Makoto Tamai

HLF/HIF-2α is a key factor in retinopathy of prematurity in association with erythropoietin

An HLF (HIF‐1α‐like factor)/HIF‐2α‐knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF‐knockdown (HLFkd/kd) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLFkd/kd mice expressing 80–20% reduction, depending on the tissue, in wild‐type HLF mRNA were fertile and apparently normal. Hyperoxia–normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild‐type mice, but not in HLFkd/kd mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLFkd/kd mice due to poor vascularization. Cre‐mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLFkd/kd mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLFkd/kd mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.

A comparative study of the polymerase chain reaction and local antibody production in acute retinal necrosis syndrome and cytomegalovirus retinitis.

Abstract• Background: It has been thought that herpes viruses may play an important role in acute retinal necrosis syndrome (herpes simplex and varicella-zoster viruses) as well as in cytomegalovirus retinitis and it would be useful to know the specificity of the methods for detecting the viruses. • Methods: Amplification of the herpetic viral genome DNA by polymerase chain reaction (PCR) in aqueous and vitreous humor was compared with Goldmann-Witmer coefficients against herpetic antigens in five patients with acute retinal necrosis syndrome and in two patients with cytomegalovirus retinitis, using vitreous samples to determine the specificity of these diagnostic methods. • Results: The varicella-zoster virus genome DNA was amplified by PCR in four of the five patients with acute retinal necrosis syndrome, and cytomegalovirus genome DNA was enhanced in both patients with cytomegalovirus retinitis. Four patients who exhibited the varicella-zoster viral genome showed marked increase of the Goldmann-Witmer coefficient against varicella-zoster virus. Conversely, the two patients with cytomegalovirus retinitis showed no remarkable changes among the antigens. • Conclusion: Our results showed that the amplification of the viral genome DNA in the samples by PCR is specific in both diseases, and that the increased level of local antibody production also is specific in varicella-zoster retinitis. In cytomegalovirus retinitis, however, antibody production against cytomegalovirus does not show increase of the Goldmann-Witmer coefficient.

Pitavastatin prevents NMDA‐induced retinal ganglion cell death by suppressing leukocyte recruitment

Excitotoxicity is a major cause of retinal ganglion cell (RGC) death during ischemic diseases such as vessel occlusion and diabetic retinopathy. However, the underlying mechanisms are not well understood. Statins, inhibitors of the HMG‐CoA reductase, have neuroprotective effects in addition to their original role in lowering cholesterol. We hypothesize that pitavastatin, a recently introduced potent statin, is protective against N‐methyl‐d‐aspartic acid (NMDA)‐induced RGC death. Pitavastatin, administered by gavage, abolished NMDA‐induced loss of RGCs. To elucidate the mechanisms underlying the neuroprotective effect of pitavastatin, we investigated its impact on inflammation. NMDA increased the expression of interleukin‐1β and TNF‐α, and endothelial adhesion molecules, including ICAM‐1, and induced leukocyte accumulation in the retinal vessels. Pitavastatin significantly reduced NMDA‐induced leukocyte accumulation and up‐regulation of endothelial adhesion molecules, whereas cytokine expression was unaffected. Systemic blockade of ICAM‐1 in wild‐type mice or absence of CD18 in gene‐deficient (CD18–/–) mice significantly suppressed NMDA‐induced leukocyte accumulation and RGC death. These findings suggest a novel and causative role for inflammatory leukocyte recruitment in NMDA‐induced excitotoxicity. Furthermore, we show the novel neuroprotective effect of statins against excitotoxicity‐induced RGC death. Statins or other anti‐inflammatory agents may thus have therapeutic benefits in excitotoxicity‐associated neuronal diseases through blockade of leukocyte recruitment.

POINT MUTATIONS OF RHODOPSIN GENE FOUND IN JAPANESE FAMILIES WITH AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA (ADRP)

SummaryThe mutations of codon 17, 23, 58, and 347 of rhodopsin gene were investigated in 24 unrelated Japanese families including 33 patients with autosomal dominant retinitis pigmentosa (ADRP). A patient with codon 17 mutation (Thr-17-Met, ACG→ATG) and a family including 4 patients with codon 347 mutation (Pro-347-Leu, CCG→CTG) were detected among them. Their clinical findings were extremely different between the two mutations. The former showed type 2 and the latter showed type 1 ADRP. No mutation of codon 23 and 58 was detected in any families so far analyzed in the present study. Clinical findings associated with the mutation in codon 17 and 347 of the rhodopsin gene show an existence of allelic heterogeneity.

Intrinsic activation of PI3K/Akt signaling pathway and its neuroprotective effect against retinal injury.

Purpose. The aim of this study was to determine whether the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway can function as a neuroprotective pathway following induced retinal injury. Methods. The activation of Akt was assessed by immunoblot analysis, and the role of PI3K/Akt pathway was evaluated by TUNEL staining and counting the number of retrogradelylabeled retinal ganglion cells (RGCs) in the whole retina at 168 h after injury with or without PI3K specific inhibitor, LY294002. Results. Akt was induced within one hr and reached a maximum 6hrs after optic nerve clamping. The activation was observed in the RGC layer including RGCs, the inner plexiform layer, inner nuclear layer, and in the photoreceptor outer segments. The number of surviving RGCs was decreased significantly 168 hrs after injury. LY294002 partially inhibited the activation of Akt, and significantly decreased the number of surviving RGCs as compared with that of injury alone. Conclusions. These results indicate that the PI3K/Akt signaling pathway is activated intrinsically and has a neuroprotective effect on injured RGCs.

Molecular genetic analysis of optineurin gene for primary open-angle and normal tension glaucoma in the Japanese population.

Purpose:To determine whether mutations in the optineurin (OPTN) gene are associated with the incidence of primary open-angle glaucoma (POAG) and normal tension glaucoma (NTG) in the Japanese. Methods:Eighty-nine unrelated Japanese patients with POAG and 65 unrelated patients with NTG were studied. Genomic DNA was extracted from leukocytes of the peripheral blood, and thirteen exons of the OPTN gene were amplified by polymerase chain reaction (PCR) and directly sequenced. Results:Sequence alterations in exons 4 (His26Asp), 5 (Met98Lys), and 16 (Arg545Gln) were found. The His26Asp and Arg545Gln mutations were not detected in 100 ethnically matched controls. The frequency of the missense Met98Lys variant was higher in the POAG and NTG groups than in the control group (16.9% versus 5%, 15.4% versus 5%; P = 0.009 and P = 0.029, and odds ratio 3.85 and 3.45, respectively, for the dominant effect of the OPTN A allele). Polymorphisms in exons 4 and 12, and in introns 6 and 7 were also detected. Conclusions:The association of the allelic variation (Met98Lys) in the OPTN gene and the prevalence of POAG and NTG in unrelated Japanese patients suggest that they are involved in the pathogenesis of POAG and NTG.

Abnormal migration and distribution of neural crest cells in Pax6 heterozygous mutant eye, a model for human eye diseases

PAX6/Pax6 gene encodes a transcription factor that is crucially required for eye development. Pax6 heterozygous mutant mouse (Pax6Sey/+) shows various ocular defects, especially in the anterior segment. It has been well known that the induction of the lens and development of the cornea and retina are dependent on PAX6/Pax6 in a cell‐autonomous fashion, although the influence of PAX6/Pax6 on the other tissues derived from the ocular mesenchyme is largely unknown. Using transgenic mouse lines in which neural crest cells are genetically marked by LacZ or EGFP, we revealed the extensive contribution of neural crest derived cells (NCDCs) to the ocular tissues. Furthermore, various eye defects in Pax6Sey/+ mouse were accompanied by abnormal distribution of NCDCs from early developmental stages to the adult. In Pax6Sey/+ mouse mice, neural crest cells abnormally migrated into the developing eye in a cell nonautonomous manner at early embryonic stages. These results indicate that normal distribution and integration of NCDCs in ocular tissues depend on a proper dosage of Pax6, and that Pax6Sey/+ eye anomalies are caused by cell autonomous and nonautonomous defects due to Pax6 haploinsufficiency.

Neuroprotective effect of nipradilol on axotomized rat retinal ganglion cells

Purpose. To determine whether nipradilol, a new antiglaucoma drug, can protect retinal ganglion cells (RGCs) from secondary cell death caused by transection of the optic nerve (ON). Methods. The ON was transected 0.7mm from its exit from the eye in Sprague Dawley rats. Nipradilol (1 × 10 -8 – 10 -3 M), timolol, prazosin, or sodium nitroprusside (SNP)(1 × 10 -6 - 10 -4 M) was injected intravitreally fifteen-minutes before the ON transection. Control eyes received the same amount of phosphate buffered (PB). The RGCs were labeled retrogradely by placing gelfoam soaked in fluoro-gold (FG) on the stump of ON. RGCs density was determined by counting the FG-labeled RGCs in flat-mounted retinas 3 to 14 days post-transection. To determine whether the neuroprotective action of nipradilol was due to its NO-donor property, carboxy-PTIO, a NO-scavenger, or KT5832, a protein kinase G inhibitor, was injected with the nipradilol. Results. After ON transection, the number of surviving RGCs after intravitreal injection of 1 × 10 -4 M nipradilol was significantly higher than that following PB injection. This protective activity was dose-dependent. Neither timolol nor prazosin had a neuroprotective effect but SNP protected RGCs in a dose-dependent manner. Carboxy-PTIO and KT5832 decreased the neuroprotective effect of nipradilol. Conclusions. These results indicate that nipradilol has a possibility of neuroprotective effect on axotomized RGCs, and the effect depended mainly on its NO-donor property.

CHANGES IN GABA METABOLISM IN STREPTOZOTOCIN-INDUCED DIABETIC RAT RETINAS

We examined the pathogenesis of reduced amplitude in electroretinogram (ERG) oscillatory potentials (OPs) in diabetes, in relation to possible changes in the metabolisms involving retinal amino acid neurotransmitters. With use of streptozotocin diabetic rats, flash ERGs were recorded and quantitative analyses of retinal free amino acids were performed. Immunocytochemical localizations of retinal glycine and GABA were examined. In addition, activities of glutamic acid decarboxylase (GAD) and GABA transaminase (GABA-T) were measured. Our results revealed that the amplitudes of OP 1 and OP 2 decreased, and retinal glycine and GABA content significantly increased in the diabetic rats. An increased immunoreactivity of GABA was observed in Müller cells in the diabetic rat retinas, while no apparent changes were found in glycine immunoreactivity. Finally, increased activations of GAD with reduced activities of GABA-T were observed in the diabetic rat retinas. Thus, reduced amplitudes of OPs were associated with changes in content, localization, and enzyme activities related to GABA in the retinas, implying that changes in GABA metabolism can be a candidate for the pathogenesis of the abnormal OPs in diabetes.

Transplantation of retinal pigment epithelium using viable cryopreserved cells

Transplantation of retinal pigment epithelium (RPE) may have potential clinical application for the surgical treatment of RPE-specific retinal degeneration, including age-related macular degeneration. The feasibility of an RPE storage bank has been investigated by experimenting with transplantation using viable, cryopreserved RPE cells. Fresh and cultured fetal human and bovine RPE cells were cryopreserved in 90% fetal bovine serum containing 10% dimethyl sulfoxide. The viability of the cells before and after cryopreservation was evaluated by trypan blue dye exclusion test, microculture tetrazolium assay (MTA), tissue culture, and transplantation after cryopreservation. The origin of RPE cells before and after cryopreservation was assessed by immunocytochemistry, immunoblotting, and indirect ELISA of RPE-marker protein using cytokeratin for cultured fetal human RPE cells and by immunocytochemistry of cellular retinaldehyde-binding protein (CR-ALBP) for cultured bovine RPE cells. Freshly isolated and cryopreserved uncultured bovine RPE cells were transplanted by posterior transscleral approach into the subretinal spaces of adult albino rabbits and 23-day-old Royal College of Surgeons (RCS) rats with a 33 gauge Hamilton syringe. Following surgery, artificial retinal blebs were confirmed by fundus examination. Morphologic examination was performed postoperatively by light and electron microscopy in albino rabbits and by light microscopy in RCS rats up to 3 mo. Control subretinal injections using vehicle solution also were performed in RCS rats. Cultured fetal human and bovine RPE cells after cryopreservation were found to be viable, based on the results of trypan blue dye exclusion test, MTA, tissue culture, and transplantation. Expression and reexpression of cytokeratin intermediate filaments in cultured fetal human RPE were demonstrated by immunocytochemistry, immunoblotting, and indirect ELISA before and after cryopreservation. Immunocytochemistry of CRALBP before and after cryopreservation in uncultured bovine RPE cells disclosed expression and reexpression of RPE cell marker protein. No uncultured fetal human RPE cells showed proliferation in tissue culture after cryopreservation. In rabbits, light and electron microscopy disclosed xenografted RPE cells residing on Bruchs membrane of the host retina. No sign of graft vs. host reaction was observed. No morphologic difference was noted between the fresh and 10-day-old cryopreserved RPE cells in situ following transplantation at day 25. In RCS rats, subretinal injection of 3-wk-old cryopreserved bovine RPE cells partially rescued photoreceptor cells locally at the transplanted area observed at 3 mo postoperatively. The retinal photoreceptors at the inferior hemisphere of the transplanted eye and the eye injected with vehicle solution showed no rescue effect. We found that cryopreserved cultured fetal human RPE cells and uncultured and cultured bovine RPE cells can be used for RPE transplantation studies. The ability to create an RPE storage bank as a source of donor cells may result in several clinical advantages.