Seita Morishita

Systemic Simvastatin Rescues Retinal Ganglion Cells from Optic Nerve Injury Possibly through Suppression of Astroglial NF-κB Activation

Neuroinflammation is involved in the death of retinal ganglion cells (RGCs) after optic nerve injury. The purpose of this study was to determine whether systemic simvastatin can suppress neuroinflammation in the optic nerve and rescue RGCs after the optic nerve is crushed. Simvastatin or its vehicle was given through an osmotic minipump beginning one week prior to the crushing. Immunohistochemistry and real-time PCR were used to determine the degree of neuroinflammation on day 3 after the crushing. The density of RGCs was determined in Tuj-1 stained retinal flat mounts on day 7. The effect of simvastain on the TNF-α-induced NF-κB activation was determined in cultured optic nerve astrocytes. On day 3, CD68-positive cells, most likely microglia/macrophages, were accumulated at the crushed site. Phosphorylated NF-κB was detected in some astrocytes at the border of the lesion where the immunoreactivity to MCP-1 was intensified. There was an increase in the mRNA levels of the CD68 (11.4-fold), MCP-1 (22.6-fold), ET-1 (2.3-fold), GFAP (1.6-fold), TNF-α (7.0-fold), and iNOS (14.8-fold) genes on day 3. Systemic simvastatin significantly reduced these changes. The mean ± SD number of RGCs was 1816.3±232.6/mm2 (n = 6) in the sham controls which was significantly reduced to 831.4±202.5/mm2 (n = 9) on day 7 after the optic nerve was crushed. This reduction was significantly suppressed to 1169.2±201.3/mm2 (P = 0.01, Scheffe; n = 9) after systemic simvastatin. Simvastatin (1.0 µM) significantly reduced the TNF-α-induced NF-κB activation in cultured optic nerve astrocytes. We conclude that systemic simvastatin can reduce the death of RGCs induced by crushing the optic nerve possibly by suppressing astroglial NF-κB activation.

Changes in expression of aquaporin-4 and aquaporin-9 in optic nerve after crushing in rats.

The purpose of this study was to determine the temporal and spatial changes in the expression of AQP4 and AQP9 in the optic nerve after it is crushed. The left optic nerves of rats were either crushed (crushed group) or sham operated (sham group), and they were excised before, and at 1, 2, 4, 7, and 14 days later. Four optic nerves were pooled for each time point in both groups. The expression of AQP4 and AQP9 was determined by western blot analyses. Immunohistochemistry was used to determine the spatial expression of AQP4, AQP9, and GFAP in the optic nerve. Optic nerve edema was determined by measuring the water content in the optic nerve. The barrier function of the optic nerve vessels was determined by the extravasated Evans blue dye on days 7 and 14. The results showed that the expression of AQP4 was increased on day 1 but the level was significantly lower than that in the sham group on days 4 and 7 (P<0.05). In contrast, the expression of AQP9 gradually increased, and the level was significantly higher than that in the sham group on days 7 and 14 (P<0.05, Tukey-Kramer). The down-regulation of AQP4 was associated with crush-induced optic nerve edema, and the water content of the nerve was significantly increased by 4.3% in the crushed optic nerve from that of the untouched fellow nerve on day 7. The expression of AQP4 and GFAP was reduced at the crushed site where AQP4-negative and AQP9-positive astrocytes were present. The barrier function was impaired at the crushed site on days 7 and 14, restrictedly where AQP4-negative and AQP9-positive astrocytes were present. The presence of AQP9-positive astrocytes at the crushed site may counteract the metabolic damage but this change did not fully compensate for the barrier function defect.

Nitric Oxide Increases the Expression of Aquaporin-4 Protein in Rat Optic Nerve Astrocytes through the Cyclic Guanosine Monophosphate/Protein Kinase G Pathway

Aims: Nitric oxide (NO) is associated with neuroinflammation in the central nervous system. We determined whether NO increases the expression of aquaporin-4 (AQP4) in optic nerve astrocytes of rats. Methods: Isolated astrocytes were incubated under normoxic or hypoxic conditions with or without glucose (5.5 mM). The astrocytes were also exposed to different concentrations of S-nitroso-N-acetyl-DL-penicillamine (SNAP, 1.0-100 μM), an NO donor. The expression of AQP4 was determined by Western blot analyses, and NO formation was measured by the Griess reaction. The changes in astrocytic cellular volumes were determined by flow cytometry. Results: Hypoxia and glucose deprivation increased AQP4 expression and NO formation. Inhibition of NO synthetase (NOS) significantly suppressed these changes. SNAP caused a significant increase in AQP4 expression, and the increase was significantly suppressed by carboxy-PTIO, a scavenger of NO. Incubation with 8-Br-cyclic guanosine monophosphate (cGMP) mimicked the effects of SNAP, while the addition of either 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; inhibitor of soluble guanylate cyclase) or KT5823 (protein kinase G inhibitor) suppressed the SNAP-induced increase in AQP4 significantly. SNAP also caused a significant increase in astrocytic cellular volume through the AQP4 channels. Conclusions: NO increased the AQP4 expression of optic nerve astrocytes through the cGMP/protein kinase G pathway and enlarged their volume.

Treatment of massive subretinal hematoma associated with age-related macular degeneration using vitrectomy with intentional giant tear.

The purpose of this study was to report the surgical outcomes after creating a 120° intentional giant retinal tear for use in removing hemorrhage and subretinal proliferative tissue in patients with polypoidal choroidal vasculopathy (PCV) or age-related macular degeneration (ARMD). This study involved 12 eyes of 12 patients (10 eyes: PCV, 2 eyes: ARMD). After removal of the lens in phakic eyes, we performed a vitrectomy with artificial posterior vitreous detachment. Subsequently, a 120° intentional giant retinal tear was created in the temporal periphery, the retina was then turned, and the subretinal hemorrhage and proliferative tissue were removed. In order to preserve as much of the retinal pigment epithelium (RPE) as possible, we used a bimanual technique under direct visualization. After stretching the retina by use of perfluorocarbon liquid (PFCL), we performed endophotocoagulation around the tear followed by PFCL/silicone oil exchange. Except for 1 eye in which extensive loss of the RPE occurred, the fundus findings and the visual acuity (VA) improved in all patients. In addition, postoperative VA improved to ≥20/50 in 3 eyes in which the macular RPE was preserved. This surgical procedure is an effective treatment for PCV or ARMD patients with extensive subretinal hemorrhage and proliferative tissue.

Treatment of systemic hypertension is important for improvement of macular edema associated with retinal vein occlusion

Background We report our findings in three cases of unilateral macular edema associated with retinal vein occlusion (RVO) that improved after successful treatment of systemic hypertension alone. Methods All three cases had systemic hypertension but no diabetes mellitus or other ocular diseases associated with macular edema. All patients were treated only with medication for systemic hypertension. Optical coherence tomography was performed to determine the foveal thickness before and after treatment. Results Case one was a 72-year-old woman with a central RVO who had macular edema in her left eye and a visual acuity (VA) of 20/50. Her blood pressure (BP) was 169/96 mmHg. One month after the initiation of a calcium blocker to treat her systemic hypertension, her BP was decreased, macular edema was reduced, and her VA improved to 20/20. Case two was a 62-year-old woman with branch RVO. Her VA was 20/40 and her BP was 165/97 mmHg. Six weeks after initiation of medication to treat her systemic hypertension, her RVO-related macular edema had decreased and her VA improved to 20/20. Case three was a 71-year-old man with branch RVO. His VA was 20/50 and his BP was 165/87 mmHg. One month after initiation of treatment for systemic hypertension, his RVO-related macular edema had disappeared and his VA improved to 20/20. All three cases had nonischemic RVO by fluorescein angiography, and they did not develop ischemic changes for at least 1 year. Conclusion The reduction of macular edema following a decrease in the systemic hypertension suggests that the edema was most likely caused by leakage of fluids from the blood vessels. We recommend that the blood pressure should be measured in all patients with macular edema before initiating intravitreal anti-VEGF therapy.

Implication of VEGF and aquaporin 4 mediating Müller cell swelling to diabetic retinal edema

PurposeAquaporin 4 (AQP4), a water channel protein, is known to be expressed in retinal Müller cells. The purpose of this study was to determine the effects of VEGF and AQP4 channels on the volumetric changes in Müller cells.MethodsRetinas from diabetic rats and a cultured Müller cell line, TR-MUL5, were used in this study. Intravitreal injections of VEGF or PBS were performed on either streptozotocin (STZ)-induced diabetic or normoglycemic rats. Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-AQP4, and anti-VEGF. VEGF protein levels from collected retinas were determined by western blot analysis. Volumetric changes and nitric oxide (NO) levels in cultured Müller cells were determined using flow cytometry (FACS), in the presence or absence of VEGF and TGN-020, a selective AQP4 inhibitor.ResultsIn the diabetic rat retina, VEGF immunoreactivity was concentrated in the internal retinal layers, and AQP4 immunoreactivity was higher than controls. The expressions of AQP4 were colocalized with GFAP. Protein levels of VEGF in the hyperglycemic rat retina were significantly higher than controls. FACS analyses showed that exposure to VEGF enlarged Müller cells, while exposure to TGN-020 suppressed the enlargement. Intracellular levels of NO were increased after exposure to VEGF, which was suppressed following the addition of TGN-020.ConclusionThe observed Müller cell swelling is mediated by VEGF and AQP4.

Cotton Wool Spots after Anti-Vascular Endothelial Growth Factor Therapy for Macular Edema Associated with Central Retinal Vein Occlusion

Purpose: To report a case series, whereby we encountered a transient increase in retinal cotton wool spots (CWS) following anti-vascular endothelial growth factor (anti-VEGF) therapy for the treatment of macular edema secondary to central retinal vein occlusion (CRVO). Methods: Eighteen eyes were treated with intravitreal aflibercept (IVA), and 5 were treated with intravitreal ranibizumab (IVR). Fundus photographs obtained 1 month after initial IVA or IVR injections were retrospectively evaluated for the presence of CWS. Results: Twenty-one (91.3%) patients had the following systemic diseases: hypertension, diabetes mellitus without retinopathy, dyslipidemia, or chronic renal failure requiring dialysis. One month after treatment, reduced macular edema was observed in 21 (91.3%) eyes. Initial injections facilitated complete resolution in 14 eyes, and CWS gradually became fainter with additional injections. Conclusion: Some eyes with CRVO-related macular edema can show a transient increase in CWS after initial anti-VEGF therapy; however, macular edema, retinal hemorrhage, and visual acuity were improved in almost every case.

Vitrectomy for Tractional Retinal Detachment with Twin Retinal Capillary Hemangiomas in a Patient with Von Hippel-Lindau Disease: A Case Report.

Purpose: The purpose of this study was to report a case of Von Hippel-Lindau disease (VHL) with twin retinal capillary hemangiomas that was successfully treated by vitreous surgery for tractional retinal detachment following laser photocoagulation. Case: A 44-year-old male presented at our university hospital after noticing decreased visual acuity in his right eye. The patient had previously undergone multiple operations for cerebellar, thoracic, and lumbar spine hemangioblastomas when he was approximately 19 years old. Upon initial examination, ocular findings revealed twin connected retinal capillary hemangiomas around the temporal upper area of the patient’s right eye. The patient was subsequently diagnosed with VHL based on his medical history and current observations of the ocular fundus. Tractional retinal detachment had occurred as the result of the formation of proliferative membranes following laser photocoagulation. The patient underwent vitreous surgery to treat the tractional retinal detachment, resulting in a successful postoperative outcome. Conclusion: The findings of this study show the possibility that proliferative changes and tractional retinal detachment can arise following photocoagulation for retinal capillary hemangiomas in patients with VHL.

Vitreous estrogen levels in patients with an idiopathic macular hole.

Purpose Estrogen, a female hormone, activates collagenase and might be associated with the pathogenesis of vitreoretinal collagen fiber disease. The purpose of the present study was to investigate the vitreous levels of estrone (E1) and estradiol (E2) in subjects with an idiopathic macular hole (IMH). Methods Vitreous samples were obtained from ten female patients with an IMH and from nine female patients with other retinal diseases (six with rhegmatogenous retinal detachment and three with age-related macular degeneration) as a control at the time of vitreous surgery. E1 and E2 levels in the vitreous samples were then determined using the Coat-A-Count® Estradiol Radioimmunoassay (RIA) Kit and the DSL-70 Estrone RIA Kit, respectively. Results The mean vitreous levels of E1 and E2 in the subjects with IMH were 1.83±2.00 pg/mL and 7.03±2.97 pg/mL, respectively, whereas in the control subjects they were 2.42±1.25 pg/mL and 4.90±2.90 pg/mL, respectively. Thus, the vitreous E2 levels in the subjects with IMH were significantly higher than in the controls (P<0.05). Conclusion The findings of this study suggest that E2 might be associated with the pathogenesis of IMH, but further investigation is needed to elucidate that association.

A comparison of sex steroid concentration levels in the vitreous and serum of patients with vitreoretinal diseases

The purpose of this study was to compare steroid hormone concentration levels in the vitreous and serum of vitreoretinal disease patients to elucidate the possibility of neurosteroid production in the retina. Serum and vitreous samples were collected from vitrectomy patients, and estradiol (E2) and testosterone (T) concentrations were measured using electro-chemiluminescence immunoassay. We measured E2 in epiretinal membrane (ERM, n = 14), macular hole (MH, n = 18), proliferative diabetic retinopathy (PDR, n = 20), and retinal detachment (RD, n = 19) cases, and T in ERM (n = 14), MH (n = 17), PDR (n = 13), and RD (n = 17) cases. No statistically significant age differences existed among the groups. Mean respective E2 concentrations (pg/ml) in the male/female vitreous were ERM: 6.67±4.04/18.82±7.10, MH: 10.3±7.02/17.00±4.8, PDR: 4.2±3.05/15.83±3.46, and RD: 10.00±4.58/16.06±4.57, while those in serum were ERM: 31.67±5.51/5.82±1.08, MH: 21.00±8.89/7.53±3.2, PDR: 29.20±7.07/12.75±10.62, and RD: 24.33±6.51/7.5±4.42. E2 concentrations were significantly higher (P<0.001) in the male serum than vitreous, yet significantly higher in the female vitreous than serum. Mean respective T concentrations (ng/ml) in the male/female vitreous were ERM: 0.15±0.03/0.15±0.01, MH: 0.15±0.01/0.15±0.01, PDR: 0.15±0.03/0.16±0.12, and RD: 0.14±0.01/0.17±0.08, while those in serum were ERM: 4.54±1.46/0.16±0.01, MH: 8.04±2.29/0.16±0.10, PDR: 5.14±1.54/0.22±0.11, and RD: 3.24±0.75/0.17±0.10. T concentrations were high in the male serum, yet extremely low in the male and female vitreous and female serum. High concentrations of E2 were found in the vitreous, and women, in particular, exhibited significantly higher concentrations in the vitreous than in the serum. This finding suggests the possibility that in vitreoretinal disease cases, the synthesis of E2 is increased locally only in female eyes.