Haruhiko Yamada

Bone Marrow-Derived Stem Cells Can Differentiate into Retinal Cells in Injured Rat Retina

It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether bone marrow stem cells can differentiate into retinal neural cells in adult rats. PKH‐67‐labeled stem cell‐enriched bone marrow cells (BMCs) were injected into the vitreous space of eyes in which the retinas had been mechanically injured using a hooked needle. Two weeks after the injection of these cells, immunohistochemical examinations were carried out. The stem cell‐enriched BMCs had been incorporated and had differentiated into retinal neural cells in the injured retina. The stem cell‐enriched BMCs had accumulated mainly in the outer nuclear layer around the injured sites. The incorporated cells expressed glial fibrillary acidic protein, calbindin, rhodopsin, and vimentin. These results raise the possibility that stem cell‐enriched BMCs have the ability to differentiate into retinal neural cells, and that the injection of stem cell‐enriched BMCs into the retina would help repair damaged retinal cells.

Spontaneous closure of traumatic Macular hole

PURPOSE To investigate the mechanism by which traumatic macular hole closes spontaneously. DESIGN Consecutive observational case series of three patients with unilateral traumatic macular hole who consulted medical staff at the Kansai Medical University between 1997 and 2000. METHODS Three patients who sustained unilateral blunt trauma to the eye and developed traumatic macular hole were followed with ophthalmic examination, fundus photography, fluorescein angiography, and optical coherence tomography (OCT). RESULTS Case 1 was an 11-year-old boy. He had neither a macular hole nor prominent macular edema at his first consultation, but a macular hole opened 3 weeks later. OCT showed macular edema and a full-thickness macular hole. The tissue around the edge of the macular hole protruded inward toward the center and finally closed spontaneously 18 weeks later. Case 2 was a 19-year-old man. He had a tiny rough-edged macular hole at his first consultation with a local ophthalmologist. OCT showed macular hole enlargement and worsening of the macular edema during follow up. The macular hole finally closed 4 months after injury. Case 3 was a 15-year-old boy. He had a tiny rough-edged macular hole at his first consultation with a local ophthalmologist. The macular hole finally closed 6 months after injury. CONCLUSION Macular hole can be a result of severe damage from ocular concussion or damage to the retina. For 6 months following injury, traumatic macular hole should probably be observed rather than surgically repaired, because of the possibility that the macular hole may close spontaneously.

Expression of basic fibroblast growth factor mRNA in developing choroidal neovascularization

PURPOSE Basic fibroblast growth factor (bFGF) is an angiogenic peptide that may be important in the pathogenesis of choroidal neovascularization. We attempted to determine the transcription of the bFGF gene during the development of experimentally induced choroidal neovascularization. METHODS Rat bFGF cDNA was inserted in the pBluescript to prepare antisense and sense riboprobes. Multiple krypton laser burns were applied to the posterior poles of the eyes of pigmented rats according to a protocol described for producing subretinal neovascularization in these animals. At intervals of up to 4 weeks after photocoagulation, the eyes were removed and cut into thin sections. The sections were subjected to histopathological analysis, cell proliferation study, or in situ hybridization with digoxigenin (DIG)-labeled single-strand riboprobes synthesized from rat bFGF cDNA. RESULTS In normal adult rat retinas, bFGF mRNA expression was mainly observed in the ganglion cell layer and the inner nuclear layer. After laser photocoagulation, proliferation of RPE cells, fibroblast-like cells and cells in the choroid in the lesions were observed. Expression of bFGF mRNA was observed in the lesions 3 days to 2 weeks after laser treatment. Signals of bFGF mRNA were detected in the proliferating RPE-like cells, choroidal vascular endothelial cells and fibroblast-like cells, all of which are essential for neovascularization. However, bFGF mRNA expression was no longer detectable in these cells 4 weeks after photocoagulation. CONCLUSIONS Our findings indicate that bFGF is normally transcribed in ganglion cells and the inner nuclear cell layer. During the neovascularization that followed laser photocoagulation, bFGF mRNA expression was detected within the laser lesions. It is thus probable that bFGF acts as a mediator in the neovascularization process.

Choroidal Neovascularization Is Provided by Bone Marrow Cells

Choroidal neovascularization (CNV) is a known cause of age‐related macular degeneration (ARMD). Moreover, the most common cause of blindness in the elderly in advanced countries is ARMD with CNV. It has recently been shown that bone marrow cells (BMCs) can differentiate into various cell lineages in vitro and in vivo. Adults maintain a reservoir of hematopoietic stem cells included in BMCs that can enter the circulation to reach various organs in need of regeneration. It has recently been reported that endothelial progenitor cells (EPCs) included in BMCs are associated with neovascularization. We examine the role of BMCs in CNV using a model of CNV in adult mice. Using methods consisting of fractionated irradiation (6.0 Gy × 2) followed by bone marrow transplantation (BMT), adult mice were engrafted with whole BMCs isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP). Three months after BMT, we confirmed that the hematopoietic cells in the recipients had been completely replaced with donor cells. We then carried out laser photocoagulation to induce CNV in chimeric mice (donor cells >95%). Two weeks after the laser photocoagulation, by which time CNV had occurred, immunohistochemical examination was carried out. The vascular wall cells of the CNV expressed both EGFP and CD31. These findings indicate that newly developed blood vessels in the CNV are derived from the BMCs and suggest that the inhibition of EPC mobilization from the bone marrow to the eyes could be a new approach to the fundamental treatment of CNV in ARMD.

Retinal neovascularisation without ischaemia in the spontaneously diabetic Torii rat

Aims/hypothesisThe spontaneously diabetic Torii (SDT) rat has recently been established as a model of type 2 human diabetes mellitus. Male SDT rats develop severe diabetic ocular complications. This study investigated the nature of the ocular complications in this model and addressed the question of whether the SDT rat is a good model of human proliferative diabetic retinopathy.MethodsMale SDT rats aged 50 weeks were studied for a period of 8 months. Under deep anaesthesia, one eye of each animal was enucleated following perfusion with fluorescein dextran and a retinal flat mount was prepared to study vascular structure. The other eye was enucleated and investigated histologically by haematoxylin–eosin and azan staining and by immunohistochemistry using antibodies against vascular endothelium (Griffonia simplicifolia isolectin B4 antibody) and vascular endothelial growth factor (VEGF).ResultsFrom the vascular structure study, 17 of 32 rats (53%) showed proliferative retinopathy without vascular non-perfusion. The histological study revealed traction retinal folds in rats with proliferative retinopathy. Azan staining showed some proliferative matrix in rats with normal retinal structure and those with proliferative retinopathy compared with normoglycaemic controls. Staining with Griffonia simplicifolia isolectin B4 antibody showed no specific vascular changes in any of the rats, while VEGF staining revealed higher immunoreactivity in the retina of rats with normal retinal structure and those with proliferative retinopathy, but only low immunoreactivity in the control animals.Conclusions/interpretationThere appear to be differences between the SDT rat model of diabetic retinopathy and human proliferative diabetic retinopathy, as the SDT rat develops retinal neovascularisation without retinal ischaemia. This very unique display of ocular neovascularisation may be caused by increased expression of VEGF.

Immunolocalization of basic fibroblast growth factor during wound repair in rat retina after laser photocoagulation.

Abstract•Background: Basic fibroblast growth factor (bFGF) stimulates the mitogenesis of various cells and plays a key role in wound repair. We studied the immunohistochemical localization of bFGF during wound repair in the rat retina after laser photocoagulation.• Methods: Krypton laser photocoagulation was performed on the eyes of pigmented rats. The eyes were enucleated on days 1, 3, 7, 14 and 28 after the photocoagulation, and the immunohistochemical localization of bFGF was assessed. Two different monoclonal antibodies and one polyclonal antibody against bFGF as first antibodies were used.• Results: Marked immunoreactivity for bFGF was found in the ganglion cell layer, and weak immunoreactivity for bFGF was found in the retinal pigment epithelial (RPE) cells of the normal adult rat retina. On day 3 after laser photocoagulation, the nuclei and cytoplasm of proliferating RPE cells at the center of the photocoagulated lesion showed intense bFGF immunoreactivity. The nuclei of RPE cells around the lesion showed intense bFGF immunoreactivity. Macrophages that migrated into the lesion showed positive staining for bFGF. These immunoreactivity decreased with time. Controls (0.05 M Tris-HCl buffer, normal serum, or these same antibodies preabsorbed with bFGF) did not show positive staining.• Conclusion: The finding of an elevated expression of bFGF immunoreactivity in the photocoagulated lesion suggests that bFGF may play a role in wound repair in the rat retina after laser photocoagulation.

Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. By using a retrovirus-mediated signal sequence trap method, we identified a previously undescribed gene, termed ARIA (apoptosis regulator through modulating IAP expression), which regulates endothelial apoptosis and angiogenesis. ARIA was expressed in blood vessels during mouse embryogenesis, as well as in endothelial cells both in vitro and in vivo. ARIA is a unique protein with no homology to previously reported conserved domain structures. Knockdown of ARIA in HUVECs by using small interfering RNA significantly reduced endothelial apoptosis without affecting either cell migration or proliferation. ARIA knockdown significantly increased inhibitor of apoptosis (cIAP)-1 and cIAP-2 protein expression, although their mRNA expression was not changed. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the antiapoptotic effect of ARIA knockdown. Using yeast 2-hybrid screening, we identified the interaction of ARIA with 20S proteasome subunit α-7. Thereafter, we found that cIAP-1 and cIAP-2 were degraded by proteasomes in endothelial cells under normal condition. Overexpression of ARIA significantly reduced cIAP-1 expression, and this reduction was abolished by proteasomal inhibition in BAECs. Also, knockdown of ARIA demonstrated an effect similar to proteasomal inhibition with respect to not only expression but also subcellular localization of cIAP-1 and cIAP-2. In vivo angiogenesis studied by Matrigel-plug assay, mouse ischemic retinopathy model, and tumor xenograft model was significantly enhanced by ARIA knockdown. Together, our data indicate that ARIA is a unique factor regulating endothelial apoptosis, as well as angiogenesis, presumably through modulating proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells.

Immunolocalization of transforming growth factor β during wound repair in rat retina after laser photocoagulation

Abstract• Background: Scatter photocoagulation induces regression of retinal neovascularization, but the mechanism of its therapeutic effect is incompletely understood. To elucidate the mechanism of therapeutic effect of photocoagulation is the main focus of our research. We have already demonstrated basic fibroblast growth factor (bFGF) immunolocalization during retinal wound repair following laser photocoagulation. Transforming growth factor beta (TGF β) reportedly inhibits endothelial cell growth and bFGF-induced cell proliferation in vitro. In the present study, we evaluated the immunohistochemical localization of TGF-β1 and -β2 during wound repair in the rat retina following laser photocoagulation. • Methods: Krypton laser photocoagulation was performed on the eyes of pigmented rats. The eyes were then enucleated on day 1, 3, 7, 14, 28 or 56 following the photocoagulation and enrolled into the analysis of immunohistochemical localization of TGF-β1 and -β2. • Results: Immunoreactivity for TGF-β1 and -β2 was present in the ganglion cell layer and photoreceptor outer segments of the normal adult rat retina. The cytoplasm of RPE cells at the photocoagulated lesion showed intense TGF-β1 and -β2 immunoreactivity on day 3 after laser photocoagulation. Macrophages that migrated into the lesion lacked positive staining for TGF-β1 and -β2. TGF-β immunoreactivity in RPE cells continued to be upregulated for more than 1 month compared with that in normal RPE cells. Controls did not exhibit any positive staining. • Conclusion: An elevated expression of TGF-β immunoreactivity for a longer period of time than bFGF was observed in RPE cells at the photocoagulated lesion in vivo. In the late phase of retinal wound repair, TGF-β may inhibit cell proliferation induced by mitogens, introduce an end stage of cellular events, and induce extracellular matrix induction.

Expression of basic fibroblast growth factor and its receptor mRNA in retinal tissue following ischemic injury in the rat

Abstract · Background: Our purpose was to determine the time-dependent changes of expression of basic fibroblast growth factor (bFGF) and its receptor in pressure-induced retinal ischemia in rats. · Methods: Retinal ischemia was induced in Wistar rats by increasing the intraocular pressure to 110 mmHg for 45 min by cannulation into the eyes. At the end of the ischemic period, reperfusion of the retinal vasculature was confirmed. Localization of bFGF and FGF receptor-1 (FGF-R) mRNAs were evaluated by in situ hybridization at various times after reperfusion. The reverse-transcription polymerase chain reaction (RT-PCR) method was used to detect the expression of bFGF and FGF-R mRNA in the sensory retina. · Results: In normal sensory retina, bFGF and FGF-R mRNAs were observed in the ganglion cell layer and inner nuclear layer. bFGF gene expression in the sensory retina increased within 24 h, particularly at 6–12 h. FGF-R gene expression increased earlier than that of bFGF. By RT-PCR, expression of bFGF gene reached a peak at 6–24 h, and FGF-R reached a peak at 3–12 h. These RT-PCR results are comparable to those of in situ hybridization. · Conclusions: These results demonstrate that transient retinal ischemia leads to the induction of bFGF mRNA synthesis, and suggest that bFGF has a protective role, e.g., a defense mechanism for the sensory retina.

Long term follow up in a case of successfully treated idiopathic retinal vasculitis, aneurysms, and neuroretinitis (IRVAN)

The idiopathic retinal vasculitis, aneurysm, and neuroretinitis (IRVAN) syndrome is a rare clinical entity characterised by peripheral retinal vascular occlusion, retinal vasculitis, and multiple posterior retinal arterial aneurysms.1,2 In most reported cases, visual acuity was aggravated.2–4 We report a case of successfully treated IRVAN syndrome in which good long term visual acuity has been maintained. A 36 year old woman visited the Kansai Medical University Hospital on January 1999, complaining of flies flying in her right eye for the past year. She also had visual disturbance and metamorphopsia for 2 months in her right eye. Her best corrected visual acuity was 20/40 in the right eye and 20/16 in the left eye. Inflammatory cells …