Toshiaki Kubota

Clearance of apoptotic photoreceptors: Elimination of apoptotic debris into the subretinal space and macrophage-mediated phagocytosis via phosphatidylserine receptor and integrin αvβ3

The effective phagocytotic clearance of apoptotic debris is fundamental to the maintenance of neural tissues during apoptosis. Retinal photoreceptors undergo apoptosis after retinal detachment. Although their induction phase of apoptosis has been well discussed, their phagocytotic process remains quite unclear. We herein demonstrate that apoptotic photoreceptors are selectively eliminated from their physiological localization, the outer nuclear layer, to the subretinal space, and then phagocytosed by monocyte-derived macrophages. This could be shown by an ultrastructural and immunophenotypic analysis. Moreover, in chimera mice expressing transgenic green fluorescent protein in bone marrow-derived cells, the local infiltration of macrophages could be detected after retinal detachment-induced photoreceptor apoptosis. The local injection of an antibody blocking the phosphatidylserine receptor (PSR) or a peptide (GRGDSP)-blocking integrin αvβ3 revealed that phagocytotic clearance involves the PSR as well as integrin αvβ3 in vivo. Importantly, the level of blockade obtained with these reagents was different. Although anti-PSR increased the frequency of apoptotic cells that fail to bind to macrophages, GRGDSP prevented the engulfment (but not the recognition) of apoptotic photoreceptor cells by macrophages. To our knowledge, this is the first report describing the mechanisms through which apoptotic photoreceptors are selectively eliminated via a directional process in the subretinal space.

Phacoemulsification and Intraocular Lens Implantation for Angle Closure Glaucoma after the Relief of Pupillary Block

Purpose: We studied the surgical outcomes of phacoemulsification and intraocular lens (IOL) implantation for cataract and/or uncontrolled intraocular pressure (IOP) in eyes with angle closure glaucoma. Setting: Department of Ophthalmology, National Nagasaki Medical Center, Nagasaki, Japan. Methods: Eighteen eyes from 15 patients after laser iridotomy (17 eyes) or peripheral iridectomy (1 eye) had undergone surgery and were studied. We used an iris retractor in 7 eyes due to insufficient mydriasis and a capsular tension ring in 2 eyes due to phacodonesis during the operation. The patients were followed up for at least 6 months (13.8 ± 7.2 months; range: 6–36 months). Results: The mean IOP significantly decreased from 17.4 ± 8.1 to 13.5 ± 3.3 mm Hg at 6 months after surgery. The IOP was below 21 mm Hg in all eyes. The visual acuity was not worsened in any eyes and became better than 2 Snellen lines in 14 eyes. The corneal endothelial cell count decreased from 2,365 ± 517 to 1,960 ± 661/mm2 (18.3 ± 17.2%). Conclusions: Phacoemulsification and IOL implantation is useful in IOP control for angle closure glaucoma after relief of pupillary block. However, we should take care of operative complications because of a shallow anterior chamber, poor mydriasis and zonular weakness.

Aqueous and vitreous penetration of levofloxacin after topical and/or oral administration

Purpose. To investigate the aqueous and vitreous penetration of levofloxacin, the drug was administered topically and/or orally to patients undergoing vitrectomy. Methods. Thirty-six patients undergoing initial vitrectomy with phacoemulsification and aspiration (PEA) were enrolled, and were divided randomly into three groups. Group 1 was treated with topical application of levofloxacin (three times on the day before surgery and seven times on the day of surgery), Group 2 received oral administration of levofloxacin (200 mg twice on the day before surgery and 200 mg at 3 hours before surgery), and Group 3 received both topical and oral levofloxacin according to the above schedules. The concentration of levofloxacin was measured in aqueous humor and vitreous fluid samples obtained during surgery. Results. In Groups 1, 2, and 3, the mean levofloxacin concentration in aqueous humor was 0.765±0.624 μg/mL, 1.279±0.440 μg/mL, and 1.823±0.490 μg/mL, respectively, while the mean levofloxacin concentration in vitreous fluid was <0.02 μg/mL, 1.455±0.445 μg/mL, and 1.369±0.530 μg/mL, respectively. Conclusions. Oral administration of levofloxacin at a dose of 400 mg/day was sufficient for the prophylaxis of ocular infections, because the drug concentrations in both aqueous humor and vitreous fluid were higher than the MIC90 values for major ocular pathogens. Topical application of levofloxacin achieved adequate drug levels in aqueous humor, but not in vitreous fluid, while combined topical and oral administration had an additive effect on the drug concentration in aqueous humor.

Fibrinogen stimulates in vitro angiogenesis by choroidal endothelial cells via autocrine VEGF.

AbstractsBackgroundThe purpose of this study is to investigate the effect of fibrinogen on angiogenesis in vitro formed by cultured bovine choroidal endothelial cells (BCECs) and the involvement of vascular endothelial growth factor (VEGF) in this mechanism.MethodsFor in vitro tube formation assay, BCECs were seeded on collagen gel containing fibrinogen (0–1.5xa0mg/ml). After 3xa0days of cultivation, the total length of the tubular structure was measured using Macscope Analyzer. Total RNA and conditioned media were collected after fibrinogen treatment and subjected to Northern and Western blot analyses, respectively. Transcription factor HIF-1α was also analyzed by Western blot analysis using cytosolic and nuclear fraction of BCECs. Involvement of VEGF in fibrinogen-dependent in vitro tube formation was evaluated using anti-VEGF neutralizing antibody or VEGF receptor 2-selective inhibitor (SU5416).ResultsFormation of the tubular structure was enhanced 20~50 times in fibrinogen-containing gel in a concentration-dependent manner. The treatment of BCECs with fibrinogen resulted in a significant increase in VEGF gene and protein expression. Accumulation of HIF-1α protein in the nuclear fraction was also detected after the treatment with fibrinogen. Finally, fibrinogen-induced tube formation was significantly inhibited in the presence of anti-VEGF-neutralizing antibody (52.0% inhibition at the concentration of 1xa0μg/ml, P<0.05) or SU5416 (54.8% inhibition at the concentration of 3xa0μΜ,nP<0.05).ConclusionsExtravasated fibrinogen might play an important role in the development of choroidal neovascularization associated with age-related macular degeneration, at least in part, through the function of VEGF in an autocrine manner. Transcription factor HIF-1 appears to be involved in fibrinogen-induced VEGF expression.

The ultrastructure of parapapillary chorioretinal atrophy in eyes with secondary angle closure glaucoma

Abstract• Background: The present study was performed to investigate the ultrastructure of deep retinal layers and choroid corresponding to the parapapillary chorioretinal atrophy in eyes with secondary angle-closure glaucoma.• Methods: The glaucomatous eyes included two eyes enucleated due to iris ring melanoma with high intraocular pressure and one eye with neovascular glaucoma enucleated due to ocular pain. The control eyes included one eye enucleated due to choroidal malignant melanoma with normal intraocular pressure and one eye enucleated during surgery for supramandibular carcinoma. These eyes were studied with light and electron microscopy.• Results: In the region of parapapillary chorioretinal atrophy of glaucomatous eyes, the retinal pigment epithelial cells showed degenerative changes, such as loss of basal in foldings and microvilli, degenerated mitochondria, vacuolar degeneration and irregular distribution of melanin granules. The photoreceptors were decreased in number in this area of glaucomatous eyes. The lumen of the choriocapillary vessels adjacent to the optic nerve was collapsed.• Conclusion: These results elucidate the fine structures of deep retina and choroid in the region of parapapillary chorioretinal atrophy of glaucomatous eyes, and suggest that the reduced choroidal perfusion might be the pathogenetic mechanism of glaucomatous parapapillary chorioretinal atrophy.

Cytopathology of early cellular reaction on implant lenses in monkeys A transmission electron microscopic study

We carried out extracapsular lens extraction with the implantation of a modified C-loop posterior chamber lens in eight monkey eyes. The animals were killed after varying survival of up to 14 days. The intraocular lenses (IOLs) were removed and examined by transmission electron microscopy. Three different types of cells, namely, polymorphonuclear leukocytes, macrophages, and multinucleated giant cells, were observed on the surface of the IOLs. A thin, membrane-like structure composed of finely granular material covered the lens surface. These cell components were not attached directly to the lens surface but, rather, were located on the membrane-like structure.

Functional role of Egr-1 mediating VEGF-induced tissue factor expression in the retinal capillary endothelium

AbstractnPurpose. To investigate the causal relationship between VEGF and tissue factor (TF) expression, and its intracellular signaling in the retinal capillary endothelium both in vitro and in vivo.nMethods. TF mRNA and protein expression in cultured bovine retinal capillary endothelial cells (BRECs) were detected by RT-PCR and western blotting. The expression and subcellular localization of Egr-1 were analyzed by immunocytochemistry and western blotting. Involvement of p44/p42 MAPK pathway in this signaling was assessed using PD98059. Electrophoretic mobility shift assay (EMSA) was performed using human TF Egr-1/Sp-1 overlapping promoter region (–85 to –70). Decoy oligonucleotide was transfected into BRECs to clarify the critical transcription factor mediating VEGF-induced TF gene expression. To evaluate the importance of GC rich region in VEGF-induced TF protein expression in rat retinas, Mithramycin was intraperitoneally administered.nResults. VEGF stimulated TF mRNA and protein expression in cultured BRECs, reaching maximal effect after 4xa0h and 10xa0h, respectively. VEGF activated transcription factor Egr-1 within 60xa0min. Inactivation of Egr-1 by PD98059 resulted in the prohibition of VEGF-induced TF gene expression. EMSA revealed the increment of Egr-1 binding with TF promoter region by displacing Sp1 after treatment with VEGF. Transfection of the Egr-1/Sp-1 overlapping decoy into BRECs inhibited VEGF-dependent TF gene expression. Mithramycin almost completely suppressed VEGF-induced TF protein expression in retinal capillary system in vivo (80%, p<0.01).nConclusion. Transcription factor Egr-1, which lies downstream of p44/p42 MAPK, critically mediates VEGF-dependent TF expression in the retinal capillary endothelium.

Neovascular tissue in the intertrabecular spaces in eyes with neovascular glaucoma.

AIMS/BACKGROUND: To investigate histological changes in the trabecular meshwork in eyes with neovascular glaucoma. METHODS: Light and electron microscopic studies were carried out on the trabecular meshwork of three enucleated eyes with neovascular glaucoma. The presence and distribution of factor VIII in the trabecular meshwork was assessed using the ABC method. RESULTS: Peripheral anterior synechiae covering the trabecular meshwork were detected in two eyes, which would explain the rise in intraocular pressure. In the third the angle was not completely closed by peripheral anterior synechiae. The spaces between the trabecular beams were lined by a single layer of vascular endothelium, and were filled with red blood cells in this patient. Factor VIII was positively stained in the endothelial cells, lining both these spaces and Schlemms canal. A basal lamina and microfibrils were detected just beneath the newly formed vascular endothelial cells. CONCLUSION: The neovascular tissue found in the trabecular spaces might be one of the factors responsible for intraocular pressure elevation in eyes with neovascular glaucoma.

Lamellar excimer laser keratoplasty: Reproducible photoablation of corneal tissue

We examined the depth of ablation of the recipient bed with different counts of oscillations of excimer laser beam, to determine the correlation between planned and real depth. The ablation rate per oscillation was tested preoperatively by blackened photographic paper of defined thickness and thus was calculated to be 5 μm. Forty pig eyes were used for the first study. Each eight eyes were ablated in the planned depth 100 μm, 200 μm, 300 μm, 400 μm and 500 μm. The corneal thickness was measured with an ultrasonic pachymeter before and after the procedure. The depth measured after the photoablation was 99.4 ± 36.4 μm for 100 μm planned depth, 186.7 ± 55.3 μm for 200 μm, 298.4 ± 68.5 μm for 300 μm, 373.9 ± 65.7 μm for 400 μm and 480.1 ± 59.3 μm for 500 μm. Comparing the depth measured after the photoablation to planned depth, there was a significant correlation (correlation coefficient: R = 0.93; p < 0.0001). Five other corneas trephinated from pig cadaver eyes were ablated from the endothelial side to the desired thickness (100 to 500 μm) of lamellar graft. In a second step a donor mask was placed onto the cornea and a laser light spot was led until perforating on all sides. The lamellar keratoplasty was completed by suturing the corneal graft into the bed. Macroscopic and microscopic examination of sutured eyes after fixation showed a good fit of wound margins and stromal interface. These results indicate that excimer laser is useful for reproducible corneal photoablation in lamellar keratoplasty.

Histopathological changes in iridocorneal angle of inherited glaucoma in rabbits.

Abstractu2002· Background: We examined morphologically the angular region of eyes affected by inherited glaucoma in rabbits genetically developed by crossbreeding in order to investigate the etiologic changes in the iridocorneal angle and to establish whether this strain of rabbit is a suitable animal model of goniodysgenetic glaucoma in humans. · Methods: The angular regions of both normal and glaucomatous eyes from four rabbits having unilateral inherited glaucoma were observed with light and electron microscopy. · Results: In the glaucomatous eyes angular region, the aqueous plexus corresponding to Schlemm’s canal in humans was open and located far peripherally to the peripheral margin of the anterior chamber angle, although the plexus of one glaucomatous eye was poorly developed with a small lumen. In the angular meshwork, which corresponds to the trabecular meshwork in humans, a thick abnormal tissue with round cells embedded in the extracellular matrix was located just beneath the plexus. A large amount of extracellular matrix of basal lamina-like material was observed in the thick tissue. In the normal eyes, the angular region consisted of well-developed trabecular sheets with neither a thick tissue nor accumulations of extracellular matrix in the angular meshwork. · Conclusion: The findings observed in the glaucomatous eyes are much the same as those observed in goniodysgenetic glaucoma in humans, suggesting that this strain of inherited glaucoma rabbits is a suitable animal model of goniodysgenetic glaucoma in humans. The present study also supports the hypothesis that the presence of a thick subcanalicular tissue due to maldevelopment of the iridocorneal angle is one of the main causes of this type of glaucoma.