Toshinori Murata

The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization

Choroidal neovascularization (CNV) is directly related to visual loss in some eye diseases, such as age‐related macular degeneration. Although several human histological studies have suggested the participation of macrophages in CNV formation, the precise mechanisms are still not fully understood. In this study, we elucidated the role of ocular‐infiltrating macrophages in experimental CNV using CCR2 knockout (KO) mice, wild‐type mice, and C57BL/6 (B6) mice. CCR2 is the receptor of monocyte chemoattractant protein‐1, and the number of infiltrating macrophage and the area of CNV were significantly reduced in CCR2 KO mice. Enriched ocular‐infiltrating macrophages from B6 mice actually showed angiogenic ability in a dorsal air sac assay. Moreover, their expression of class II, CD40, B7‐1 and B7‐2 molecules, and the mRNA for potential angiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor α, was also observed. Collectively, we conclude that ocular‐infiltrating macrophages play an important role in CNV generation.

Vascular Endothelial Growth Factor Plays a Role in Hyperpermeability of Diabetic Retinal Vessels

In rat diabetic retinas, we immunohistochemically looked for vascular endothelial growth factor (VEGF) which is also known as vascular permeability factor (VPF). In nondiabetic retinas, VEGF immunoreactivity was weak and restricted to the nerve fiber and ganglion cell layers. On the other hand, in diabetic retinas, VEGF immunoreactivity was markedly increased and was observed in all layers of the retina, especially in the perivascular area. Hyperpermeability of these vessels was confirmed by immunohistochemically detecting extravasation of albumin. These findings indicate that vascular endothelial growth factor plays an important role in blood-retinal barrier breakdown in diabetic retinopathy.

The relationship between accumulation of advanced glycation end products and expression of vascular endothelial growth factor in human diabetic retinas

Summary Both advanced glycation end products and vascular endothelial growth factor are believed to play a role in the pathogenesis of diabetic retinopathy. It is known that vascular endothelial growth factor causes retinal neovascularization and a breakdown of the blood-retinal barrier; how advanced glycation end products affect the retina, however, remains largely unclear. The substance Ne-(carboxymethyl)lysine is a major immunologic epitope, i. e. a dominant advanced glycation end products antigen. We generated an anti-Ne-(carboxymethyl)lysine antibody to investigate the relationship between the localization of advanced glycation end products and that of vascular endothelial growth factor in 27 human diabetic retinas by immunohistochemistry. Nine control retinas were also examined. In all 27 diabetic retinas, Ne-(carboxymethyl)lysine was located in the thickened vascular wall. In 19 of the 27 retinas, strand-shaped Ne-(carboxymethyl)lysine immunoreactivity was also observed around the vessels. In all 27 diabetic retinas, vascular endothelial growth factor revealed a distribution pattern similar to that of Ne-(carboxymethyl)lysine. Vascular endothelial growth factor was also located in the vascular wall and in the perivascular area. Neither Ne-(carboxymethyl)lysine nor vascular endothelial growth factor immunoreactivity was detected in the 9 control retinas. Vessels with positive immunoreactivity for Ne-(carboxymethyl)lysine and/or vascular endothelial growth factor were counted. A general association was noted between accumulation of Ne-(carboxymethyl)lysine and expression of vascular endothelial growth factor in the eyes with non-proliferative diabetic retinopathy (p < 0.01) and proliferative diabetic retinopathy (p < 0.05). [Diabetologia (1997) 40: 764–769]

Relocalization of Apoptosis-Inducing Factor in Photoreceptor Apoptosis Induced by Retinal Detachment in Vivo

Apoptosis-inducing factor (AIF) is a novel mediator in apoptosis. AIF is a flavoprotein that is normally confined to the mitochondrial intermembrane space, yet translocates to the nucleus in several in vitro models of apoptosis. To investigate the role of AIF in the apoptotic process in vivo, we induced retinal detachment (RD) by subretinal injection of sodium hyaluronate, either in Brown Norway rats or in C3H mice. Apoptotic DNA fragmentation, as determined by terminal nick-end labeling, was most prominent 3 days after RD. The subcellular localization of AIF was examined by immunohistochemistry and immunoelectron microscopy. In normal photoreceptor cells, AIF was present in the mitochondrion-rich inner segment. However, AIF was found in the nucleus after RD. Photoreceptor apoptosis developed similarly in C3H control mice, and in mice bearing the gld or lpr mutations, indicating that cell death occurs independently from the CD95/CD95 ligand system. Both the mitochondrio-nuclear transition of AIF localization and the nuclear DNA fragmentation were inhibited by subretinal application of brain-derived neurotrophic factor. To our knowledge, this is the first description of AIF relocalization occurring in a clinically relevant, in vivo model of apoptosis.

Ultrastructure of the vitreoretinal interface following the removal of the internal limiting membrane using indocyanine green

Purpose. To investigate the morphological changes of the vitreoretinal interface following the removal of the internal limiting membrane (ILM) using indocyanine green (ICG). Methods. In 10 primate eyes, a pars plana vitrectomy was performed followed by ICG-assisted peeling of the ILM. Morphological changes in the exposed inner surface of the retina were investigated by electronmicroscopy immediately, 3, 6 and 12 months after the ILM removal. Results. The excised ILM was associated with fragments of glial tissue. The Muller cell processes were damaged and removed at the corresponding region of the retina. Regenerative spindle-shaped Muller cell processes were observed focally showing a meshwork like configuration at 3 and 6 months. Flattened and stretched processes were observed at 12 months; however, there was no apparent ILM regeneration. Conclusions. ICG assisted ILM peeling was observed to cause mild damage to the vitreoretinal interface, which did not completely recover within 12 months.

The relative contributions of each subset of ocular infiltrated cells in experimental choroidal neovascularisation

Aim: Choroidal neovascularisation (CNV) is a major cause of blindness in adults. The aim of this study was to investigate the role of infiltrating cells in the development of experimental CNV. Methods: CNV was induced in C57BL/6 (B6) mice by laser photocoagulation (PC). After PC, the numbers of each subset of infiltrated cells were analysed by flow cytometry at multiple time points. Each subset (except for macrophages) was depleted by the specific antibodies in vivo. Thereafter, the area of CNV was compared between the control B6 mice and the specific antibody treated mice 7 days after PC. The CNV formation in neutrophil depleted CC chemokine receptor-2 (CCR2) knockout mice was also examined to minimise the effects of macrophages. Results: In the early phase of CNV formation, a large number of neutrophils and macrophages infiltrated to the eyes. Natural killer (NK) cells and T lymphocytes were barely detected while no B lymphocytes were detected. The CNV areas did not significantly change compared between the control B6 mice and the specific antibody treated mice. However, the neutrophil depleted CCR2KO mice resulted in a reduction of CNV. Conclusion: Although lymphocytes and NK cells had little effect on CNV formation, neutrophils partially contributed to CNV in the absence of macrophages.

Immunohistochemical detection of extravasated fibrinogen (fibrin) in human diabetic retina.

In diabetic retinopathy, breakdown of the blood-retinal barrier is an early functional disorder that can cause retinal edema, which in turn results in visual disturbance. Hard exudates, composed mainly of lipid and proteinaceous material, are one sign of chronic retinal edema caused by long-standing leakage from the vessels due to breakdown of the blood-retinal barrier. Utilizing diabetic retinas in which hard exudates were present, we performed immunohistochemical staining for fibrinogen. Because fibrinogen is a serum protein, its extravascular localization implies the existence of blood-retinal barrier breakdown. Our studies showed that the extravasated fibrinogen from blood-retinal barrier breakdown accumulated in the hard exudates and in areas of hemorrhage found primarily in the outer plexiform layer and was then phagocytosed by macrophages.

Abnormal retinal vascular development in IL-18 knockout mice

Recent studies have indicated that interleukin 18 (IL-18) might act as either an angiogenic or an angiostatic factor, but the true function of this protein in vascular development is unclear. We therefore investigated the role of IL-18 in the formation of retinal vessels. Development of the retinal vasculature was compared in IL-18 knockout (KO) and wild-type (WT) mice at several different time points. The formation of vessels was evaluated using angiography of flat-mounted retinal samples after inoculation with fluorescein dextran. Retinal samples from both groups were also evaluated through histological examinations, and the expression of angiogenic factors was examined using the reverse-transcription-polymerase chain reaction. The capillary retinal vessels in both WT and IL-18 KO mice had reached the peripheral retina by postnatal day (P) 7. However, IL-18 KO mice showed angiectasis and vascular leakage at P7, especially in the mid-peripheral retina. These symptoms were not observed in WT mice at any stage. Histopathological analysis confirmed abnormal vascular formation in IL-18 KO mice at P14. Interestingly, these abnormalities regressed over time and had disappeared by P84. Several angiogenesis-associated factors, including vascular endothelial growth factor (VEGF), basic fibroblast-growth factor (bFGF), platelet-derived growth factor (PDGF) and pigment epithelium-derived factor (PEDF), were overexpressed in the retinas of IL-18 KO mice compared with those of WT mice at P14. Interferon-γ was detected only in WT mouse retinas at P14. These results provide new evidence for the role of IL-18 in retinal vascular development.

Peripheral Choriovitreal Neovascularization in Proliferative Diabetic Retinopathy: Histopathologic and Ultrastructural Study

We describe the histopathologic and ultrastructural evidence of choriovitreal neovascularization in the peripheral fundus of a non-vitrectomized eye with proliferative diabetic retinopathy (PDR). One eye with PDR was surgically enucleated because of neovascular glaucoma and studied with light and electron microscopy. The eye had neovascular membranes at the ora serrata of the peripheral fundus. The newly formed vessels originated from the choroid, passed through Bruch’s membrane and the retina, and extended into the vitreous. These vessels had either developing or mature characteristics. The endothelial cells of the developing vessels contained a bulky cytoplasm with many intracytoplasmic filaments, ribosomes and rough endoplasmic reticulum. Budding endothelial cells were frequently found in the developing vessels. The endothelial cells of the mature vessels had attenuated cytoplasm and fenestrations with diaphragms. These observations suggest that choriovitreal neovascularization in the peripheral fundus is one of the features of PDR.

The diagnostic use of low molecular weight keratin expression in sebaceous carcinoma.

Sebaceous carcinoma is an infrequent skin tumor and its histological features sometimes closely resemble those of squamous cell carcinoma (SCC) and basal cell epithelioma (BCE), which often leads to a misdiagnosis. In the present immunohistochemical study, however, sebaceous carcinoma exhibited quite a different expression of keratins from SCC and BCE. We immunohistochemically examined 26 excised specimens of sebaceous carcinoma, 10 of SCC and 12 of BCE of the eyelids, using two monoclonal antibodies against high molecular weight keratins, 34 beta B4 (68kd) and 34 beta E12 (56kd, 56.5kd, 58kd), and two monoclonal antibodies against low molecular weight keratins, 35 beta H11 (54kd) and CAM5.2 (39kd, 43kd, 50kd). The cases of sebaceous carcinoma were positive with 34 beta B4 (23%), 34 beta E12 (54%), 35 beta H11 (81%) and CAM5.2 (73%). Of the four anti-keratin antibodies used in this study, 35 beta H11 was negative in all cases of SCC or BCE. These findings indicate that when sebaceous carcinoma is suspected, but no fat staining appropriate materials are available, a monoclonal antibody against low molecular weight keratin, 35 beta H11 (54kd), can be a useful tool to immunohistochemically rule out both SCC and BCE.