RESUMEN
INTRODUCTION: We previously reported that the intravitreal activities of chymase and tryptase were more increased in the patients with macular hole (MH) and epiretinal membrane (ERM) than in those with proliferative diabetic retinopathy (PDR) and that the source of these serine proteases might be mast cells in the bursa premacularis (BPM). The purpose of this study was to compare the density of mast cells in BPM samples obtained from MH, ERM, and PDR patients. METHODS: BPM and vitreous core samples were first collected during vitrectomy from eyes afflicted with vitreoretinal diseases (MH: 6 eyes, ERM: 3 eyes, and PDR: 9 eyes), and then were stained with hematoxylin, toluidine blue, antibodies against chymase and tryptase, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay kit. RESULTS: Hematoxylin nuclear staining showed fewer positive-staining cells in the BPM samples obtained from PDR patients than in those obtained from MH and ERM patients. Toluidine blue staining of the BPM revealed metachromasia in the mast cells of the patients with MH and ERM, but not those of the patients with PDR. In addition, immunostaining using anti-chymase and anti-tryptase antibodies showed that the BPM samples were more intensely stained than the vitreous core samples from the patients with MH and ERM and that both tissue samples were poorly stained in the patients with PDR. The apoptotic cells were more frequently observed in the BPM samples from patients with MH than in those from patients with PDR. CONCLUSIONS: These findings indicated that lower activities of chymase and tryptase in the vitreous of PDR patients appeared to be attributable to the decreased presence of mast cells in the BPM. The lack of mast cells in the BPM might be related to the pathogenesis of PDR.
Asunto(s)
Diabetes Mellitus , Retinopatía Diabética , Perforaciones de la Retina , Quimasas , Membrana Epirretinal , Hematoxilina , Humanos , Mastocitos , Cloruro de Tolonio , TriptasasRESUMEN
Tauopathies are neurodegenerative diseases characterized by abnormal metabolism of misfolded tau proteins and are progressive. Pathological phosphorylation of tau occurs in the retinal ganglion cells (RGCs) after optic nerve injuries. Cyclin-dependent kinase-5 (Cdk5) causes hyperphosphorylation of tau. To determine the roles played by Cdk5 in retinal degeneration, roscovitine, a Cdk5 inhibitor, was injected intravitreally after optic nerve crush (ONC). The neuroprotective effect of roscovitine was determined by the number of Tuj-1-stained RGCs on day 7. The change in the levels of phosphorylated tau, calpain-1, and cleaved α-fodrin was determined by immunoblots on day 3. The expression of P35/P25, a Cdk5 activator, in the RGCs was determined by immunohistochemistry. The results showed that roscovitine reduced the level of phosphorylated tau by 3.5- to 1.6-fold. Calpain-1 (2.1-fold) and cleaved α-fodrin (1.5-fold) were increased on day 3, suggesting that the calpain signaling pathway was activated. P35/P25 was accumulated in the RGCs that were poorly stained by Tuj-1. Calpain inhibition also reduced the increase in phosphorylated tau. The number of RGCs decreased from 2191 ± 178 (sham) to 1216 ± 122 cells/mm2 on day 7, and roscovitine preserved the level at 1622 ± 130 cells/mm2. We conclude that the calpain-mediated activation of Cdk5 is associated with the pathologic phosphorylation of tau.
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Quinasa 5 Dependiente de la Ciclina/fisiología , Traumatismos del Nervio Óptico , Células Ganglionares de la Retina , Tauopatías , Proteínas tau/metabolismo , Animales , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Fosforilación , Ratas , Ratas Wistar , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Roscovitina/farmacología , Tauopatías/metabolismo , Tauopatías/patologíaRESUMEN
We previously reported that the bursa premacularis (BPM), a peculiar vitreous structure located above the macula, contains numerous cells expressing markers of lymphatic endothelial cells, such as podoplanin and LYVE-1. Herein, we examined the expression of lymphatic markers in the Berger's space (BS), BPM, and vitreous core (VC). BS, BPM, and VC specimens were selectively collected in macular hole and epiretinal membrane patients during vitrectomy and were then immunostained with antibodies for podoplanin, LYVE-1, and fibrillin-1 and -2. By visualization using triamcinolone acetonide, the BS was recognized as a sac-like structure with a septum located behind the lens as well as BPM. Those tissues adhered to the lens or retina in a circular manner by means of a ligament-like structure. Immunostaining showed intense expression of podoplanin and LYVE-1 in the BS. Both BS and BPM stained strongly positive for fibrillin-1 and -2. The VC was faintly stained with antibodies for those lymph-node markers. Our findings indicate that both BS and BPM possibly belong to the lymphatic system, such as lymph nodes, draining excess fluid and waste products into lymphatic vessels in the dura mater of the optic nerve and the ciliary body, respectively, via intravitreal canals.
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Biomarcadores/metabolismo , Vasos Linfáticos/metabolismo , Cuerpo Vítreo/anatomía & histología , Anciano , Anticuerpos/metabolismo , Femenino , Fibrilinas/metabolismo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Microvascular changes are the earliest adverse events in diabetic retinopathy, but recent studies have shown that oxidative stress induced by photoreceptors is associated with the development of the retinopathy. The purpose of this study was to determine the roles played by superoxides formed by photoreceptors under hyperglycemic conditions on autophagy. To accomplish this, we cultured 661 W cells, a transformed murine cone cell line, with 5.5 or 25 mM glucose in the presence or absence of 3 methyl adenine (3MA) or rapamycin. The superoxides were determined by flow cytometry using hydroethidine as a fluorescence probe. The autophagy activity was determined by changes in the expression of LC3B2 and P62 by immunoblotting. The degree of mitophagy was determined by the accumulation of mitochondria and lysosomes. Apoptotic changes of 661 W cells were determined by the caspase 3/7 activities. Our results showed higher levels of P62 and superoxides in cells cultured in 25 mM glucose than in 5.5 mM glucose. Addition of 3MA caused a significant increase of P62, superoxides, and caspase 3/7 activities in the 661 W cells cultured in high glucose but not in low glucose. These findings suggest that autophagy is important for the functioning and survival of 661 W cells under hyperglycemic conditions.
Asunto(s)
Retinopatía Diabética/metabolismo , Glucosa/efectos adversos , Proteínas Asociadas a Microtúbulos/metabolismo , Células Fotorreceptoras Retinianas Conos/citología , Proteína Sequestosoma-1/metabolismo , Superóxidos/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Autofagia/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Retinopatía Diabética/etiología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Modelos Biológicos , Estrés Oxidativo/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/metabolismo , Sirolimus/farmacologíaRESUMEN
PURPOSE: To investigate the effect of a selective aquaporin 4 (AQP4) inhibitor, 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020), on the expression of vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) production, as well as on the retinal edema in diabetic retina. METHODS: Intravitreal injections of bevacizumab, TGN-020, or phosphate-buffered saline (PBS) were performed on streptozotocin-induced diabetic rats. Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-AQP4, and anti-VEGF. Protein levels of VEGF from collected retinas were determined by Western blot analysis. In addition, retinal vascular leakage of Evans Blue was observed in the flat-mounted retina from the diabetic rats in the presence or absence of TGN-020. Volumetric changes of rat retinal Müller cells (TR-MUL5; transgenic rat Müller cells) and intracellular levels of ROS were determined using flow cytometry analysis of ethidium fluorescence in the presence or absence of TGN-020 or bevacizumab under physiological and high glucose conditions. RESULTS: In the diabetic retina, the immunoreactivity and protein levels of VEGF were suppressed by TGN-020. AQP4 immunoreactivity was higher than in the control retinas and the expressions of AQP4 were co-localized with GFAP. Similarly to VEGF, AQP4 and GFAP were also suppressed by TGN-020. In the Evans Blue assay, TGN-020 decreased leakage in the diabetic retinas. In the cultured Müller cells, the increase in cell volumes and intracellular ROS production under high glucose condition were suppressed by exposure to TGN-020 as much as by exposure to bevacizumab. CONCLUSION: TGN-020 may have an inhibitory effect on diabetic retinal edema.
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Acuaporina 4/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Niacinamida/análogos & derivados , Niacinamida/antagonistas & inhibidores , Retina/metabolismo , Tiadiazoles/antagonistas & inhibidores , Animales , Vasos Sanguíneos/diagnóstico por imagen , Vasos Sanguíneos/metabolismo , Retinopatía Diabética/metabolismo , Células Ependimogliales/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Ratones , Ratas , Ratas Wistar , Retina/diagnóstico por imagen , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Lattice degeneration involves thinning of the retina that occurs over time. Here we performed an immunohistological study of tissue sections of human peripheral retinal lattice degeneration to investigate if retinal pigment epithelium (RPE) cells are involved in the pathogenesis of this condition. In two cases of retinal detachment with a large tear that underwent vitreous surgery, retinal lattice degeneration tissue specimens were collected during surgery. In the obtained specimens, both whole mounts and horizontal section slices were prepared, and immunostaining was then performed with hematoxylin and antibodies against glial fibrillary acidic protein (GFAP), RPE-specific protein 65 kDa (RPE65), pan-cytokeratin (pan-CK), and CK18. Hematoxylin staining showed no nuclei in the center of the degenerative lesion, thus suggesting the possibility of the occurrence of apoptosis. In the degenerative lesion specimens, GFAP staining was observed in the center, RPE65 staining was observed in the slightly peripheral region, and pan-CK staining was observed in all areas. However, no obvious CK18 staining was observed. In a monkey retina used as the control specimen of a normal healthy retina, no RPE65 or pan-CK staining was observed in the neural retina. Our findings suggest that migration, proliferation, and differentiation of RPE cells might be involved in the repair of retinal lattice degeneration.
Asunto(s)
Proteína Ácida Fibrilar de la Glía/genética , Queratina-18/genética , Degeneración Retiniana/genética , cis-trans-Isomerasas/genética , Anciano , Femenino , Regulación de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Retina/metabolismo , Retina/patología , Degeneración Retiniana/patología , Desprendimiento de Retina/genética , Desprendimiento de Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
PURPOSE: Diabetic retinopathy (DR) involves a proliferation of vascular endothelial cells and loss of pericytes. There is a link among the action of protein kinase C (PKC) and insulin signaling. Thus, we investigated the differences between these cells in insulin receptor (IR) phosphorylation in DR. METHODS: Retinas were removed from streptozotocin-induced diabetic or healthy rats, and IR expression levels were compared by immunoblot and immunohistochemistry. In vitro assays also were performed in order to determine the expressions of phosphorylated IR in both cells cultured under 5.5 or 25 mM glucose by immunoblot. Cell viability was determined in both cells cultured under different concentrations of phorbol myristate acetate (PMA), a PKC activator. To determine the involvement of the PI3 kinase pathway of IR, PMA with or without wortmannin-induced changes in Akt was also analyzed. RESULTS: Immunoreactivity to the IR was decreased in diabetic retina. High glucose (25 mM) increased phosphorylated IR levels in endothelial cells but not in pericytes. PMA (1 nM or higher) induced death of pericytes, while endothelial cells were increased. PMA increased phosphorylated Akt in endothelial cells and decreased in pericytes. Wortmannin suppressed the PMA-induced phosphorylation of Akt in endothelial cells. CONCLUSIONS: The different responses to 25 mM glucose and PMA were observed between retinal endothelial cells and pericytes. Thus, IR phosphorylation is likely important for retinal vascular cells to survive in diabetic retina.
Asunto(s)
Diabetes Mellitus Experimental , Retinopatía Diabética/metabolismo , Endotelio Vascular/metabolismo , Proteína Quinasa C/metabolismo , Receptor de Insulina/metabolismo , Vasos Retinianos/metabolismo , Animales , Western Blotting , Proliferación Celular , Células Cultivadas , Retinopatía Diabética/patología , Endotelio Vascular/patología , Masculino , Fosforilación , Fotomicrografía , Ratas , Ratas Wistar , Vasos Retinianos/patologíaRESUMEN
PURPOSE: Whilst the pathogenesis of retinal vein occlusion (RVO) is still unclear, systemic hypertension and increased level of endothelin-1 (ET-1) are known risk factors. Therefore, we studied the influence of ET-1 on the retinal veins in hypertensive rats. METHODS: We focused on the behavior of retinal veins in spontaneous hypertensive rats (SHR). To determine whether ET-1 was associated with the blood flow in eyes of SHRs, the chorioretinal blood flow in the rats was assessed using laser speckle flowgraphy (LSFG-Micro, Softcare, Fukuoka, Japan) before and after an intravenous injection of ET-1 under general anesthesia. In addition, retinas from SHRs and age-matched normotensive Wistar-Kyoto rats (WKYs) were removed, and retinal sections were immunostained for the ET-A and ET-B receptors. The protein levels of both ET-1 receptors and hypoxia-inducible factor 1 (HIF-1) in the retinal tissues were also determined by western blot analysis. RESULTS: One of the retinal veins became exceptionally constricted and was nearly occluded, and the chorioretinal blood flow significantly decreased in the retinas of SHRs following the injection of ET-1. Immunoreactivity to ET-A receptor was higher in SHR retinas than in WKY retinas. The protein levels of ET-A receptor and HIF-1 were also significantly higher in SHR retinas than in WKY retinas. CONCLUSIONS: An increase of ET-1 in circulating blood leads to the local constriction of retinal veins and this effect is accentuated in hypertensive rats by an upregulation of ET-A receptor. It is plausible that such a constriction of retinal veins increases retinal venous pressure, and may even contribute to the pathogenesis of RVO.
Asunto(s)
Endotelina-1/administración & dosificación , Oclusión de la Vena Retiniana/etiología , Oclusión de la Vena Retiniana/fisiopatología , Vena Retiniana/fisiopatología , Animales , Coroides/irrigación sanguínea , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inyecciones Intravenosas , Flujometría por Láser-Doppler , Masculino , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Flujo Sanguíneo Regional/fisiología , Oclusión de la Vena Retiniana/metabolismo , Vasos Retinianos/fisiología , VasoconstricciónRESUMEN
PURPOSE: Aquaporin 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. METHODS: Retinas 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. RESULTS: In 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. CONCLUSION: The observed Müller cell swelling is mediated by VEGF and AQP4.
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Acuaporina 4/genética , Diabetes Mellitus Experimental , Retinopatía Diabética/genética , Células Ependimogliales/metabolismo , Regulación de la Expresión Génica , Papiledema/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Acuaporina 4/biosíntesis , Western Blotting , Células Cultivadas , Retinopatía Diabética/complicaciones , Retinopatía Diabética/patología , Células Ependimogliales/patología , Citometría de Flujo , Inmunohistoquímica , Masculino , Papiledema/diagnóstico , Papiledema/etiología , Ratas , Ratas Wistar , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
The purpose of this study was to determine whether inhibition of aquaporin 4 (AQP4) is neuroprotective or neurodestructive after crushing the optic nerve of rats. The left optic nerves of rats were crushed, and TGN-020 (5.0 mg/kg, crush TGN-020) or its vehicle (DMSO, crush placebo) was injected intraperitoneally just after the crushing. As controls, the left optic nerves were exposed but not touched in other rats (sham controls). The retinal damages were determined by the density of retinal ganglion cells (RGCs) and the ratio of BAX/Bcl-2 on day 7. The glutamate level in the optic nerve on day 1 after the crushing was determined. The expressions of glutamine synthetase, glutamate-aspartate transporter (GLAST), and AQP4 were determined on day 3 by immunoblotting. The effects of AQP4 inhibition on the glutamate-induced changes of AQP4 expression and on the glutamate uptake were determined for optic nerve astrocytes in culture. The results showed that the density of RGCs was 2040 ± 91.3 cells/mm(2) (n = 6) in the sham control, and it was significantly decreased to 1072 ± 134.3 cells/mm(2) after crushing the optic nerve (P < 0.0001, crush placebo, n = 7; Fisher). An intraperitoneal injection of TGN-020 led to a further significant (P = 0.02, Fisher) decrease of the density of RGCs to 743 ± 371 cells/mm(2) (crush TGN-020, n = 7). The mRNA level of BAX/Bcl-2 ratio was 0.37 ± 0.05 in the sham control (n = 6) which was significantly increased to 0.88 ± 0.10 after crushing the optic nerve (placebo crush, n = 7; P = 0.0001, Scheffe). TGN-020 also significantly increased the BAX/Bcl-2 ratio to 1.29 ± 0.4 (n = 6) from the crush placebo group (P = 0.04, Scheffe). Immunoblotting showed similar changes in the protein levels. The glutamate level in the optic nerve was significantly increased to 53.7 ± 6.0 µM/mg/protein on day 1 (n = 4) from the sham control level of 45.9 ± 3.1 µM/mg/protein (n = 4; P = 0.04, t test). TGN-020 significantly (P < 0.05, Scheffe) depressed the expression of glutamate metabolism-related proteins on day 3. Exposure of cultured optic nerve astrocytes to glutamate (1.0 mM, n = 4) significantly increased the expression of AQP4 (P < 0.001, Scheffe) that was depressed by TGN-020 (100 nM, n = 4). In addition, glutamate uptake was inhibited by TGN-020 at 10 nM or higher. These results indicate that an inhibition of AQP4 enhances the loss of RGCs and retinal damages after crushing the optic nerve. Inhibition of AQP4 impairs glutamate metabolism which may account in part for these neurodestructive events.
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Acuaporina 4/antagonistas & inhibidores , Ácido Glutámico/metabolismo , Niacinamida/análogos & derivados , Traumatismos del Nervio Óptico/metabolismo , Nervio Óptico/metabolismo , Células Ganglionares de la Retina/patología , Tiadiazoles/farmacología , Animales , Acuaporina 4/metabolismo , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Immunoblotting , Masculino , Niacinamida/farmacología , Nervio Óptico/efectos de los fármacos , Nervio Óptico/patología , Traumatismos del Nervio Óptico/patología , ARN/genética , Ratas , Ratas Transgénicas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Regulación hacia Arriba , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
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.
Asunto(s)
Acuaporina 4/metabolismo , Astrocitos/efectos de los fármacos , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Óxido Nítrico/fisiología , Nervio Óptico/citología , Animales , Astrocitos/metabolismo , Western Blotting , Hipoxia de la Célula/fisiología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Glucosa/farmacología , Masculino , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Wistar , S-Nitroso-N-Acetilpenicilamina/farmacología , Edulcorantes/farmacologíaRESUMEN
PURPOSE: To investigate whether the P2X7 receptor is involved in retinal ganglion cell (RGC) death after the intraocular pressure (IOP) is elevated in rats. METHODS: After the IOP was elevated to 90 mmHg for 1 h, the rats were subsequently administered oxidized adenosine triphosphate (OxATP) and brilliant blue G (BBG) as P2X7 antagonists. The rats were euthanized 7 days after IOP elevation for histologic evaluation and at 1, 3, and 7 days after IOP elevation to immunostain for the P2X7 receptor and neuron-specific class III ß-tubulin in the retina. Changes in P2X7 receptor expression were measured in total retina extracts using western blot analysis. Quantitative real-time PCR was also performed using the entire retina to determine whether the P2X7 receptor is involved in upregulating tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 at 1, 2, and 3 days after the IOP was elevated. RESULTS: RGC density and the inner plexiform layer thickness significantly decreased 7 days after IOP elevation, but were dose-dependently preserved when treated with OxATP or BBG. P2X7 immunoreactivity in the RGCs increased after IOP elevation, with the peak occurring from day 1 through day 3. Protein levels of P2X7 receptor were significantly increased 1, 2, and 3 days after IOP elevation. The messenger ribonucleic acid expression of the P2X7 receptor, TNF-α, IL-1ß, and IL-6 was significantly upregulated in the retina after IOP elevation, and was suppressed by treatment with OxATP. CONCLUSIONS: These results suggest the expression of the P2X7 receptor is upregulated in the retina after IOP elevation, leading to RGC death. Upregulation of TNF-α, IL-1ß, and IL-6 might be involved in this mechanism of RGC death. Furthermore, P2X7 antagonists may prevent RGC death after IOP elevation.
Asunto(s)
Receptores Purinérgicos P2X7/metabolismo , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Animales , Western Blotting , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Interleucinas/genética , Interleucinas/metabolismo , Presión Intraocular/efectos de los fármacos , Presión Intraocular/genética , Masculino , Antagonistas del Receptor Purinérgico P2X/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Purinérgicos P2X7/genética , Células Ganglionares de la Retina/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Modulation of enzyme activity through nitrosylation has recently been identified as a new physiological activity of nitric oxide (NO). We hypothesized that NO enhances the TNF-α-induced death of retinal neurons through a suppression of nuclear factor-κB (NF-κB) by nitrosylation. In this study, cells from the RGC-5 line were exposed to different concentrations (2.0, 10, and 50 ng/ml) of TNF-α, and the degree of TNF-α-induced cell death was determined by the WST-8 assay and by flow cytometric measurements of the externalization of phosphatidylserine. The effects of etanercept, a soluble TNFR-Fc fusion protein, and S-nitroso-N-penicillamine (SNAP), an NO donor, on the toxicity were determined. Experiments were also performed to determine whether nitric oxide synthase (NOS) was associated with the toxicity of TNF-α. The activation of NF-κB was determined by the detection of the p65 subunit in the nuclear extracts. Our results showed that exposure of RGC-5 cells to different concentrations of TNF-α significantly decreased the number of living cells in a dose-dependent way. The death was partially due to apoptosis with an externalization of phosphatidylserine, and the death was suppressed by etanercept. Exposure to TNF-α increased the activation of NF-κB and the expression of iNOS. Although NF-κB inhibitors suppressed the increase of iNOS, they also potentiated the TNF-α-induced death. Both L-NAME and aminoguanidine, both NOS inhibitors, rescued the cells from death. In contrast, addition of SNAP caused nitrosylation of the inhibitory κB kinase, and suppressed the NF-κB activation and potentiated the TNF-α-induced neurotoxicity. These results indicate that NO potentiates the neurotoxicity of TNF-α by suppressing NF-κB.
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FN-kappa B/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Óxido Nítrico/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Sinergismo Farmacológico , Ratones , FN-kappa B/metabolismo , Neuronas/fisiología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitrosación/efectos de los fármacos , S-Nitroso-N-Acetilpenicilamina/farmacologíaRESUMEN
Determine the impact of the mTOR inhibitor, rapamycin, on the hyperglycemia-induced expression of vascular endothelial growth factor (VEGF) and the production of reactive oxygen species (ROS) in retinal cells. Rats made hyperglycemic for 8 weeks by streptozotocin, as well as control rats, received i.p. rapamycin (1 mg/kg) for 3 days prior to immunostaining of their retinas with anti-VEGF and anti-glial fibrillary acidic protein (GFAP) and measuring retinal protein levels of VEGF and GFAP by Western blotting. In other experiments, flow cytometry analysis of ethidium fluorescence determined intracellular ROS levels in the absence or presence of rapamycin (1 µM) under normoglycemic (5.5 mM) and hyperglycemic (25 mM) conditions in a rat retinal Müller cell line (TR-MUL5) and primary human retinal microvascular endothelial cells (HRMECs). In the diabetic retina, VEGF was elevated and colocalized with the glial marker, GFAP, whose level was also elevated. Treatment with rapamycin inhibited the diabetes-induced VEGF and GFAP increases. We also found that raising extracellular glucose from 5.5 mM to 25 mM resulted in significant rapamycin-sensitive increases in the ROS levels of TR-MUL5 cells and HRMECs. In rat retina, rapamycin attenuates the diabetes-induced VEGF overexpression, and in cultured Müller cells and HRMECs, inhibits the hyperglycemia-induced boost ROS.
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Hiperglucemia/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Retina/efectos de los fármacos , Sirolimus/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Western Blotting , Línea Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Hiperglucemia/fisiopatología , Inyecciones Intraperitoneales , Masculino , Ratas Wistar , Retina/citología , Retina/metabolismo , Sirolimus/administración & dosificación , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Purpose: To determine whether tauopathies are associated with impaired autophagy and involved in the death of retinal ganglion cells (RGCs) of rats from an optic nerve crush (ONC). Methods: Short interfering RNA (siRNA) of the tau gene (si-Tau) or nontargeting siRNA (si-NC) was injected intravitreally 48 hours prior to ONC. The effects of silencing the tau gene on neuroprotection were determined by the number of Tuj-1-stained RGCs on days 7 and 14 after the ONC. The changes in the expressions of phosphorylated tau, P62, and LC3B were determined by immunoblots and immunohistochemistry on day 7. Results: Autophagy was impaired in the retina on day 7 after the ONC as the P62 level increased by 3.1-fold from the sham control level with a reduction in the ratio LC3B2/LC3B1. There was a 2.1-fold increase of phosphorylated tau (ser 396) in the retina, and si-Tau depressed the increase by 1.3-fold (n = 3 each). The expressions of tau and P62 were well colocalized. They were observed in the somas of RGCs and retinal nerve fibers (RNFs), and these expressions were increased after the ONC. Pretreatment by si-Tau showed significant protection in the number of RGCs after the ONC. Specifically, the density of RGCs was 540 ± 74.5 cells/mm2 on day 14 in the si-NC group, while the level was maintained at 1321 ± 192 cells/mm2 in the si-Tau group (n = 4 each). Conclusions: Silencing the tau gene is neuroprotective, and tauopathies may be involved in the death of RGCs after ONC. Impaired autophagy may be involved in ONC-induced tauopathies.
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Autofagia/fisiología , Compresión Nerviosa , Neuroprotección/fisiología , Traumatismos del Nervio Óptico/fisiopatología , Células Ganglionares de la Retina/fisiología , Proteínas tau/fisiología , Animales , Supervivencia Celular/fisiología , Modelos Animales de Enfermedad , Silenciador del Gen , Masculino , Ratas , Proteínas tau/metabolismoRESUMEN
The fovea centralis, an anatomically concave pit located at the center of the macula, is avascular, hypoxic, and characteristic of stem-cell niches of other tissues. We hypothesized that in the fovea, undifferentiated retinal-stem-cell-like cells may exist, and that neurogenesis may occur. Hence, we performed an immunohistological study using cynomolgus monkey retinas. After preparing frozen tissue sections of the retina including the foveal pit, immunostaining was performed for glial fibrillary acidic protein (GFAP), nestin, vimentin, neuron-specific class III ß-tubulin (Tuj-1), arrestin 4, neurofilament, CD117, CD44, Ki67, and cellular retinaldehyde-binding protein (CRALBP), followed by fluorescence and/or confocal microscopy examinations. Immunostaining of the tissue sections enabled clear observation of strongly GFAP-positive cells that corresponded to the inner-half layer of the foveolar Müller cell cone. The surface layer of the foveal slope was partially costained with GFAP and vimentin. Tuj-1-positive cells were observed in the innermost layer of the foveolar retina, which spanned to the surrounding ganglion cell layer. Moreover, colocalization of Tuj-1 and GFAP was observed at the foveal pit. The coexpression of CD117 and CD44 was found in the interphotoreceptor matrix of the fovea. The foveolar cone stained positive for both nestin and arrestin 4, however, the photoreceptor layer outside of the foveola displayed weak staining for nestin. Colocalization of nestin and vimentin was observed in the inner half of the Henle layer, while colocalization of nestin and neurofilament was observed in the outer half, predominantly. Scattered Ki67-positive cells were observed in the cellular processes of the outer plexiform layer and the ganglion cell layer around the foveola. Immunostaining for CRALBP was negative in most parts of the GFAP-positive area. The Müller cell cone was divided into GFAP-strongly positive cells, presumably astrocytes, in the inner layer and nestin-positive/GFAP-weakly positive radial glia-like cells in the outer layer. These findings indicated that groups of such undifferentiated cells in the foveola might be involved in maintaining morphology and regeneration.
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Fóvea Central/metabolismo , Retina/metabolismo , Animales , Arrestinas/metabolismo , Proteínas Portadoras/metabolismo , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Receptores de Hialuranos/metabolismo , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Macaca fascicularis , Masculino , Nestina/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismoRESUMEN
We previously reported on the elevated intravitreal activities of tryptase and chymase in association with idiopathic epiretinal membrane (ERM) and idiopathic macular hole (MH). In this present study, we investigated the potential intraocular production of these serine proteases, and measured and compared tryptase and chymase activities in the vitreous body and serum in ERM, MH, proliferative diabetic retinopathy (PDR), and rhegmatogenous retinal detachment (RRD) patients. In addition, nuclear staining with hematoxylin and eosin (H&E) and mast-cell staining with toluidine blue were performed on samples of the vitreous core and bursa premacularis (BPM) of MH. We also performed immunostaining on the above two regions of vitreous samples for MH with anti-tryptase antibody, anti-chymase antibody, anti-podoplanin antibody, anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) antibody, and anti-fibroblast antibody. Moreover, we performed immunostaining with anti-tryptase antibody and anti-chymase antibody on ERMs collected intraoperatively. Tryptase activity in the vitreous body was significantly higher in ERM and MH than in PDR. However, no significant differences were observed in the tryptase activity in the serum among these four diseases. Chymase activity in the vitreous body was significantly higher in MH than in the other three diseases, yet chymase activity in the serum was below detection limit in any of the diseases. Nuclear staining with H&E revealed an abundance of nuclei in the BPM region, but few in the surrounding area. Mast-cell staining with toluidine blue revealed that the BPM showed metachromatic staining. In immunostaining with anti-fibroblasts antibody, anti-tryptase antibody, anti-chymase antibody, anti-podoplanin antibody, and anti-LYVE-1 antibody, the BPM stained more strongly than the vitreous core. Tryptase and chymase-positive cells were also observed in ERM. These findings revealed that the presence of mast cells in the BPM potentially represent the source of these serine proteases. Moreover, the BPM, as a lymphatic tissue, may play an important role in the pathogenesis of macular disease.
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Mastocitos/patología , Enfermedades de la Retina/etiología , Anciano , Quimasas/sangre , Quimasas/metabolismo , Retinopatía Diabética/enzimología , Retinopatía Diabética/etiología , Retinopatía Diabética/patología , Membrana Epirretinal/enzimología , Membrana Epirretinal/etiología , Membrana Epirretinal/patología , Femenino , Humanos , Inmunohistoquímica , Mácula Lútea/enzimología , Mácula Lútea/patología , Masculino , Mastocitos/enzimología , Persona de Mediana Edad , Desprendimiento de Retina/enzimología , Desprendimiento de Retina/etiología , Desprendimiento de Retina/patología , Enfermedades de la Retina/enzimología , Enfermedades de la Retina/patología , Perforaciones de la Retina/enzimología , Perforaciones de la Retina/etiología , Perforaciones de la Retina/patología , Triptasas/sangre , Triptasas/metabolismo , Cuerpo Vítreo/enzimología , Cuerpo Vítreo/patologíaRESUMEN
PURPOSE: Microparticle technology enables local administration of medication. The purpose of this study was to examine the inhibitory effect of locally administered candesartan (CAN)-encapsulated microparticles on experimental choroidal neovascularization (CNV). METHODS: Laser photocoagulation was used to induce CNV in Brown Norway rats. The rats were pretreated with subconjunctival injections of CAN (5.0 mg/eye) or phosphate buffer saline for 3 days before photocoagulation. The volume of CNV was evaluated 7 days after laser injury using the lectin staining technique. The infiltration of macrophages within the CNV lesion was determined using immunofluorescent staining with an anti-CD68 antibody. mRNA levels of MCP-1, IL1-ß and VEGF in the retinal pigment epithelium/choroid complex were determined using quantitative PCR (q-PCR). RESULTS: CNV volume was significantly suppressed by the treatment with CAN compared with that in vehicle-treated eyes (P<0.05, two-tailed Student's t-test). Subconjunctival injections of CAN decreased the numbers of CD68+ cells in the CNV lesion. The increased mRNA levels of MCP-1, IL1-ß, and VEGF induced by photocoagulation was significantly suppressed following the local administration of CAN (P<0.05, two-tailed Student's t-test). CONCLUSION: Local administration of CAN inhibited experimentally induced CNV possibly through anti-inflammatory effects.
RESUMEN
Retinal vein occlusion (RVO) is a common vascular disease of the retina; however, the pathogenesis of RVO is still unclear. Branch RVO (BRVO) commonly occurs at the arteriovenous crossing and it was formerly believed that the diseased artery mechanically compresses the vein. However, it has been reported that the retinal vein runs deep beneath the artery at the arteriovenous crossing in eyes with an arterial overcrossing, and the venous lumen often appears to be preserved, even at the arteriovenous crossing, as shown by optical coherence tomography. Paques et al. [1] found venous nicking without arteriovenous contact using adaptive optics imaging. Thus, we investigated the potential role of a dysregulation of the retinal vein. While the pathogenesis of retinal vein occlusion (RVO) is still unclear, systemic hypertension and increased level of endothelin-1 (ET-1) are known risk factors (Flammer and Konieczka, 2015) [2]. We focused on the behavior of retinal veins in spontaneous hypertensive rats (SHR). Then, one of the retinal veins became exceptionally constricted and was nearly occluded (Fig. 1), and the chorioretinal blood flow significantly decreased in the retinas of SHRs following the intravenous injection of ET-1. In addition, immunoreactivity to ET-A receptor was higher in SHR retinas than in control (WKY; Wistar Kyoto rat) retinas (Fig. 2). The protein levels of ET-A receptor and HIF-1 were also significantly higher in SHR retinas than in WKY retinas (Fig. 3). We observed vasoactivity of retinal veins; a retinal venous constriction (Kida et al., 2018) [3]. This supports the hypothesis that ET-1 can constrict retinal veins, thus increasing retinal venous pressure, and that ET-1 may even contribute to the pathogenesis of RVO.
RESUMEN
Purpose: To determine whether P7C3-A20 can inhibit the phosphorylation of the mammalian target of rapamycin (mTOR), depress neuroinflammation, and protect retinal ganglion cells (RGCs) of rats from optic nerve crush (ONC). Methods: The left optic nerve was crushed, and 5.0 mg/kg/d of P7C3-A20, 1.0 mg/kg/d of rapamycin, or their vehicle was injected intraperitoneally for 3 consecutive days beginning 1 day before the ONC. The protective effects on the RGCs were determined by immunohistochemical staining for Tuj-1. The level of phosphorylated mTOR was determined by immunoblotting. The neuroinflammation in the optic nerve was determined by changes in the expression of CD68, TNF-α, MCP-1, and iNOS. Results: The density of Tuj-1-stained cells in the control was 2010 ± 81.5/mm2 and 1842 ± 80.4/mm2 on days 7 and 14 after the sham operation. These levels were lower at 995 ± 122/mm2 and 450 ± 52.4/mm2 on days 7 and 14 after the ONC, respectively. Rapamycin and P7C3-A20 preserved the density at significantly higher levels on both days (P < 0.05, Scheffe test). The level of phosphorylated mTOR increased by 1.56-fold above the control level on day 7. Rapamycin and P7C3 significantly lowered the level of phosphorylated mTOR to 0.89-fold and 0.67-fold of the control, respectively. There was an accumulation of CD68+ cells that were immunoreactive to TNF-α at the crush site. The expression of MCP-1 and iNOS was increased chiefly in the astrocytes around the lesion. These inflammatory events were suppressed by both rapamycin and P7C3. Conclusions: P7C3-A20 can inhibit mTOR phosphorylation in the crushed optic nerve, which may suppress neuroinflammation and preserve the RGCs.