic control, PDR severity, and frequency of postoperative complications were compared between the groups. RESULTS: There was no significant difference in preoperative status between the groups. In the low-VEGF group, a reoperation was required due to postoperative complications in 2 eyes (2.2%); 1 with vitreous hemorrhage (VH) and 1 with retinal detachment (RD). In contrast, a reoperation was required in 8 eyes (17.8%) in the high-VEGF group; 3 with VH, 2 with RD, and 3 with neovascular glaucoma. The difference between the groups was significant. There was a statistically lower postoperative corrected visual acuity logMAR (6 months after surgery) in the high-VEGF than in the low-VEGF group (p = 0.02, unpaired t test). CONCLUSION: Current findings indicate that careful observation is needed in patients with elevated VEGF levels.

Effects of intravitreal injection of bevacizumab on inflammatory cytokines in the vitreous with proliferative diabetic retinopathy.

BACKGROUND: To investigate the effects of preoperative intravitreal injection of bevacizumab (IVB) on the levels of 27 inflammatory cytokines, including interleukins (ILs) and vascular endothelial growth factor. METHODS: From among 200 patients who had proliferative diabetic retinopathy and underwent vitrectomy in our department from September 2009 to October 2010, 8 study subjects met the enrollment criteria in which both eyes at nearly equivalent stages underwent vitrectomy. The first vitrectomy for each patient was performed without IVB (control group), whereas the second vitrectomy on the contralateral eye was performed with IVB treatment (1.25 mg/0.05 mL) 3 days before surgery (IVB group). Undiluted vitreous fluid was collected at the start of each vitrectomy. A multiplex assay was used to simultaneously determine the levels of 27 inflammatory cytokines and growth factors. RESULTS: Mean vascular endothelial growth factor levels were significantly lower in the IVB group (519.69 pg/mL) than in the control group (11,807.44 pg/mL) (P = 0.012, Wilcoxon signed rank test). Moreover, the mean levels (IVB/control, pg/mL) of IL-1RA (38.50/62.31, P = 0.036), IL-5 (27.75/34.00, P = 0.018), IL-10 (433.63/1,995.94, P = 0.012), IL-12 (246.69/1,033.69, P = 0.012), IL-13 (707.50/1,450.38, P = 0.012), and interferon γ (71.13/84.69, P = 0.036) were significantly lower in the IVB group. No other significant differences were observed in the levels of the other 20 cytokines and growth factors between the 2 groups. CONCLUSION: Preoperative IVB reduced not only the intravitreal vascular endothelial growth factor level but also the intravitreal levels of other inflammatory cytokines, including IL-1RA, IL-5, IL-10, IL-12, IL-13, and interferon γ. These results indicate the interaction of some cytokines in the vitreous fluid of proliferative diabetic retinopathy patients and suggest the possibility that preoperative IVB may not only reduce vascular proliferation by its direct antivascular endothelial growth factor effect but also modulate the inflammatory response through putative cytokine networks. None of the other cytokines examined were elevated after IVB.

Exclusion of influences of ARMS2 polymorphisms on the central visual field in retinitis pigmentosa.

PURPOSE: To investigate the effects of polymorphisms of the age-related maculopathy susceptibility 2 (ARMS2) gene on the central visual field defects in retinitis pigmentosa (RP). SUBJECTS AND METHODS: The visual field was evaluated using the 10-2 Swedish Interactive Threshold Algorithm Fast Program and mean deviation (MD) slope, and regression coefficients of average sensitivity of the central 4 points (Cent4) were compared between each genetic subgroup. RESULTS: The MD slope (right/left) was as follows: GG, -1.37 ± 2.18/ -0.89 ± 1.15; GT, -0.56 ± 1.40/-0.77 ± 1.04; TT, -0.75 ± 0.64/ -0.38 ± 0.92 dB/year. The Cent4 was as follows: GG, -1.34 ± 2.37/-1.60 ± 3.21; GT, -1.15 ± 2.08/1.07 ± 1.80; TT, -1.20 ± 0.91/-0.65 ± 1.37 dB/year. No significant differences in the degree of progression were observed when comparing groups. CONCLUSIONS: These data suggest that polymorphisms of the ARMS2 do not modify the progression of the central field of vision in RP patients.

Decrease of ATP by mitochondrial m-calpain inhibitory peptide in the rat retinas.

Activations of mitochondrial calpains cause apoptosis-inducing factor-dependent apoptosis of retinal photoreceptor cells in the Royal College of Surgeons (RCS) rat, an animal model of retinitis pigmentosa. In the present study, we attempted to develop specific inhibitors of mitochondrial calpains that would prevent the retinal degeneration. We examined the inhibitory potency of 20-mer peptides of the m-calpain for mitochondrial calpains activity, determined the inhibitory regions, and conjugated the cell-penetrating peptides (CPP). The cytotoxicity and delivery of the peptide was evaluated using mouse photoreceptor-derived 661W cells. After intravitreal injection of the peptide in RCS rats, we examined the peptide delivery to the retina, photoreceptor cell death numbers, responses of the electroretinogram (ERG), concentrations of intracellular ATP, and changes of retinal morphology. Results showed that one of the peptides inhibited the activity of the mitochondrial m-calpain. The HIV-1 tat-conjugated m-calpain peptide, HIV-Nm, could preserve the inhibitory potency of the mitochondrial m-calpain, and penetrate into the 661W cells. While intravitreal injection of HIV-Nm made it possible to deliver to the retina, it did not prevent photoreceptor cell death. Furthermore, it caused the ERG attenuation and the decrease in the intracellular ATP only a day after the injection. Although HIV-Nm did not cause histological change of the retina after 1 or 2 days of the administration, the morphological abnormality of the retina was observed after 3-14 days. Our results demonstrated that HIV-Nm failed to prevent the photoreceptor cell death, but rather caused the attenuation of ERG response and the decrease of ATP.

Intravitreal injection or topical eye-drop application of a µ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa.

Mitochondrial µ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial µ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of µ-calpain. Two µ-calpain peptides N2 and N9 inhibited mitochondrial µ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50µM of both µ-calpain peptides caused specific degradation of mitochondrial µ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of µ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial µ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50µM HIV-conjugated µ-calpain N2-10-2 peptide (HIV-Nµ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nµ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nµ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nµ, a peptide inhibitor of mitochondrial µ-calpain, offers a new modality for treating RP.

Cisplatin binding and inactivation of mitochondrial glutamate oxaloacetate transaminase in cisplatin-induced rat nephrotoxicity.

Cisplatin is a widely used chemotherapeutic agent, but its use is limited by nephrotoxicity associated with mitochondrial dysfunction. Because its mechanisms are poorly understood, we aimed to identify the mitochondrial proteins targeted by cisplatin. We isolated renal mitochondrial proteins from Sprague-Dawley (SD) rats and performed cisplatin-affinity column chromatography. The proteins eluted were detected on SDS-PAGE and subjected to in-gel tryptic digestion and LC-MS/MS analysis. We identified glutamate oxaloacetate transaminase (GOT) and mitochondrial malate dehydrogenase (MDH). Next, we administered cisplatin intraperitoneally to SD rats to induce nephrotoxicity and assayed the activities of the enzymes. The results indicated that cisplatin caused a severe decrease in mitochondrial GOT activity on day 1 after cisplatin administration. Three d later, we also identified a decrease in mitochondrial MDH activity. Our results indicate that cisplatin binds to mitochondrial GOT and inhibits its activity, causing mitochondrial dysfunction and subsequent nephrotoxicity.

M-opsin protein degradation is inhibited by MG-132 in Rpe65⁻/⁻ retinal explant culture.

PURPOSE: The 65 kDa retinal pigment epithelium-specific protein, RPE65, is an essential enzyme for 11-cis-retinal synthesis in the eye. Mutations of the RPE65 gene in humans result in severe vision loss, and Rpe65(-/-) mice show early cone photoreceptor degeneration. We used an explant culture system to evaluate whether posttranslational downregulation of M-opsin protein in Rpe65(-/-) mice is caused by proteolytic degradation. METHODS: The eyes of three-week-old Rpe65(-/-) mice were incubated in culture medium. Western blot analysis was used to evaluate the level of M-opsin protein, and immunofluorescence was used for protein localization. The transcriptional level of M-opsin was evaluated with real-time reverse-transcriptase-PCR. RESULTS: Degradation of the M-opsin protein in Rpe65(-/-) mouse retina was inhibited by the proteasome inhibitor MG-132 but not by the lysosomal inhibitor pepstatin A and E64d. 9-cis-retinal, used as an analog of 11-cis-retinal, increased M-opsin protein but did not increase M-opsin mRNA. Moreover, 9-cis-retinal did not change the transcriptional levels of photoreceptor specific genes. CONCLUSIONS: Our data suggest that M-opsin protein was degraded through a proteasome pathway and that M-opsin degradation was suppressed with 9-cis-retinal treatment in Rpe65(-/-) mice to some extent.

A case of herpes zoster ophthalmicus with optic neuritis of the total length of the optic nerve in the orbital space and ischemic optic neuropathy.

Purpose: We herein report a case of optic neuritis and ischemic optic neuropathy associated with herpes zoster ophthalmicus and decreased visual acuity. Observations: A 65-year-old man with no special medical history had a headache on the right side in December 2019, and a few days later, a facial rash appeared on the same side. A dermatologist diagnosed him with herpes zoster ophthalmicus and started antiviral drug therapy. On the same day, he was referred to a local ophthalmologist and was found to have inflammatory signs in his right cornea and conjunctiva. The next day, when he visited the ophthalmologist again, he had decreased visual acuity, optic disc swelling, and fundus hemorrhaging in his right eye, so he was referred to our department. At the first visit to our department, his best-corrected visual acuity was light sense OD, 1.0 OS. His right fundus showed optic disc swelling, spotted fundus hemorrhaging, and dilation/tortuosity of the retinal vein. Fluorescein angiography showed the near absence of optic disc filling as well as delayed retinal vein perfusion in his right eye, and magnetic resonance imaging confirmed high signals in the total length of the right optic nerve in the orbital space using the short inversion-time inversion recovery method. Based on these findings, we diagnosed him with optic neuritis and ischemic optic neuropathy associated with inflammation of the orbital part caused by herpes zoster ophthalmicus. We started systemic administration of antiviral drugs (acyclovir) and oral steroid. However, after treatment, his visual acuity improved only to hand motion OD, and the fundus appearance was ultimately optic atrophy OD. Conclusion and Importance: Various complications can occur with herpes zoster ophthalmicus, however, few reports have described cases of herpes zoster ophthalmicus associated with optic neuritis and ischemic optic neuropathy. Therefore, there is no consensus concerning the ideal treatment for these conditions. By referencing cases involving issues such as orbital apex syndrome and optic neuritis caused by herpes zoster ophthalmicus, antiviral drugs and oral steroids were administered, but the prognosis of the visual acuity was poor.

A Case of Anti-BP180-type Mucous Membrane Pemphigoid with IgG and IgA Autoantibodies Showing Distinct Reactivities.

Dear Editor, Mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by erosive mucosal lesions mainly on the oral and ocular mucosae (1). We report a case of oral and ocular anti-BP180-type MMP with variable IgG and IgA reactivities and underlying dementia. An 84-year-old Japanese man presented with a 4-year history of erosions in the oral cavity and on the conjunctivae, with progressive vision impairment. The medical history included benign prostatic hyperplasia, cataract, sinusitis, and dementia. Physical examination revealed erosions and white atrophic scars along the gingival mucosa and on the hard palate (Figure 1, a, b). Conjunctival inflammation and corneal scarring were also observed only on the left eye (Figure 1, c, d). No lesions were observed on the skin or on any other mucosae. A skin biopsy from the patient's oral mucosa showed lymphocytic infiltration in the superficial dermis without apparent subepithelial blister. Direct immunofluorescence showed linear depositions of IgG, IgA, and C3 at the epithelial basement membrane zone (Figure 1, e-g). Circulating IgG and IgA autoantibodies were not detected by indirect immunofluorescence of normal human skin, while circulating IgA, but not IgG, autoantibodies were bound to the epidermal side of 1M NaCl-split normal human skin at 1:10 serum dilution (Figure 1, h, i). Commercially available IgG enzyme-linked immunosorbent assays (ELISAs) of BP180 NC16a domain, BP230, and type VII collagen (MBL, Nagoya, Japan) showed negative results. IgG and IgA immunoblotting analyses of six different antigen sources, including BP180 C-terminal domain recombinant protein, were all negative. However, ELISA of full-length BP180 was slightly positive for IgG antibodies (index = 5.79; cut-off <4.64). Immunoblotting analysis of full-length BP180 was negative for both IgG and IgA antibodies (Figure 1, j, k). Immunoblotting analysis of hemidesmosome-rich fraction was negative for both IgG and IgA antibodies to integrin ß4 (Figure 1, l). Based mainly on the clinical and immunological findings, we established a diagnosis of MMP with IgG and IgA autoantibodies, likely reactive with BP180. Because the patient refused systemic treatments, we prescribed a mouth rinse sodium gualenate hydrate and eyedrops of fluorometholone and purified sodium hyaluronate, which did not improve the oral and ocular mucosal symptoms during the 8 month follow-up period (Figure 1, m, n). Both IgG and IgA autoantibodies in anti-BP180-type MMP tend to react with the C-terminal domain of BP180 (2), and IgG autoantibodies in 39.7% of MMP patients reactive with the epidermal side of split skin were reported to be positive with BP180 C-terminal domain (3). The full-length BP180 ELISA shows excellent sensitivity for diagnosing BP180-type MMP (4). The different IgG and IgA reactivities among various methods used in the present study may be attributed either to different methodologies (i.e., immunoblotting or ELISA) or to the different substrates, since BP180-type MMP targets various regions of BP180, including the NC16a domain, the C-terminal domain, and the intracytoplasmic region (5). Precise diagnosis for MMP by various immunological methods is critical, because urgent and extensive treatments are necessary for the ocular and laryngeal lesions, which may result in loss of eyesight and airway obstruction, respectively. Acknowledgments: We express our gratitude to Ms. Mako Mine and Dr. Daisuke Hayashi, Department of Dermatology, Osaka City University Graduate School of Medicine in Osaka, Japan for the HD-rich fraction immunoblotting analysis, and Dr. Yoshiaki Hirako, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Aichi, Japan for the preparation of the HD-rich fraction sample. This work was supported by JSPS KAKENHI Grant Number JP20k08684 and the Hirosaki University Research Support System.

Effect of nilvadipine on central visual field in retinitis pigmentosa: a 30-month clinical trial.

PURPOSE: To assess the effects of nilvadipine on the progression of central visual field defect in retinitis pigmentosa (RP). DESIGN: Prospective, randomized, nonmasked, single-center trial. METHODS: Patients with RP were randomly divided into a treated group receiving oral nilvadipine at 4 mg/day for ≥30 months and a control group receiving tocopherol nicotinate at 300 mg/day, helenien at 15 mg/day or no medication for the same periods. Progression of RP was evaluated using the 10-2 SITA Fast Program of the Humphrey Visual Field Analyzer, and regression coefficients calculated from the time courses of mean deviation (MD slope) were compared between groups. RESULTS: Nineteen patients in the treated group and 14 patients in the control group completed the follow-up for ≥30 months. The mean (±standard deviation) duration of observation was 48.8 ± 11.8 months (median 48 months, range 30-66 months) for the treated group and 49.2 ± 18.1 months (median 48 months, range 30-90 months) for the control group (p = 0.94). Mean (±standard error of the mean, SEM) regression coefficients of the averaged MD values for the initial 30 months were -0.35 ± 0.17 dB/year in the treated group and -0.75 ± 0.06 dB/year in the control group (p < 0.01). Mean (±SEM) MD slopes for total observational periods were -0.49 ± 0.17 dB/year in the treated group and -0.89 ± 0.16 dB/year in the control group (mean ± SEM, p = 0.042). CONCLUSION: Nilvadipine at 4 mg/day significantly retarded progression of central visual field defects in RP in this small patient series.

A Case of Cyclodialysis after Microhook Trabeculotomy Treated with Vitreous Surgery.

We report a case of cyclodialysis with decreased visual acuity after microhook trabeculotomy (mTLO) successfully treated by vitreous surgery. A 41-year-old man had been medically treated for primary open-angle glaucoma in both eyes. He was scheduled to undergo mTLO due to progression of visual field impairment and unstable intraocular pressure in his right eye. His preoperative best-corrected visual acuity (BCVA) was 0.4 OD, and the intraocular pressure was unstable, ranging from 12 to 27 mm Hg. On the day after the operation, a shallow anterior chamber developed, and a low intraocular pressure occurred. His visual acuity continued to decrease, and cyclodialysis was confirmed by ultrasonic biomicroscopy. No improvement was obtained with medical treatment, and his BCVA dropped to 0.08 OD, while his intraocular pressure remained at 2-3 mm Hg. Three months later, a second surgery was performed by combining cataract surgery with intraocular lens implantation, vitrectomy, cryopexy for the pars plana of the ciliary body, and 20% SF6 gas tamponade. Two weeks after the reoperation, the intraocular pressure had been normalized to 12 mm Hg, and the BCVA had returned to 0.3. We successfully treated cyclodialysis as a complication after mTLO by vitreous surgery that led to the recovery of the visual acuity and intraocular pressure.

Racemose hemangioma complicated with macular macroaneurysm rupture.

PURPOSE: To describe a rare case of racemose hemangioma which developed spontaneous macular macroaneurysm (MA) rupture and vitreaous hemorrhage. OBSERVATIONS: A 29-year-old healthy asian female visited our hospital and a racemose hemangioma was found in the left eye. At presentation, the best corrected visual acuity (BCVA) was 30/20 in her left eye. At 9 years after the first visit, MA-like lesion was noted in the macular area. After that, vitreous and subretinal hemorrhage appeared in the left eye. The patient underwent simultaneous vitrectomy and cataract surgery, but vitreous re-hemorrhage occurred two days after the operation. To avoid re-hemorrhage, silicone oil (SO) tamponade was added in the second vitrectomy. Two years after the second operation, SO was removed and postoperative BCVA in the left eye was 20/200 without re-bleeding in the vitreous. CONCLUSIONS AND IMPORTANCE: Although retinal hemorrhages have been reported in the patients with a racemose hemangioma, in our case the macular MA rupture occurred at 9 years after the first visit. Congenital retinal arteriovenous anastomosis can show a change in vascular shape in some cases, thus it is important to observe carefully.

S-opsin protein is incompletely modified during N-glycan processing in Rpe65(-/-) mice.

Retinal pigment epithelium-specific protein 65 kDa (RPE65) is a key enzyme for the visual cycle in the eye. Rpe65(-/-) mice lack 11-cis-retinal, and show early cone degeneration and mislocalization of cone opsins. The present study investigated whether abnormal modification of cone opsins at the protein level is present in Rpe65(-/-) mice. Retina-RPE-choroids of Rpe65(-/-) mice at 3, 5 and 7 weeks old were used. Immunohistochemistry of opsins was performed using cryosections and retinal flatmounts. We evaluated levels of mRNA for cone and rod opsin genes by RT-PCR and levels of proteins by western blotting. To examine modification patterns of N-glycan in Rpe65(-/-) mice, cone opsins were digested with peptide-N-glycosidase (PNGase) F. S-opsin protein was detected at approximately 40-kDa as a major band in wild-type mice, whereas approximately 42-kDa S-opsin protein was detected in Rpe65(-/-) mice. After PNGase F treatment, mobility of S-opsin protein in wild-type and Rpe65(-/-) mice on SDS-PAGE was similar. In addition, approximately 25-kDa S-opsin polypeptide was notably detected in Rpe65(-/-) mice. Conversely, M-opsin proteins were not observed by immunohistochemistry or western blotting in Rpe65(-/-) mice, but expression of M-opsin mRNA in Rpe65(-/-) mice did not differ significantly from that in wild-type mice at 3 and 5 weeks. Mobility of M-opsin protein in Rpe65(-/-) mice was unchanged. Our data suggest that S-opsin protein is incompletely modified during N-glycan processing in Rpe65(-/-) mice, whereas M-opsin protein is severely reduced by posttranslational degradation in the absence of incomplete N-glycan processing in Rpe65(-/-) mice.

Inhibitory effects of trehalose on fibroblast proliferation and implications for ocular surgery.

Trehalose is a disaccharide which plays an important role in preserving cells from completely dehydrated circumstances. In this study, we investigated effects of trehalose on proliferative activity of fibroblasts and epithelial cells both in vitro and in vivo. As in vitro assessment, normal human dermal fibroblasts and normal human epidermal keratinocytes were cultured in media containing various concentrations of trehalose. Growth activities of cells were evaluated with MTT assay and diff-quick™ staining. Expressions of vimentin and α smooth muscle actin (α-SMA) changed by trehalose were semiquantitatively measured by Western blot. As an in vivo study, 5% or 10% trehalose was topically instilled onto rabbit eyes after simple conjunctival incision or trabeculectomy. Condition of the surgical wound was evaluated by morphologically and immunohistochemically using isolectin B4 and antibodies specific for vimentin and α-SMA. Intraocular pressures (IOPs) after trabeculectomy were compared between eyes treated with trehalose and 0.04% mitomycin C (MMC). Results obtained by in vitro experiments showed that growth activities of cultured fibroblasts and keratinocytes were inhibited by trehalose in a dose-dependent manner. Fibroblasts were strongly inhibited by trehalose concentrations ≧ 5% of trehalose, whereas keratinocytes were less inhibited compared to fibroblasts. Expressions of vimentin and α-SMA were reduced by trehalose. With in vivo experiments, postoperative application of trehalose resulted in less firm adhesion between conjunctiva and sclera compared to controls. Immunohistochemical studies showed reduced staining of isolectin B4, vimentin and α-SMA in conjunctival wounds treated by topical trehalose. Also, after trabeculectomy, IOP remained in a low range during instillation of topical trehalose solution. We concluded that trehalose has inhibitory effects on proliferation of fibroblasts and vascular tissues, partially due to inhibition of transformation of fibroblasts into myofibroblasts in wound tissues. The present results imply that trehalose can be a potential agent for preventing postoperative fibrous scar formation after ocular surgery such as glaucoma filtration surgery.

Activation of mitochondrial calpain and release of apoptosis-inducing factor from mitochondria in RCS rat retinal degeneration.

The present study was performed to investigate changes of cytosolic and mitochondrial calpain activities, and effects of intravitreously injected calpain inhibitor on photoreceptor apoptosis in Royal College of Surgeon's (RCS) rats. Time courses of activities for both cytosolic and mitochondrial calpains and amount of calpastatin in RCS rat retina were analyzed by subcellular fractionation, calpain assay and western blotting. Calpain assay was colorimetrically performed using Suc-LLVY-Glo as substrate. Effects of intravitreously injected calpain inhibitor (ALLN and PD150606) on RCS rat retinal degeneration were analyzed by TUNEL staining. Effects of mitochondrial calpain activity on activation and translocation of apoptosis-inducing factor (AIF) were analyzed by western blotting. Mitochondrial calpain started to be significantly activated at postnatal (p) 28 days in RCS rat retina, whereas cytosolic micro-calpain was activated at p 35 days, although specific activity of mitochondrial calpain was 13% compared to cytosolic micro-calpain. Intravitreously injected ALLN and PD150606 effectively inhibited photoreceptor apoptosis only when injected at p 25 days, but did not inhibit photoreceptor apoptosis when injected at p 32 days. Parts of AIF were truncated/activated by mitochondrial calpains and translocated to the nucleus. These results suggest that 1), calpain presents not only in the cytosolic fraction but also in the mitochondrial fraction in RCS rat retina; 2), mitochondrial calpain is activated earlier than cytosolic calpain during retinal degeneration in RCS rats; 3), photoreceptor apoptosis may be regulated by not only calpain systems but also other mechanisms; 4), mitochondrial calpain may activate AIF to induce apoptosis; and 5), calpain inhibitors may be partially effective to inhibit photoreceptor apoptosis in RCS rats. The present study provides new insights into the molecular basis for photoreceptor apoptosis in RCS rats and the future possibility of new pharmaceutical treatments for retinitis pigmentosa.

Clinicopathological roles of aberrantly expressed recoverin in malignant tumor cells.

PURPOSE: Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions toward recoverin, a retina-specific calcium binding protein, and other retinal antigens aberrantly expressed in tumor cells are elicited. As a consequence, photoreceptor cell degeneration is induced. To elucidate the pathological role of the aberrantly expressed recoverin, we studied the recoverin expression levels in various malignant tumors and the effects of the expressed recoverin on the sensitivity to anti-cancer drugs. METHODS: Recoverin expression levels were determined by immunohistochemistry with anti-human recoverin monoclonal antibody on multiple tissue arrays obtained from several lung, stomach, colon and other cancers. In the presence of several anti-cancer drugs, including anti-tumor antibiotics, plant alkaloids and anti-metabolites, cytotoxicity assay was performed using recoverin-positive or recoverin-negative A549 cells originating from human lung adenocarcinoma. RESULTS: Immunofluorescence labeling revealed that recoverin immunoreactivities were detected at approximately 10-40% of malignant tumor tissues. Cytotoxic effects by anti-cancer drugs were higher in recoverin-positive A549 cells as compared to recoverin-negative cells. CONCLUSION: The present data may correlate with the previous observation that recoverin-expressing cancer cells induced tumor immunity and a favorable prognosis for primary cancer in CAR patients.

[A case of hypertrophic pachymeningitis impeding differential diagnosis of mycosis of orbital apex syndrome].

PURPOSE: Mycosis of the orbital apex is often fatal. We report case of orbital apex syndrome, which appeared to have been caused by hypertrophic pachymeningitis with positive systemic mycosis antigens treated with systemic corticosteroid combined with antimicrobiotic therapy. CASE: A 57-year-old woman presented with disturbed ocular motility and visual disturbance of her left eye. She had rheumatoid arthritis. Magnetic resonance imaging(MRI) showed an abnormal blush in the left orbital apex and meningeal thickening in the cranial base, leading to the diagnosis of hypertrophic pachymeningitis. She also had candidemia. Systemic corticosteroids combined with antimicrobiotic therapy resulted in improved clinical ocular manifestations. She died 13 months later due to interstitial pneumonia. CONCLUSION: The diagnosis of this case was difficult because of the candidemia. But the effective steroid response indicates that the probable cause was hypertrophic pachymeningitis due to RA. To avoid complications, hypertrophic pachymeningitis associated with fungemia should be treated with corticosteroids combined with antimicrobiotic therapy.

The qualitative assessment of optical coherence tomography and the central retinal sensitivity in patients with retinitis pigmentosa.

PURPOSE: To analyze the relationships between qualitative and quantitative parameters of spectral-domain optical coherence tomography (SD-OCT) and the central retinal sensitivity in patients with retinitis pigmentosa (RP). MATERIALS AND METHODS: Ninety-three eyes of 93 patients were finally enrolled, with a median age (quartile) of 58 (24.5) years. We assessed the patients using SD-OCT and the 10-2 program of a Humphry Field Analyzer (HFA). As a qualitative parameter, two graders independently classified the patients' SD-OCT images into five severity grades (grades 1-5) based on the severity of damage to the photoreceptor inner and outer segments (IS/OS) layer. As quantitative parameters, we measured the IS-ellipsoid zone (IS-EZ) width, IS/OS thickness, outer nuclear layer (ONL) thickness, central macular thickness (CMT, 1 and 3 mm) and macular cube (6 × 6 mm) volume and thickness. The central retinal sensitivity was defined by the best-corrected visual acuity (BCVA; logMAR), average sensitivities of the central 4 (foveal sensitivity [FS]) and 12 (macular sensitivity [MS]) points of the HFA 10-2 program and the mean deviation (MD) of the 10-2 program. Spearman's correlation was used to assess the association between both qualitative and quantitative parameters and variables of the central retinal sensitivity. In addition, we performed a multiple regression analysis using these parameters to identify the parameters most strongly influencing the central retinal sensitivity. RESULTS: The IS/OS severity grade was significantly correlated with the BCVA (ρ = 0.741, P < 0.001), FS (ρ = -0.844, P < 0.001), MS (ρ = -0.820, P < 0.001) and MD (ρ = -0.681, P < 0.001) and showed stronger correlations to them than any other quantitative parameters including the IS-EZ width, IS/OS thickness, ONL thickness, CMTs and macular cube volume/thickness. Furthermore, a step-wise multiple regression analysis indicated that the IS/OS severity grade was more strongly associated with the BCVA (ß = 0.659, P < 0.001), FS (ß = -0.820, P < 0.001), MS (ß = -0.820, P < 0.001) and MD (ß = -0.674, P < 0.001) than any other quantitative parameters. The intraclass correlation coefficient between two graders indicated substantial correlation (κ = 0.70). DISCUSSION: The qualitative grading of OCT based on the severity of the IS/OS layer was simple and strongly correlated with the central retinal sensitivity in patients with RP. It may be useful to assess the central visual function in patients with RP, although there is some variation in severity within the same severity grade.