RESUMEN
As a prototypical member of the IL-17 family, interleukin-17A (IL-17A) has received increasing attentions for its potent proinflammatory role as well as potential to be a key therapeutic target in human autoimmune inflammatory diseases; however, its roles in other pathological scenarios like neuroinflammations are not fully elucidated yet but appear essentially correlating and promising. Glaucoma is the leading cause of irreversible blindness with complicated pathogenesis still to be understood, where neuroinflammation was reported to be critically involved in its both initiation and progression. Whether IL-17A takes part in the pathogenesis of glaucoma through interfering neuroinflammation due to its potent proinflammatory effect is still unknown. In the present study, we investigated the role of IL-17A in the pathological process of glaucoma neuropathy as well as its relationship with the predominant immune inflammation mediator microglia in retina, trying to elucidate the underlying mechanisms from the view of inflammation modulation. In our study, RNA sequencing was performed for the retinas of chronic ocular hypertension (COH) and control mice. Western blot, RT-PCR, immunofluorescence, and ELISA were used to evaluate the microglial activation and proinflammatory cytokines release at conditioned levels of IL-17A, along with assessment of optic nerve integrity including retinal ganglion cells (RGCs) counting, axonal neurofilament quantification, and flash visual-evoked potential (F-VEP) examination. And the possibly involved signaling pathways were screened out to go through further validation in scenarios with conditioned IL-17A. Subsequently, IL-17A was found to be significantly upregulated in COH retina. Furthermore, suppression of IL-17A effectively diminished the loss of RGCs, improved axonal quality, and F-VEP performance in COH mice. Mechanistically, IL-17A promoted microglial activation and proinflammatory cytokines release along with enhanced phenotypic conversion of activated microglia to M2-type in early stage and to M1-type in late stage in glaucomatous retinas. Microglia elimination decreased the proinflammatory factors secretion, enhanced the RGCs survival and axonal quality mediated by IL-17A. Furthermore, IL-17A-induced the overactivation of microglia in glaucomatous condition was alleviated after blocking the p38 MAPK pathway. Taken together, IL-17A is involved in the regulation of retinal immune response and RGCs cell death in experimental glaucoma by essentially promoting retinal microglial activation via p38 MAPK signaling pathway. IL-17A dynamically regulates the phenotypic conversion of retinal microglia in experimental glaucoma partly depending on the duration of elevated intraocular pressure. Suppression of IL-17A contributes to alleviate glaucoma neuropathy and exhibits promising potential as an innovative target for therapeutic strategy in glaucoma.
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Glaucoma , Hipertensión Ocular , Ratones , Humanos , Animales , Interleucina-17/metabolismo , Microglía/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Enfermedades Neuroinflamatorias , Glaucoma/metabolismo , Retina/metabolismo , Hipertensión Ocular/etiología , Inflamación/metabolismo , Citocinas/metabolismo , Modelos Animales de EnfermedadRESUMEN
PURPOSE: To determine whether short-latency changes in multifocal electroretinography (mfERG) observed in experimental glaucoma (EG) are secondary solely to retinal ganglion cell (RGC) loss or whether there is a separate contribution from elevated intraocular pressure (IOP). METHODS: Prior to operative procedures, a series of baseline mfERGs were recorded from six rhesus macaques using a 241-element unstretched stimulus. Animals then underwent hemiretinal endodiathermy axotomy (HEA) by placing burns along the inferior 180° of the optic nerve margin in the right eye (OD). mfERG recordings were obtained in each animal at regular intervals following for 3-4 months to allow stabilization of the HEA effects. Laser trabecular meshwork destruction (LTD) to elevate IOP was then performed; first-order kernel (K1) waveform root-mean-square (RMS) amplitudes for the short-latency segment of the mfERG wave (9-35 ms) were computed for two 7-hexagon groupings-the first located within the superior (non-axotomized) macula and the second within the inferior (axotomized) macula. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was done. RESULTS: By 3 months post HEA, there was marked thinning of the inferior nerve fiber layer as measured by optical coherence tomography. Compared with baseline, no statistically significant changes in 9-35 ms K1 RMS amplitudes were evident in either the axotomized or non-axotomized portions of the macula. Following LTD, mean IOP in HEA eyes rose to 46 ± 9 compared with 20 ± 2 mmHg (SD) in the fellow control eyes. In the HEA + EG eyes, statistically significant increases in K1 RMS amplitude were present in both the axotomized inferior and non-axotomized superior portions of the OD retinas. No changes in K1 RMS amplitude were found in the fellow control eyes from baseline to HEA epoch, but there was a smaller increase from baseline to HEA + EG. Upregulation of GFAP in the Müller cells was evident in both non-axotomized and axotomized retina in eyes with elevated IOP. CONCLUSIONS: The RMS amplitudes of the short-latency mfERG K1 waveforms are not altered following axotomy but undergo marked increases following elevated IOP. This suggests that the increase in mfERG amplitude was not solely a result of RGC loss and may reflect photoreceptor and bipolar cell dysfunction and/or changes in Müller cells.
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Glaucoma , Células Ganglionares de la Retina , Animales , Células Ganglionares de la Retina/fisiología , Electrorretinografía/métodos , Axotomía , Macaca mulatta/fisiología , Glaucoma/diagnóstico , Retina , Presión IntraocularRESUMEN
Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 µm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13-15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.
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Antagonistas de Leucotrieno/uso terapéutico , Microglía/metabolismo , Hipertensión Ocular/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Leucotrienos/metabolismo , Células Ganglionares de la Retina/fisiología , Acetatos/uso terapéutico , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Proteínas de Unión al Calcio/metabolismo , Supervivencia Celular/fisiología , Ciclopropanos/uso terapéutico , Modelos Animales de Enfermedad , Electrorretinografía , Femenino , Regulación de la Expresión Génica/fisiología , Presión Intraocular/fisiología , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Hipertensión Ocular/fisiopatología , Quinolinas/uso terapéutico , ARN Mensajero/genética , Ratas , Ratas Endogámicas BN , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/metabolismo , Retina/fisiopatología , Sulfuros/uso terapéutico , Tonometría Ocular , Factor de Transcripción Brn-3B/metabolismoRESUMEN
Somatostatin plays important roles in modulating neuronal functions by activating the five specific G-protein coupled receptors (sst1-sst5). Previous studies have demonstrated that sst5 were expressed in retinal ganglion cells (RGCs) and sst5 agonist attenuated the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid-induced retinal neurotoxicity. In this study, we investigated effects and underlying mechanisms of the sst5 agonist L-817,818 on RGC injury induced by elevated intraocular pressure (COH) in experimental glaucoma. Our results showed that intraperitoneal administration of L-817,818 significantly reduced RGC loss and decreased the number of terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL)-positive RGCs in COH retinas, suggesting that L-817,818 may attenuate RGC apoptosis. Consistently, in COH retinas with L-817,818 administration, both the down-regulated mRNA and protein levels of anti-apoptotic Bcl-2 and the up-regulated mRNA and protein levels of pro-apoptotic Bax were partially reversed. L-817,818 administration downregulated the expression of apoptosis-related proteins caspase-9 and caspase-3 in COH retinas. In addition, L-817,818 administration reduced the concentrations of reactive oxygen species/reactive nitrogen species and malondialdehyde, and ameliorated the functions of mitochondrial respiratory chain complex (MRCC). Our results imply that administration of the sst5 agonist L-817,818 reduces RGC loss in COH rats through decreasing RGC apoptosis, which is mediated by regulating Bcl-2/Bax balance, reducing oxidative stress and rescuing activities of MRCC. Activation of sst5 may provide neuroprotective roles for RGCs in glaucoma.
Asunto(s)
Amidas/farmacología , Modelos Animales de Enfermedad , Glaucoma/patología , Naftalenos/farmacología , Fármacos Neuroprotectores/farmacología , Receptores de Somatostatina/agonistas , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular , Peróxido de Hidrógeno/metabolismo , Etiquetado Corte-Fin in Situ , Inyecciones Intraperitoneales , Masculino , Malondialdehído/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Superóxido Dismutasa/metabolismo , Proteína X Asociada a bcl-2/genéticaRESUMEN
Glaucoma is a neurodegenerative disease characterized by the loss of retinal ganglion cells (RGCs). An increase in the intraocular pressure is the principal risk factor for such loss, but controlling this pressure does not always prevent glaucomatous damage. Activation of immune cells resident in the retina (microglia) may contribute to RGC death. Thus, a substance with anti-inflammatory activity may protect against RGC degeneration. This study investigated the neuroprotective and anti-inflammatory effects of a hydrophilic saffron extract standardized to 3% crocin content in a mouse model of unilateral, laser-induced ocular hypertension (OHT). Treatment with saffron extract decreased microglion numbers and morphological signs of their activation, including soma size and process retraction, both in OHT and in contralateral eyes. Saffron extract treatment also partially reversed OHT-induced down-regulation of P2RY12. In addition, the extract prevented retinal ganglion cell death in OHT eyes. Oral administration of saffron extract was able to decrease the neuroinflammation associated with increased intraocular pressure, preventing retinal ganglion cell death. Our findings indicate that saffron extract may exert a protective effect in glaucomatous pathology.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/farmacología , Crocus/química , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Animales , Biomarcadores , Modelos Animales de Enfermedad , Glaucoma/tratamiento farmacológico , Glaucoma/etiología , Glaucoma/metabolismo , Glaucoma/fisiopatología , Interacciones Hidrofóbicas e Hidrofílicas , Presión Intraocular/efectos de los fármacos , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patologíaRESUMEN
Glaucoma is a group of neurodegenerative eye diseases characterized by progressive impairment of visual function due to loss of retinal ganglion cells (RGC). As hydrogen sulfide (H2S) was reported to play a role in the process of glaucomatous neuropathy and improve RGC survival in experimental glaucoma, the authors aimed to investigate the anti-apoptosis effect of H2S supplement in a rat glaucoma model, and further tried to explore the involved factors in the neuroprotection. A chronic ocular hypertension (COH) rat model induced by intracameral injection of cross-linking hydrogel was employed to simulate glaucoma and 288 rats were subjected to experimental procedures in the present study. After 4 weeks of sodium hydrosulfide (NaHS) administration following COH induction, the apoptosis of RGC isolated from experimented rats as well as apoptosis of neurons in ganglion cell layer (GCL), intrinsic apoptotic pathway activity, mitochondrial function, glial activation, NF-κB pathway activity, NADPH oxidase activity, autophagy activity and TNF-α level in retina were evaluated. The results showed that H2S supplement effectively attenuated the apoptosis of RGC in experimental glaucoma, and the neuroprotection by H2S might correlate with preservation of mitochondrial function, attenuation of oxidative stress, suppression of glial activation, inhibition of inflammatory pathways and downregulation of autophagy. Our study indicated that H2S supplement resulted in significant neuroprotection through attenuation of RGC apoptosis which might be linked with multiple factors in experimental glaucoma. The new therapeutic strategy might potentially contribute to benefit glaucoma treatment, which is worth further concerns.
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Apoptosis/efectos de los fármacos , Glaucoma/tratamiento farmacológico , Sulfuro de Hidrógeno/farmacología , Fármacos Neuroprotectores/farmacología , Hipertensión Ocular/tratamiento farmacológico , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Autofagia/efectos de los fármacos , Modelos Animales de Enfermedad , Glaucoma/metabolismo , Masculino , Mitocondrias/efectos de los fármacos , NADPH Oxidasas/metabolismo , Neuroglía/efectos de los fármacos , Hipertensión Ocular/metabolismo , Ratas , Retina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The immune system plays an important role in glaucomatous neurodegeneration. Retinal microglial reactivation associated with ganglion cell loss could reportedly contribute to the glaucoma progression. Recently we have described signs of microglia activation both in contralateral and ocular hypertension (OHT) eyes involving all retinal layers 15 days after OHT laser induction in mice. However, no works available have analyzed the microglial activation at earliest time points after OHT induction (24 h) in this experimental model. Thus, we seek to describe and quantify signs of microglia activation and differences depending on the retinal layer, 24 h after unilateral laser-induced OHT. Two groups of adult Swiss mice were used: age-matched control (naïve) and lasered. In the lasered animals, OHT eyes as well as contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1 and MHC-II. We quantified the number of microglial cells in the photoreceptor layer (OS), outer plexiform layer (OPL), and inner plexiform layer (IPL); the number of microglial vertical processes connecting the OPL and OS; the area of the retina occupied by Iba-1+ cells (Iba1-RA) in the nerve fiber layer-ganglion cell layer (NFL-GCL), the total arbor area of microglial cells in the OPL and IPL and; Iba-1+ cell body area in the OPL, IPL and NFL-GCL. In contralateral and OHT eyes the morphological features of Iba-1+ cell activation were: migration, enlargement of the cell body, higher degree of branching and reorientation of the processes, radial disposition of the soma and processes toward adjacent microglial plexuses, and presence of amoeboid cells acting as macrophages. These signs were more pronounced in OHT eyes. Most of Iba-1+ cells did not express MHC-II; rather, only dendritic and rounded cells expressed it. In comparison with naïve eyes, in OHT eyes and contralateral eyes no significant differences were found in the microglial cell number; but there was a significant increase in Iba1-RA. The total arbor area of microglial cells was significantly decreased in: i) OHT eyes with respect contralateral eyes and naïve-eyes in IPL; ii) OHT eyes with respect to naïve eyes in OPL. The number of microglial vertical processes connecting the OPL and OS were significantly increased in contralateral eyes compared with naïve-eyes and OHT eyes. In OPL, IPL and NFL-GCL, the cell body area of Iba-1+ cells was significantly greater in OHT eyes than in naïve and contralateral eyes, and greater in contralateral eyes than in naïve eyes. A non-proliferative microglial reactivation was detected both in contralateral eyes and in OHT eyes in an early time after unilateral laser-induced OHT (24 h). This fast microglial activation, which involves the contralateral eye, could be mediated by the immune system.
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Modelos Animales de Enfermedad , Microglía/metabolismo , Hipertensión Ocular/metabolismo , Retina/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Recuento de Células , Técnica del Anticuerpo Fluorescente Indirecta , Antígenos de Histocompatibilidad Clase II/metabolismo , Presión Intraocular/fisiología , Coagulación con Láser/efectos adversos , Masculino , Ratones , Proteínas de Microfilamentos/metabolismo , Microglía/patología , Fibras Nerviosas/metabolismo , Hipertensión Ocular/etiología , Hipertensión Ocular/patología , Retina/patología , Células Ganglionares de la Retina/metabolismo , Tonometría OcularRESUMEN
A shift or displacement of the retinal blood vessels (RBVs) with neuroretinal rim thinning indicates the progression of glaucomatous optic neuropathy. In chronic open angle glaucoma, individuals with RBV positional shifts exhibit more rapid visual field loss than those without RBV shifts. The retinal vessels reportedly move onto the optic nerve head (ONH) in response to glaucoma damage, suggesting that RBVs are pulled toward the ONH in response to increased cupping. Whether this phenomenon only applies to RVBs located in the vicinity or inside the ONH or, more generally, to RBVs also located far from the ONH, however, is unclear. The aim of this study was to evaluate the movement of RBVs located relatively far from the ONH edge after increasing intraocular pressure (IOP) in an experimental monkey model of glaucoma. Fundus photographs were obtained in 17 monkeys. High IOP was induced in the monkeys by laser photocoagulation burns applied uniformly with 360° irradiation around the trabecular meshwork of the left eye. The right eye was left intact and used as a non-treated control. Considering the circadian rhythm of IOP, it was measured in both eyes of each animal at around the same time-points. Then, fundus photographs were obtained. Using Image J image analysis software, an examiner (N.E.) measured the fundus photographs at two time-points, i.e. before laser treatment (time 1) and the last fundus photography after IOP elevation (time 2). The following parameters were measured (in pixels): 1) vertical diameter of the ONH (DD), 2) distance from the ONH edge to the first bifurcation point of the superior branch of the central retinal vein (UV), 3) distance from the ONH edge to the first bifurcation point of the inferior branch of the central retinal vein (LV), 4) ONH area, and 5) surface area of the cup of the ONH. We calculated the ratios of UV to DD (UV/DD), LV to DD (LV/DD), and the cup area to disc area ratio (C/D). The mean UV/DD at time 1 (0.656 ± 0.233) was decreased at time 2 (0.542 ± 0.192) (p < 0.01), and the mean LV/DD at time 1 (0.642 ± 0.151) was decreased at time 2 (0.534 ± 0.171) (p < 0.01). The mean C/D at time 1 (0.303 ± 0.035) was increased at time 2 (0.556 ± 0.110) (p < 0.01). The mean IOP at time 1 was 19.8 ± 2.5 and that at time 2 was 54.2 ± 15.8. The amount and rate of the change in LV/DD and C/D between time 1 and time 2 were significantly correlated (r = -0.654 and -0.536, p = 0.004 and 0.026, respectively). Therefore, in an experimental monkey model of glaucoma, RBVs located relatively far from the ONH were pulled toward the ONH as cupping increased.
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Glaucoma/fisiopatología , Presión Intraocular/fisiología , Disco Óptico/irrigación sanguínea , Vasos Retinianos/fisiopatología , Animales , Modelos Animales de Enfermedad , Glaucoma/diagnóstico , Glaucoma/etiología , Haplorrinos , Masculino , Vasos Retinianos/diagnóstico por imagenRESUMEN
AIMS: We investigated the relationship between elevated intraocular pressure (IOP) and changes in global and peripapillary sector retinal nerve fiber layer (RNFL) thickness around the optic nerve head (ONH) in the laser-induced ocular hypertension monkey model. METHODS: To induce high IOP, green laser photocoagulation burns were applied around the trabecular meshwork of 1 eye from each of 12 cynomolgus monkeys. The animals had been acclimated to IOP measurement under conscious conditions for more than 2 months, and IOP was chronologically measured. RNFL thickness was measured for 6 peripapillary sectors and global area using spectral-domain optical coherence tomography. RESULTS: After model induction, marked IOP elevation and enlarged optic disk cupping were observed. Thinning of the RNFL associated with elevated IOP was observed around the ONH from 6 until 9 weeks after laser treatment, and the degree of reduction in RNFL thickness varied between the peripapillary sectors. Correlations between cumulative IOP elevation and RNFL thickness reduction were statistically significant for the temporal-superior (p = 0.024), nasal-inferior (p = 0.044), and temporal (p = 0.049) sectors, and global RNFL (p = 0.018). CONCLUSIONS: These results suggest that this model reflected the pathology of clinical glaucoma in terms of the specific pattern of RNFL thinning around the ONH.
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Presión Intraocular/fisiología , Fibras Nerviosas/patología , Hipertensión Ocular/fisiopatología , Disco Óptico/patología , Células Ganglionares de la Retina/patología , Tomografía de Coherencia Óptica/métodos , Animales , Modelos Animales de Enfermedad , Rayos Láser/efectos adversos , Macaca fascicularis , Masculino , Hipertensión Ocular/diagnósticoRESUMEN
Glaucoma is a neurodegenerative disease that leads to irreversible retinal ganglion cell (RGC) loss and is one of the main causes of blindness worldwide. The pathogenesis of glaucoma remains unclear, and novel approaches for neuroprotective treatments are urgently needed. Previous studies have revealed significant down-regulation of α-crystallin B as an initial reaction to elevated intraocular pressure (IOP), followed by a clear but delayed up-regulation, suggesting that this small heat-shock protein plays a pathophysiological role in the disease. This study analyzed the neuroprotective effect of α-crystallin B in an experimental animal model of glaucoma. Significant IOP elevation induced by episcleral vein cauterization resulted in a considerable impairment of the RGCs and the retinal nerve fiber layer. An intravitreal injection of α-crystallin B at the time of the IOP increase was able to rescue the RGCs, as measured in a functional photopic electroretinogram, retinal nerve fiber layer thickness, and RGC counts. Mass-spectrometry-based proteomics and antibody-microarray measurements indicated that a α-crystallin injection distinctly up-regulated all of the subclasses (α, ß, and γ) of the crystallin protein family. The creation of an interactive protein network revealed clear correlations between individual proteins, which showed a regulatory shift resulting from the crystallin injection. The neuroprotective properties of α-crystallin B further demonstrate the potential importance of crystallin proteins in developing therapeutic options for glaucoma.
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Glaucoma/metabolismo , Fármacos Neuroprotectores/metabolismo , Cadena B de alfa-Cristalina/metabolismo , Animales , Recuento de Células , Modelos Animales de Enfermedad , Regulación hacia Abajo , Electrorretinografía , Glaucoma/patología , Glaucoma/fisiopatología , Presión Intraocular , Espectrometría de Masas , Mapas de Interacción de Proteínas , Proteómica , Células Ganglionares de la Retina/patología , Neuronas Retinianas/metabolismo , Neuronas Retinianas/patología , Regulación hacia ArribaRESUMEN
Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, and is the second leading cause of blindness worldwide. Elevated intraocular pressure is a well known risk factor for the development of glaucomatous optic neuropathy and pharmacological or surgical lowering of intraocular pressure represents a standard procedure in glaucoma treatment. However, the treatment options are limited and although lowering of intraocular pressure impedes disease progression, glaucoma cannot be cured by the currently available therapy concepts. In an acute short-term ocular hypertension model in rat, we characterize RGC loss, but also microglial cell activation and vascular alterations of the retina at certain time points. The combination of these three parameters might facilitate a better evaluation of the disease progression, and could further serve as a new model to test novel treatment strategies at certain time points. Acute ocular hypertension (OHT) was induced by the injection of magnetic microbeads into the rat anterior chamber angle (n = 22) with magnetic position control, leading to constant elevation of IOP. At certain time points post injection (4d, 7d, 10d, 14d and 21d), RGC loss, microglial activation, and microvascular pericyte (PC) coverage was analyzed using immunohistochemistry with corresponding specific markers (Brn3a, Iba1, NG2). Additionally, the tightness of the retinal vasculature was determined via injections of Texas Red labeled dextran (10 kDa) and subsequently analyzed for vascular leakage. For documentation, confocal laser-scanning microscopy was used, followed by cell counts, capillary length measurements and morphological and statistical analysis. The injection of magnetic microbeads led to a progressive loss of RGCs at the five time points investigated (20.07%, 29.52%, 41.80%, 61.40% and 76.57%). Microglial cells increased in number and displayed an activated morphology, as revealed by Iba1-positive cell number (150.23%, 175%, 429.25%,486.72% and 544.78%) and particle size analysis (205.49%, 203.37%, 412.84%, 333.37% and 299.77%) compared to contralateral control eyes. Pericyte coverage (NG2-positive PC/mm) displayed a significant reduction after 7d of OHT in central, and after 7d and 10d in peripheral retina. Despite these alterations, the tightness of the retinal vasculature remained unaltered at 14 and 21 days after OHT induction. While vascular tightness was unchanged in the course of OHT, a progressive loss of RGCs and activation of microglial cells was detected. Since a significant loss in RGCs was observed already at day 4 of experimental glaucoma, and since activated microglia peaked at day 10, we determined a time frame of 7-14 days after MB injection as potential optimum to study glaucoma mechanisms in this model.
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Barrera Hematorretinal/patología , Modelos Animales de Enfermedad , Microglía/patología , Hipertensión Ocular/patología , Células Ganglionares de la Retina/patología , Enfermedad Aguda , Animales , Antígenos/metabolismo , Biomarcadores/metabolismo , Barrera Hematorretinal/metabolismo , Proteínas de Unión al Calcio/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Presión Intraocular , Masculino , Proteínas de Microfilamentos/metabolismo , Microglía/metabolismo , Microscopía Confocal , Hipertensión Ocular/etiología , Hipertensión Ocular/metabolismo , Proteoglicanos/metabolismo , Ratas , Ratas Endogámicas BN , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Ganglionares de la Retina/metabolismo , Factores de Tiempo , Factor de Transcripción Brn-3A/metabolismoRESUMEN
In this study, we utilized yellow-wavelength laser treatment and measured aqueous outflow facility to establish a model for chronic glaucoma in rhesus monkeys. We then compared the effects of photocoagulation resulting from exposure to the yellow laser or to a green laser. Twelve rhesus monkeys were used to establish the model, and the yellow and green lasers were utilized for 360° photocoagulation in the anterior-chamber angles of the right eye in all subjects. After certain periods of time before and after the creation of the glaucoma model, the cornea, aqueous humor, optic cup, intraocular pressure (IOP), outflow facility, retinal nerve fiber layer (RNFL), and pathology of the trabecular meshwork were analyzed. Both the yellow and green lasers caused an increase in IOP compared with before photocoagulation (18.6 ± 2.6 mm Hg and 16.1 ± 1.8 mm Hg, respectively), with an average photocoagulation from the yellow and green lasers of 39.2 ± 7.9 mm Hg and 30.3 ± 4.7 mm Hg, respectively (P < 0.01). However, the success rate of a second photocoagulation treatment in the yellow laser group was significantly higher than in the green laser group (P < 0.05). After the increase in IOP, both groups exhibited an inflammatory response in the anterior segment, enlarged cupping, and a decrease in the average thickness of the RNFL. However, the yellow laser caused less corneal edema than the green laser (P < 0.05), and the outflow facility of the two groups (0.33 ± 0.09 and 0.30 ± 0.07 µl/min/mm Hg for the yellow and green lasers, respectively) showed different degrees of differences (0.05 ± 0.02 and 0.07 ± 0.02 µl/min/mm Hg for the yellow and green lasers, respectively) into the abnormal range after photocoagulation. Pathological examination revealed that the depth of destruction of the trabecular meshwork appeared to be deeper in the yellow laser group than in the green laser group. In conclusion, application of a yellow laser combined with measuring aqueous outflow facility produced a glaucoma model with a minor inflammatory response and few IOP fluctuations.
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Glaucoma/terapia , Presión Intraocular , Coagulación con Láser/métodos , Malla Trabecular/cirugía , Animales , Humor Acuoso/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Glaucoma/fisiopatología , Macaca mulatta , Malla Trabecular/patología , Resultado del TratamientoRESUMEN
The purpose of this report is to summarize the current strengths and weaknesses of the non-human primate (NHP) experimental glaucoma (EG) model through sections devoted to its history, methods, important findings, alternative optic neuropathy models and future directions. NHP EG has become well established for studying human glaucoma in part because the NHP optic nerve head (ONH) shares a close anatomic association with the human ONH and because it provides the only means of systematically studying the very earliest visual system responses to chronic intraocular pressure (IOP) elevation, i.e. the conversion from ocular hypertension to glaucomatous damage. However, NHPs are impractical for studies that require large animal numbers, demonstrate spontaneous glaucoma only rarely, do not currently provide a model of the neuropathy at normal levels of IOP, and cannot easily be genetically manipulated, except through tissue-specific, viral vectors. The goal of this summary is to direct NHP EG and non-NHP EG investigators to the previous, current and future accomplishment of clinically relevant knowledge in this model.
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Glaucoma/fisiopatología , Presión Intraocular/fisiología , Nervio Óptico/patología , Células Ganglionares de la Retina/patología , Animales , Modelos Animales de Enfermedad , Glaucoma/patología , PrimatesRESUMEN
To investigate the long-term effects of laser-photocoagulation (LP)-induced ocular hypertension (OHT) in the innermost and outermost (outer-nuclear and outer segment)-retinal layers (ORL). OHT was induced in the left eye of adult rats. To investigate the ganglion cell layer (GCL) wholemounts were examined at 1, 3 or 6 months using Brn3a-immunodetection to identify retinal ganglion cells (RGCs) and DAPI-staining to detect all nuclei in this layer. To study the effects of LP on the ORL up to 6 months, retinas were: i) fresh extracted to quantify the levels of rod-, S- and L-opsin; ii) cut in cross-sections for morphometric analysis, or; iii) prepared as wholemounts to quantify and study retinal distributions of entire populations of RGCs (retrogradely labeled with fluorogold, FG), S- and L-cones (immunolabeled). OHT resulted in wedge-like sectors with their apex on the optic disc devoid of Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. The levels of all opsins diminished by 2 weeks and further decreased to 20% of basal-levels by 3 months. Cross-sections revealed focal areas of ORL degeneration. RGC survival at 15 days represented approximately 28% and did not change with time, whereas the S- and L-cone populations diminished to 65% and 80%, or to 20 and 35% at 1 or 6 months, respectively. In conclusion, LP induces in the GCL selective RGCs loss that does not progress after 1 month, and S- and L-cone loss that progresses for up to 6 months. Thus, OHT results in severe damage to both the innermost and the ORL.
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Coagulación con Láser/efectos adversos , Hipertensión Ocular/patología , Retina/patología , Animales , Western Blotting , Recuento de Células , Modelos Animales de Enfermedad , Femenino , Hipertensión Ocular/etiología , Opsinas/metabolismo , Ratas , Ratas Sprague-Dawley , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/efectos de la radiaciónRESUMEN
Macroglia (astrocytes and Müller glia) may play an important role in the pathogenesis of glaucoma. In a glaucoma mouse model, we studied the effects of unilateral laser-induced ocular hypertension (OHT) on macroglia in OHT and contralateral eyes at different time points after laser treatment (1, 3, 5, 8 and 15 days) using anti-GFAP and anti-MHC-II, analyzing the morphological changes, GFAP-labelled retinal area (GFAP-PA), and GFAP and MHC-II immunoreactivity intensities ((GFAP-IRI and MHC-II-IRI)). In OHT and contralateral eyes, with respect to naïve eyes, at all the time points, we found the following: (i) astrocytes with thicker somas and more secondary processes, mainly in the intermediate (IR) and peripheral retina (PR); (ii) astrocytes with low GFAP-IRI and only primary processes near the optic disc (OD); (iii) an increase in total GFAP-RA, which was higher at 3 and 5 days, except for at 15 days; (iv) an increase in GFAP-IRI in the IR and especially in the PR; (v) a decrease in GFAP-IRI near the OD, especially at 1 and 5 days; (vi) a significant increase in MHC-II-IRI, which was higher in the IR and PR; and (vii) the Müller glia were GFAP+ and MHC-II+. In conclusion, in this model of glaucoma, there is a bilateral macroglial activation maintained over time involved in the inflammatory glaucoma process.
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Age and intraocular pressure (IOP) are the two most important risk factors for the development and progression of open-angle glaucoma. While IOP is commonly considered in models of experimental glaucoma (EG), most studies use juvenile or adult animals and seldom older animals which are representative of the human disease. This paper provides a concise review of how retinal ganglion cell (RGC) loss, the hallmark of glaucoma, can be evaluated in EG with a special emphasis on serial in vivo imaging, a parallel approach used in clinical practice. It appraises the suitability of EG models for the purpose of in vivo imaging and argues for the use of models that provide a sustained elevation of IOP, without compromise of the ocular media. In a study with parallel cohorts of adult (3-month-old, equivalent to 20 human years) and old (2-year-old, equivalent to 70 human years) mice, we compare the effects of elevated IOP on serial ganglion cell complex thickness and individual RGC dendritic morphology changes obtained in vivo. We also evaluate how age modulates the impact of elevated IOP on RGC somal and axonal density in histological analysis as well the density of melanopsin RGCs. We discuss the challenges of using old animals and emphasize the potential of single RGC imaging for understanding the pathobiology of RGC loss and evaluating new therapeutic avenues.
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Glaucoma de Ángulo Abierto , Glaucoma , Animales , Modelos Animales de Enfermedad , Glaucoma/patología , Humanos , Presión Intraocular , Ratones , Tonometría OcularRESUMEN
Glaucoma represents a group of chronic neurodegenerative diseases, constituting the second leading cause of blindness worldwide. To date, chronically elevated intraocular pressure has been identified as the main risk factor and the only treatable symptom. However, there is increasing evidence in the recent literature that IOP-independent molecular mechanisms also play an important role in the progression of the disease. In recent years, it has become increasingly clear that glaucoma has an autoimmune component. The main focus nowadays is elucidating glaucoma pathogenesis, finding early diagnostic options and new therapeutic approaches. This review article summarizes the impact of different antibodies and proteins associated with glaucoma that can be detected for example by microarray and mass spectrometric analyzes, which (i) provide information about expression profiles and associated molecular signaling pathways, (ii) can possibly be used as a diagnostic tool in future and, (iii) can identify possible targets for therapeutic approaches.
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Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of acute IOP elevation, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss and improved functional outcomes in a mouse model of acute IOP elevation. These results provide evidence that Annexin V-conjugated cubosomes-based LM22A-4 delivery may be a useful targeted approach to prevent the progression of RGCs loss in glaucoma. STATEMENT OF SIGNIFICANCE: Recent studies suggest that the therapy of effectively delivering neurotrophic factors to the injured retinal ganglion cells (RGCs) could promote the survival of RGCs in glaucoma. Our present work has for the first time used cubosomes as an active targeted delivery system and have successfully delivered a neuroprotective drug to the damaged RGCs in vivo. Our new cubosomal formulation can protect apoptotic cell death in vitro and in vivo, showing that cubosomes are a promising drug carrier system for ocular drug delivery and glaucoma treatment. We have further found that by controlling cubosomes in Pn3m phase we can facilitate delivery of neuroprotective drug through apoptotic membranes. This data, we believe, has important implications for future design and formulation of cubosomes for therapeutic applications.
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Glaucoma , Disco Óptico , Animales , Benzamidas , Modelos Animales de Enfermedad , Glaucoma/tratamiento farmacológico , Presión Intraocular , Ratones , Células Ganglionares de la RetinaRESUMEN
Glaucoma filtration surgery plays an important role in achieving intraocular pressure (IOP) reduction in patients who have high IOP despite maximum medical therapy. Preclinical experimental models of glaucoma filtration surgery contribute a great deal to our knowledge of the wound healing processes that predispose to scarring and may lead to poor outcomes. However, this research needs to be interpreted in the light of the specific study design, animal model and methods used. We review the existing literature addressing various models of experimental glaucoma filtration surgery, discuss the considerations in assessing these models and describe future steps in evaluating potential therapeutics and bleb characteristics that could impact translational research in this field.
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Cirugía Filtrante/métodos , Glaucoma/cirugía , Presión Intraocular/fisiología , Modelos Teóricos , Animales , Glaucoma/fisiopatología , Humanos , Cicatrización de HeridasRESUMEN
Glaucoma is a neurodegenerative disease characterized by the progressive loss of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is a primary risk factor for the development and progression of glaucoma. Rodent models of glaucoma have greatly improved our understanding of the pathophysiology of glaucoma and served as a useful tool to investigate neuroprotective agents. An ideal glaucoma animal model should be easy to induce, reproducible, biologically plausible and predictable. Of the available animal models of glaucoma, rodents are commonly studied because they have a relatively short life span and can be genetically altered. A successful hypertensive glaucoma model should induce structural glaucomatous changes: including loss of retinal nerve fibres, retinal ganglion cells and optic-disc cupping along with IOP elevation. The level and duration of IOP elevation should be titratable depending on the targeted glaucomatous damage. This review summarizes the outcomes of induced rodent hypertensive glaucoma models including intracameral injection of microbeads, laser photocoagulation, episcleral vein cauterization, injection of hypertonic saline and hyaluronic acid. We aim to provide a detailed overview of each of the models with a focus on parameters that defines a successful glaucoma model. The induced IOP elevation and duration of elevation varied among the different models and strain of rodent; nonetheless, they all achieved a sustainable raised IOP with corresponding RGC loss. The limitations of each model are discussed.