Jie Hyun Kim

Retinal ganglion cell death induced by endoplasmic reticulum stress in a chronic glaucoma model

This study investigated whether endoplasmic reticulum (ER) stress induced retinal ganglion cell (RGC) death in chronic ocular hypertension, one of the RGC death mechanisms, using an experimental glaucoma rat model. Glaucoma was induced in adult male Sprague-Dawley rats by cauterizing three episcleral veins. The intraocular pressure (IOP) remained elevated in the cauterized eyes for the 8-week experiment, whereas it was not elevated in the contralateral control eyes. The average number of RGCs decreased significantly, and TUNEL-positive cells were detected in the ganglion cell layer. In western blotting, Bip, the phosphorylated form of PKR (p-PERK), and C/EBP-homologous protein (CHOP) were significantly expressed at 1 or 2 weeks, and this persisted throughout the 8-week experiment. Phosphorylated eukaryotic initiation factor 2 (p-eIF2alpha) began to increase at 1 week, was sustained through 4 weeks, and decreased slightly at 8 weeks. In cauterized eyes, strong p-PERK and CHOP immunoreactivity was observed in ganglion cells after 8 weeks of IOP elevation. Taken together, in the experimental chronic glaucoma model, ER stress is involved in RGC death, and the PERK-p-eIF2alpha-CHOP pathway plays a role in the RGC apoptosis associated with ER stress. This might be a good therapeutic target to protect RGCs from ER stress injury in glaucoma.

Retinal glial cell responses and Fas/FasL activation in rats with chronic ocular hypertension.

Responses in the retina post injury provoke glial reactions that are not completely understood. This study investigated the reaction of retinal glial cells and the expression and localization of the Fas and Fas-ligand (FasL) in rats with chronic ocular hypertension. Experimental glaucoma was induced in one eye of 60 Sprague-Dawley rats by cauterizing three episcleral vessels. It caused a moderate intraocular pressure (IOP) elevation and significant retinal ganglion cell (RGC) loss for at least 6 weeks in all animals. Immunohistochemical analysis revealed that the expression of GFAP and OX-42 increased in the injured retinae. Fas/FasL immunoreactivity was elevated in the microglia, and we also observed an incremental increase in Fas associated death domain (FADD) immunoreactivity in Müller glial cells and RGCs in the IOP-elevated retinae. The activation of glial cells and upregulation of Fas and FasL suggest that glial cells may contribute to Fas-mediated cell death in the neurodegeneration process of chronic ocular hypertensive retinal insult.

Neuronal cell death in the inner retina and the influence of vascular endothelial growth factor inhibition in a diabetic rat model.

To inhibit vascular changes in diabetic retinopathy, inhibiting vascular endothelial growth factor (VEGF) has become a mainstay of the treatment of diabetic retinopathy. However, its effects on neuronal cells remain to be elucidated. We aimed to evaluate the effect of VEGF inhibition on neuronal cells in a streptozotocin-induced diabetic rat retina. VEGF inhibition was performed by intravitreal VEGF-A antibody injection. After anti-VEGF treatment, apoptosis in retinal ganglion cells (RGCs) increased, and novel apoptosis in amacrine and bipolar cells of the inner nuclear layer was observed by TUNEL staining. Phosphorylated Akt expression was significantly higher in RGCs but was decreased in neuronal cells of the inner nuclear layer after anti-VEGF treatment by Western blot analysis and immunohistochemical staining. These results demonstrate that VEGF inhibition significantly increased RGC apoptosis and neuronal cell apoptosis in the inner nuclear layer of a diabetic retina, which seems to consist primarily of amacrine and bipolar cells. The phosphorylated Akt pathway, which plays a neuroprotective role via VEGF, was significantly affected by VEGF inhibition in the inner nuclear layer, suggesting that neurotrophic factor deprivation is the main mechanism for neuronal cell death after inhibiting VEGF. The results of this study show that inhibiting VEGF may have detrimental effects on the apoptosis of neuronal cells in the inner layers of the diabetic retina.

Glial cell response and iNOS expression in the optic nerve head and retina of the rat following acute high IOP ischemia–reperfusion

Acute high IOP ischemia-reperfusion induces the loss of retinal ganglion cells, supporting the hypothesis that the condition of ischemia-reperfusion contributes to the induction and progression of glaucoma. This study investigated morphological changes, glial cell response, and expression of inducible nitric oxide synthase (iNOS) in the optic nerve head and retina of the rat following acute high IOP ischemia-reperfusion. A 60-min ischemic period was administered to the rat eye by raising the IOP, followed by a reperfusion period lasting 2, 5, or 7 days. Histological examination showed that acute high IOP ischemia-reperfusion injury produced optic nerve head and retina damage. In immunohistochemical staining, GFAP and OX-45 were limited to the ganglion cell layer (GCL) or inner nuclear layer (INL) of the control retina and increased to nearly all layers of the retina after acute high IOP ischemia-reperfusion. GFAP and OX-42 were detected at the control optic nerve heads and increased after acute high IOP ischemia-reperfusion. After acute high IOP ischemia-reperfusion, expression of iNOS increased, mostly at the GCL and INL of the retina and at the optic nerve head. Western blot analysis showed that expression of iNOS increased significantly, compared with the control, in the retina and optic nerve head after acute high IOP ischemia-reperfusion. Activation of glial cells and the up-regulation of iNOS may contribute to the damage of the retina and optic nerve head of the rat following acute high IOP ischemia-reperfusion.

Stem cell-based delivery of brain-derived neurotrophic factor gene in the rat retina

As an alternative to a viral vector, the application of stem cells to transfer specific genes is under investigation in various organs. Using this strategy may provide more effective method to supply neurotrophic factor to the neurodegenerative diseases caused by neurotrophic factor deprivation. This study investigated the possibility and efficacy of stem cell-based delivery of the brain-derived neurotrophic factor (BDNF) gene to rat retina. Rat BDNF cDNA was transduced into rat bone marrow mesenchymal stem cells (rMSCs) using a retroviral vector. Its incorporation into the experimental rat retina and the expression of BDNF after intravitreal injection or subretinal injection were detected by real-time PCR, western blot analysis, and immunohistochemical staining. For the incorporated rMSCs, retinal-specific marker staining was performed to investigate the changes in morphology and the characteristics of the stem cells. Transduction of the rMSCs by retrovirus was effective, and the transduced rMSCs expressed high levels of the BDNF gene and protein. The subretinal injection of rMSCs produced rMSC migration and incorporation into the rat retina (about 15.7% incorporation rate), and retinal BDNF mRNA and protein expression was increased at 4 weeks after transplantation. When subretinal injection of rMSCs was applied to axotomized rat retina, it significantly increased the expression of BDNF until 4 weeks after transplantation. Some of the transplanted rMSCs exhibited morphological changes, but the retinal-specific marker stain was not sufficient to indicate whether neuronal differentiation had occurred. Using mesenchymal stem cells to deliver the BDNF gene to the retina may provide new treatment for glaucoma.

VEGF Induces TGF-β1 Expression and Myofibroblast Transformation after Glaucoma Surgery

Subconjunctival fibrosis at the surgical site determines the outcome of glaucoma surgery. Myofibroblast transformation has a significant role in fibrosis, and vascular endothelial growth factor (VEGF) is reported to trigger myofibroblast transformation by inducing transforming growth factor (TGF)-β1. In the present study, we used IHC, Western blot analysis, enzyme-linked immunosorbent assay, and electron microscopy to determine the contribution of VEGF to myofibroblast transformation in subconjunctival fibrosis after glaucoma surgery. A rabbit trabeculectomy model was generated, and VEGF stimulation or VEGF inhibition was performed during surgery. VEGF stimulation induced TGF-β1 expression in a dose-dependent manner. Down-regulation of epithelial markers (E-cadherin and β-catenin) and up-regulation of mesenchymal marker (α-smooth muscle actin) were observed in the subconjunctival layers after trabeculectomy with VEGF stimulation. Up-regulations of Smad and Snail, which play a central role in myofibroblast transformation, were observed in the conjunctival and subconjunctival layers at the site of trabeculectomy. Electron microscopy revealed changes of the conjunctival epithelial cells, especially the presence of myofilaments and increased rough endoplasmic reticulum in the cytoplasm. Myofibroblast transformation was activated by VEGF stimulation and decreased by VEGF inhibition. These findings suggest that VEGF potentially affected the TGF-β1/Smad/Snail pathway, thereby triggering myofibroblast transformation. Therapeutic approaches modulating VEGF may control myofibroblast transformation and reduce subconjunctival fibrosis after glaucoma surgery.

Alteration of retinal intrinsic survival signal and effect of alpha2-adrenergic receptor agonist in the retina of the chronic ocular hypertension rat.

The purpose of this study is to examine the retinal expression of intrinsic cell survival molecules and to elucidate the effect of an alpha2-adrenergic receptor agonist in the chronic ocular hypertensive rat model. Chronic ocular hypertension was induced in both eyes of each rat by episcleral vein cauterization. Two five-microliter drops of the selective alpha2-adrenoceptor agonist brimonidine 0.2% (Alphagan; Allergan Inc., Irvine, CA, USA) were topically administered twice daily for up to eight weeks in one eye. The fellow eye received balanced salt solution as a control. Protein and mRNA expression were evaluated at 1, 4, and 8 weeks after injury. Retinal expression of BDNF, Akt, and GFAP was assessed using immunohistochemistry. Retinal levels of mRNA for BDNF, bcl-2, and bcl-xL were determined using semi-quantitative RT-PCR. Retinal ganglion cell (RGC) density was evaluated after retrograde labeling with 4-Di-10-ASP (DiA). A significant decrease in RGC density was observed in ocular hypertensive eyes. Cauterized eyes showed an increase in GFAP expression from one week after injury, and the expression of bcl-2, bcl-xL, and BDNF mRNA was also increased. Treatment of ocular hypertensive eyes with brimonidine resulted in a reduction in RGC loss, a decrease in the level of GFAP immunoreactivity, and an increment in BDNF mRNA and p-Akt expression. Brimonidine appears to protect RGCs from neurodegeneration through mechanisms involving alpha2-adrenergic receptor mediated survival signal activation and up-regulation of endogenous neurotrophic factor expression in the chronic ocular hypertensive rat retina.

Differential expression of two glutamate transporters, GLAST and GLT-1, in an experimental rat model of glaucoma.

Glutamate is the major excitatory neurotransmitter of the mammalian retina, and excessive glutamate has been implicated in the pathogenesis of glaucoma. It is well known that glutamate transport, mainly via GLAST and GLT-1, is cardinal mechanism for maintaining glutamate homeostasis in normal and pathological conditions, including ischemia in the brain. In an effort to understand the role of glutamate and the glutamate regulation system of the retina in the pathogenesis of glaucoma, we examined changes in the expression of two glutamate transporters, GLAST and GLT-1, by Western blot analysis and immunocytochemistry in a rat glaucoma model. GLT-1 was expressed in cone photoreceptors and some cone bipolar cells and the levels of expression were significantly increased in the cauterized eyes throughout the entire experimental period. In contrast, GLAST expression, which occurred in Müller cells, the main retinal glial cells, remained stable during the experimental period. These results suggest that GLT-1 may be a prerequisite for the maintenance of glutamate homeostasis in the retina undergoing glaucoma.

Level of Vascular Endothelial Growth Factor in Tenon Tissue and Results of Glaucoma Surgery

OBJECTIVE To determine the levels of vascular endothelial growth factor (VEGF) in both the aqueous humor and Tenon tissue in patients with primary open angle glaucoma (POAG) and the associations between the VEGF and outcomes of glaucoma surgery. METHODS The study involved 19 patients with POAG who were scheduled to undergo glaucoma surgery owing to uncontrolled intraocular pressure (IOP) and 17 control subjects who were scheduled to undergo cataract surgery. At the time of surgery, about 0.1 mL of aqueous humor was collected through an anterior chamber paracentesis and a 44-mm Tenon tissue sample was cut from the eye. Concentrations of VEGF were analyzed by enzyme-linked immunosorbent assay. Spearman correlation and regression analysis were used to assess the relationship with VEGF level to the clinical characteristics and postoperative IOP. RESULTS The VEGF in Tenon tissue was significantly elevated in patients with POAG compared with control subjects(P=.001). When patients with POAG were divided into success or failure groups 1 year following surgery, the VEGF levels were significantly higher in the Tenon tissue of the failure group compared with the success group(P=.014). The preoperative IOP was significantly related to the VEGF level in Tenon tissue in both the univariate(P=.001) and multivariate (P=.012) regressions. CONCLUSIONS The VEGF level in Tenon tissue at the time of surgery was significantly related to 1-year surgical outcomes of glaucoma surgery, and it was significantly associated with the final IOP in patients with POAG.

Neuroprotective Effects of Cilostazol on Retinal Ganglion Cell damage in Diabetic Rats

Neurodegeneration is an important component of diabetic retinopathy, with increasing evidence that retinal ganglion cell (RGC) death occurs early in diabetes. We investigated the effects of cilostazol, which has been widely used to manage diabetic complications, on retinal ganglion cell death in the diabetic retina. Four-week-old Otsuka Long-Evans Tokushima fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) rats as matched nondiabetic controls were treated with daily oral cilostazol at 30 mg/kg or 0.9% saline solution. In OLETF rats at the age of 40 weeks, glial fibrillary acidic protein (GFAP) immunofluorescence staining was upregulated in vertical sections, and showed a more ramified pattern in whole-mount retinas compared with that in LETO rats. Vascular endothelial growth factor (VEGF) expression was limited to the ganglion cell layer in LETO rats, but extended into the outer plexiform layer in OLETF rats. Immunofluorescence staining and Western blotting demonstrated that cilostazol treatment reduced GFAP and VEGF expression in the retinas of OLETF rats. Terminal deoxynucleotidyl transferase-mediated terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) staining revealed an increase in the RGC layer in OLETF compared with LETO rats (P < 0.05), and cilostazol treatment reduced the number of TUNEL-positive cells in OLETF rats (P < 0.05). Relieving retinal ischemia by systemic cilostazol treatment had a noticeable protective effect on RGCs in diabetic rats. Cilostazol treatment may be useful for the management of diabetic retinal vascular dysfunction and neuronal degeneration.