RESUMEN

Zebrafish maintain a remarkable ability to regenerate their neural retina following rapid and extensive loss of retinal neurons. This is mediated by Müller glial cells (MG), which re-enter the cell cycle to produce amplifying progenitor cells that eventually differentiate into the lost retinal neurons. For example, exposing adult albino zebrafish to intense light destroys large numbers of rod and cone photoreceptors, which are then restored by MG-mediated regeneration. Here, we describe an updated method for performing these acute phototoxic lesions to adult zebrafish retinas. Next, we contrast this method to a chronic, low light lesion model that results in a more muted and sustained damage to photoreceptors and does not trigger a MG-mediated regeneration response. Thus, these two methods can be used to compare and contrast the genetic and morphological changes associated with acute and chronic methods of photoreceptor degeneration.

Asunto(s)

Modelos Animales de Enfermedad , Degeneración Retiniana , Pez Cebra , Animales , Degeneración Retiniana/patología , Degeneración Retiniana/genética , Células Ependimogliales/patología , Células Ependimogliales/metabolismo , Luz , Células Fotorreceptoras de Vertebrados/patología , Retina/patología , Retina/metabolismo

RESUMEN

Purpose: This study aims to develop a mathematical model to elucidate fluid circulation in the retina, focusing on the movement of interstitial fluid (comprising water and albumin) to understand the mechanisms underlying exudative macular edema (EME). Methods: The model integrates physiological factors such as retinal pigment epithelium (RPE) pumping, osmotic pressure gradients, and tissue deformation. It accounts for spatial variability in hydraulic conductivity (HC) across the retina and incorporates the structural role of Müller cells (MCs) in maintaining retinal stability. Results: The model predicts that tissue deformation is maximal at the center of the fovea despite fluid exudation from blood capillaries occurring elsewhere, aligning with clinical observations. Additionally, the model suggests that spatial variability in HC across the thickness of the retina plays a protective role against fluid accumulation in the fovea. Conclusions: Despite inherent simplifications and uncertainties in parameter values, this study represents a step toward understanding the pathophysiology of EME. The findings provide insights into the mechanisms underlying fluid dynamics in the retina and fluid accumulation in the foveal region, showing that the specific conformation of Müller cells is likely to play a key role.

Asunto(s)

Líquido Extracelular , Edema Macular , Epitelio Pigmentado de la Retina , Humanos , Edema Macular/fisiopatología , Edema Macular/metabolismo , Líquido Extracelular/metabolismo , Líquido Extracelular/fisiología , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/fisiopatología , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Modelos Teóricos , Retina/fisiopatología , Retina/metabolismo , Tomografía de Coherencia Óptica , Fóvea Central/patología , Presión Osmótica

RESUMEN

Microglia are resident immune cells in the central nervous system, including the retina that surveil the environment for damage and infection. Following retinal damage, microglia undergo morphological changes, migrate to the site of damage, and express and secrete pro-inflammatory signals. In the zebrafish retina, inflammation induces the reprogramming and proliferation of Müller glia and the regeneration of neurons following damage or injury. Immunosuppression or pharmacological ablation of microglia reduce or abolish Müller glia proliferation. We evaluated the retinal architecture and retinal regeneration in adult zebrafish irf8 mutants, which have significantly depleted numbers of microglia. We show that irf8 mutants have normal retinal structure at 3 months post fertilization (mpf) and 6 mpf but fewer cone photoreceptors by 10 mpf. Surprisingly, light-induced photoreceptor ablation induced Müller glia proliferation in irf8 mutants and cone and rod photoreceptor regeneration. Light-damaged retinas from both wild-type and irf8 mutants show upregulated expression of mmp-9, il8, and tnfß pro-inflammatory cytokines. Our data demonstrate that adult zebrafish irf8 mutants can regenerate normally following acute retinal injury. These findings suggest that microglia may not be essential for retinal regeneration in zebrafish and that other mechanisms can compensate for the reduction in microglia numbers.

Asunto(s)

Factores Reguladores del Interferón , Microglía , Retina , Pez Cebra , Animales , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Microglía/metabolismo , Retina/metabolismo , Retina/patología , Mutación , Regeneración , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Proliferación Celular , Luz , Células Ependimogliales/metabolismo , Células Ependimogliales/patología

RESUMEN

Ischemic retinopathies including diabetic retinopathy are major causes of vision loss. Inner blood-retinal barrier (BRB) breakdown with retinal vascular hyperpermeability results in macular edema. Although dysfunction of the neurovascular unit including neurons, glia, and vascular cells is now understood to underlie this process, there is a need for fuller elucidation of the underlying events in BRB dysfunction in ischemic disease, including a systematic analysis of myeloid cells and exploration of cellular cross-talk. We used an approach for microglia depletion with the CSF-1R inhibitor PLX5622 (PLX) in the retinal ischemia-reperfusion (IR) model. Under non-IR conditions, PLX treatment successfully depleted microglia in the retina. PLX suppressed the microglial activation response following IR as well as infiltration of monocyte-derived macrophages. This occurred in association with reduction of retinal expression of chemokines including CCL2 and the inflammatory adhesion molecule ICAM-1. In addition, there was a marked suppression of retinal neuroinflammation with reduction in expression of IL-1b, IL-6, Ptgs2, TNF-a, and Angpt2, a protein that regulates BRB permeability. PLX treatment significantly suppressed inner BRB breakdown following IR, without an appreciable effect on neuronal dysfunction. A translatomic analysis of Müller glial-specific gene expression in vivo using the Ribotag approach demonstrated a strong suppression of Müller cell expression of multiple pro-inflammatory genes following PLX treatment. Co-culture studies of Müller cells and microglia demonstrated that activated microglia directly upregulates Müller cell-expression of these inflammatory genes, indicating Müller cells as a downstream effector of myeloid cells in retinal IR. Co-culture studies of these two cell types with endothelial cells demonstrated the ability of both activated microglia and Müller cells to compromise EC barrier function. Interestingly, quiescent Müller cells enhanced EC barrier function in this co-culture system. Together this demonstrates a pivotal role for myeloid cells in inner BRB breakdown in the setting of ischemia-associated disease and indicates that myeloid cells play a major role in iBRB dysregulation, through direct and indirect effects, while Müller glia participate in amplifying the neuroinflammatory effect of myeloid cells.

Asunto(s)

Barrera Hematorretinal , Células Ependimogliales , Células Mieloides , Barrera Hematorretinal/efectos de los fármacos , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/patología , Animales , Ratones , Células Ependimogliales/metabolismo , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/patología , Células Mieloides/metabolismo , Células Mieloides/efectos de los fármacos , Ratones Endogámicos C57BL , Enfermedades de la Retina/patología , Enfermedades de la Retina/metabolismo , Isquemia/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Masculino , Microglía/metabolismo , Microglía/efectos de los fármacos , Compuestos Orgánicos

RESUMEN

Subretinal fibrosis is a major untreatable cause of poor outcomes in neovascular age-related macular degeneration. Mouse models of subretinal fibrosis all possess a degree of invasiveness and tissue damage not typical of fibrosis progression. This project characterises JR5558 mice as a model to study subretinal fibrosis. Fundus and optical coherence tomography (OCT) imaging was used to non-invasively track lesions. Lesion number and area were quantified with ImageJ. Retinal sections, wholemounts and Western blots were used to characterise alterations. Subretinal lesions expand between 4 and 8 weeks and become established in size and location around 12 weeks. Subretinal lesions were confirmed to be fibrotic, including various cell populations involved in fibrosis development. Müller cell processes extended from superficial retina into subretinal lesions at 8 weeks. Western blotting revealed increases in fibronectin (4 wk and 8 wk, p < 0.001), CTGF (20 wks, p < 0.001), MMP2 (12 wks and 20 wks p < 0.05), αSMA (12 wks and 20 wks p < 0.05) and GFAP (8 wk and 12 wk, p ≤ 0.01), consistent with our immunofluorescence results. Intravitreal injection of Aflibercept reduced subretinal lesion growth. Our study provides evidence JR5558 mice have subretinal fibrotic lesions that grow between 4 and 8 weeks and confirms this line to be a good model to study subretinal fibrosis development and assess treatment options.

Asunto(s)

Modelos Animales de Enfermedad , Fibrosis , Retina , Tomografía de Coherencia Óptica , Animales , Ratones , Tomografía de Coherencia Óptica/métodos , Retina/patología , Retina/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Fibronectinas/metabolismo , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Degeneración Macular/patología , Degeneración Macular/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Inyecciones Intravítreas , Proteína Ácida Fibrilar de la Glía/metabolismo , Actinas/metabolismo , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión

RESUMEN

Purpose: The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. Methods: The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Results: Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000 µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Conclusions: Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. Translational Relevance: This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.

Asunto(s)

Membrana Basal , Colágeno Tipo IV , Modelos Animales de Enfermedad , Ratones Noqueados , Nefritis Hereditaria , Animales , Nefritis Hereditaria/patología , Nefritis Hereditaria/genética , Nefritis Hereditaria/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Colágeno Tipo IV/deficiencia , Ratones , Membrana Basal/metabolismo , Membrana Basal/patología , Membrana Basal/ultraestructura , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Microscopía Electrónica de Transmisión , Ratones Endogámicos C57BL , Cápsula del Cristalino/metabolismo , Cápsula del Cristalino/patología , Cápsula del Cristalino/ultraestructura , Epitelio Corneal/patología , Epitelio Corneal/ultraestructura , Epitelio Corneal/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Retina/patología , Retina/metabolismo , Retina/ultraestructura , Autoantígenos/genética , Autoantígenos/metabolismo , Células Ependimogliales/patología , Células Ependimogliales/metabolismo , Células Ependimogliales/ultraestructura , Inmunohistoquímica , Masculino

RESUMEN

Augmenting the natural melanocortin pathway in mouse eyes with uveitis or diabetes protects the retinas from degeneration. The retinal cells are protected from oxidative and apoptotic signals of death. Therefore, we investigated the effects of a therapeutic application of the melanocortin alpha-melanocyte-stimulating hormone (α-MSH) on an ischemia and reperfusion (I/R) model of retinal degenerative disease. Eyes were subjected to an I/R procedure and were treated with α-MSH. Retinal sections were histopathologically scored. Also, the retinal sections were immunostained for viable ganglion cells, activated Muller cells, microglial cells, and apoptosis. The I/R caused retinal deformation and ganglion cell loss that was significantly reduced in I/R eyes treated with α-MSH. While α-MSH treatment marginally reduced the number of GFAP-positive Muller cells, it significantly suppressed the density of Iba1-positive microglial cells in the I/R retinas. Within one hour after I/R, there was apoptosis in the ganglion cell layer, and by 48 h, there was apoptosis in all layers of the neuroretina. The α-MSH treatment significantly reduced and delayed the onset of apoptosis in the retinas of I/R eyes. The results demonstrate that therapeutically augmenting the melanocortin pathways preserves retinal structure and cell survival in eyes with progressive neuroretinal degenerative disease.

Asunto(s)

Apoptosis , Homeostasis , Daño por Reperfusión , Retina , Células Ganglionares de la Retina , alfa-MSH , Animales , Ratones , alfa-MSH/farmacología , alfa-MSH/metabolismo , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Células Ependimogliales/metabolismo , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/patología , Homeostasis/efectos de los fármacos , Ratones Endogámicos C57BL , Microglía/metabolismo , Microglía/efectos de los fármacos , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Retina/metabolismo , Retina/efectos de los fármacos , Retina/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/tratamiento farmacológico , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología

RESUMEN

Diabetic retinopathy (DR) affects over 140 million people globally. The mechanisms that lead to blindness are still enigmatic but there is evidence that sustained inflammation and hypoxia contribute to vascular damage. Despite efforts to understand the role of inflammation and microglia in DR's pathology, the contribution of astrocytes to hypoxic responses is less clear. To investigate the role of astrocytes in hypoxia-induced retinopathy, we utilized a 7-day systemic hypoxia model using the GFAP-CreERT2:Rosa26iDTR transgenic mouse line. This allows for the induction of inflammatory reactive astrogliosis following tamoxifen and diphtheria toxin administration. We hypothesize that DTx-induced astrogliosis is neuroprotective during hypoxia-induced retinopathy. Glial, neuronal, and vascular responses were quantified using immunostaining, with antibodies against GFAP, vimentin, IBA-1, NeuN, fibrinogen, and CD31. Cytokine responses were measured in both the brain and serum. We report that while both DTx and hypoxia induced a phenotype of reduced microglia morphological activation, DTx, but not hypoxia, induced an increase in the Müller glia marker vimentin. We did not observe that the combination of DTx and hypoxic treatments exacerbated the signs of reactive glial cells, nor did we observe a significant change in the expression immunomodulatory mediators IL-1ß, IL2, IL-4, IL-5, IL-6, IL-10, IL-18, CCL17, TGF-ß1, GM-CSF, TNF-α, and IFN-γ. Overall, our results suggest that, in this hypoxia model, reactive astrogliosis does not alter the inflammatory responses or cause vascular damage in the retina.

Asunto(s)

Modelos Animales de Enfermedad , Células Ependimogliales , Gliosis , Ratones Transgénicos , Microglía , Animales , Ratones , Astrocitos/metabolismo , Astrocitos/patología , Astrocitos/efectos de los fármacos , Citocinas/metabolismo , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Toxina Diftérica , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Células Ependimogliales/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/patología , Gliosis/metabolismo , Gliosis/inducido químicamente , Hipoxia/metabolismo , Hipoxia/patología , Microglía/metabolismo , Microglía/patología , Microglía/efectos de los fármacos , Retina/metabolismo , Retina/patología , Retina/efectos de los fármacos , Vimentina/metabolismo , Vimentina/genética

RESUMEN

Diabetic retinopathy (DR), a leading cause of visual impairment, demands a profound comprehension of its cellular mechanisms to formulate effective therapeutic strategies. Our study presentes a comprehensive single-cell analysis elucidating the intricate landscape of Müller cells within DR, emphasizing their nuanced involvement. Utilizing scRNA-seq data from both Sprague-Dawley rat models and human patients, we delineated distinct Müller cell clusters and their corresponding gene expression profiles. These findings were further validated through differential gene expression analysis utilizing human transcriptomic data. Notably, certain Müller cell clusters displayed upregulation of the Rho gene, implying a phagocytic response to damaged photoreceptors within the DR microenvironment. This phenomenon was consistently observed across species. Additionally, the co-expression patterns of RHO and PDE6G within Müller cell clusters provided compelling evidence supporting their potential role in maintaining retinal integrity during DR. Our results offer novel insights into the cellular dynamics of DR and underscore Müller cells as promising therapeutic targets for preserving vision in retinal disorders induced by diabetes.

Asunto(s)

Retinopatía Diabética , Células Ependimogliales , Ratas Sprague-Dawley , Análisis de la Célula Individual , Retinopatía Diabética/patología , Retinopatía Diabética/genética , Células Ependimogliales/patología , Células Ependimogliales/metabolismo , Análisis de la Célula Individual/métodos , Animales , Humanos , Ratas , Transcriptoma

RESUMEN

Diabetic retinopathy (DR) is a common microvascular complication that causes visual impairment or loss. Aquaporin 4 (AQP4) is a regulatory protein involved in water transport and metabolism. In previous studies, we found that AQP4 is related to hypoxia injury in Muller cells. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a non-selective cation channel protein involved in the regulation of a variety of ophthalmic diseases. However, the effects of AQP4 and TRPV4 on ferroptosis and oxidative stress in high glucose (HG)-treated Muller cells are unclear. In this study, we investigated the functions of AQP4 and TRPV4 in DR. HG was used to treat mouse Muller cells. Reverse transcription quantitative polymerase chain reaction was used to measure AQP4 mRNA expression. Western blotting was used to detect the protein levels of AQP4, PTGS2, GPX4, and TRPV4. Cell count kit-8, flow cytometry, 5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolyl carbocyanine iodide staining, and glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits were used to evaluate the function of the Muller cells. Streptozotocin was used to induce DR in rats. Haematoxylin and eosin staining was performed to stain the retina of rats. GSH, SOD, and MDA detection kits, immunofluorescence, and flow cytometry assays were performed to study the function of AQP4 and TRPV4 in DR rats. Results found that AQP4 and TRPV4 were overexpressed in HG-induced Muller cells and streptozotocin-induced DR rats. AQP4 inhibition promoted proliferation and cell cycle progression, repressed cell apoptosis, ferroptosis, and oxidative stress, and alleviated retinal injury in DR rats. Mechanistically, AQP4 positively regulated TRPV4 expression. Overexpression of TRPV4 enhanced ferroptosis and oxidative stress in HG-treated Muller cells, and inhibition of TRPV4 had a protective effect on DR-induced retinal injury in rats. In conclusion, inhibition of AQP4 inhibits the ferroptosis and oxidative stress in Muller cells by downregulating TRPV4, which may be a potential target for DR therapy.

Asunto(s)

Acuaporina 4 , Retinopatía Diabética , Células Ependimogliales , Ferroptosis , Estrés Oxidativo , Canales Catiónicos TRPV , Animales , Masculino , Ratones , Ratas , Acuaporina 4/metabolismo , Acuaporina 4/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Retinopatía Diabética/genética , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Glucosa/metabolismo , Glucosa/farmacología , Ratones Endogámicos C57BL , Ratas Sprague-Dawley , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética

RESUMEN

Exosomes are nanosized vesicles that have been reported as cargo-delivering vehicles between cells. Müller cells play a crucial role in the pathogenesis of diabetic retinopathy (DR). Activated Müller cells in the diabetic retina mediate disruption of barrier integrity and neovascularization. Endothelial cells constitute the inner blood-retinal barrier (BRB). Herein, we aim to evaluate the effect of Müller cell-derived exosomes on endothelial cell viability and barrier function under normal and hyperglycemic conditions. Müller cell-derived exosomes were isolated and characterized using Western blotting, nanoparticle tracking, and electron microscopy. The uptake of Müller cells-derived exosomes by the human retinal endothelial cells (HRECs) was monitored by labeling exosomes with PKH67. Endothelial cell vitality after treatment by exosomes under normo- and hypoglycemic conditions was checked by MTT assay and Western blot for apoptotic proteins. The barrier function of HRECs was evaluated by analysis of ZO-1 and transcellular electrical resistance (TER) using ECIS. Additionally, intracellular Ca+2 in HRECs was assessed by spectrofluorimetry. Analysis of the isolated exosomes showed a non-significant change in the number of exosomes isolated from both normal and hyperglycemic condition media, however, the average size of exosomes isolated from the hyperglycemic group showed a significant rise when compared to that of the normoglycemic group. Müller cells derived exosomes from hyperglycemic condition media markedly reduced HRECs cell count, increased caspase-3 and Annexin V, decreased ZO-1 levels and TER, and increased intracellular Ca+ when compared to other groups. However, treatment of HRECs under hyperglycemia with normo-glycemic Müller cells-derived exosomes significantly decreased cell death, preserved cellular integrity and barrier function, and reduced intracellular Ca+2. Collectively, Müller cell-derived exosomes play a remarkable role in the pathological changes associated with hyperglycemia-induced inner barrier dysfunction in DR. Further in vivo research will help in understanding the role of exosomes as therapeutic targets and/or delivery systems for DR.

Asunto(s)

Apoptosis , Barrera Hematorretinal , Supervivencia Celular , Retinopatía Diabética , Células Endoteliales , Células Ependimogliales , Exosomas , Exosomas/metabolismo , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Retinopatía Diabética/fisiopatología , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Humanos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/patología , Células Cultivadas , Proteína de la Zonula Occludens-1/metabolismo , Permeabilidad Capilar , Señalización del Calcio , Línea Celular , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Vasos Retinianos/fisiopatología

RESUMEN

Glaucoma, the leading cause of irreversible blindness worldwide, is characterized by neurodegeneration and neuroinflammation with retinal NAD/NADP and GSH decline. Nicotinamide adenine dinucleotide (NAD)/NAD phosphate (NADP) and glutathione (GSH) are two redox reducers in neuronal and glial metabolism. However, therapeutic strategies targeting NAD/NADP or GSH do not exert ideal effects, and the underlying mechanisms are still poorly understood. We assessed morphological changes in retinal ganglion cells (RGCs), the affected neurons in glaucoma, and Müller cells, the major glial cells in the retina, as well as the levels of phosphorylated p38 (p-p38) and Caspase-3 in glaucoma patients. We constructed a modified chronic ocular hypertensive rat model and an oxygen-glucose deprivation (OGD) cell model. After applying NADPH and N-acetylcysteine (NAC), a precursor to cysteine, the rate-limiting substrate in GSH biosynthesis, to cells, apoptosis, axonal damage and peroxidation were reduced in the RGCs of the NAC group and p-p38 levels were decreased in the RGCs of the NADPH group, while in stimulated Müller cells cultured individually or cocultured with RGCs, gliosis and p38/MAPK, rather than JNK/MAPK, activation were inhibited. The results were more synergistic in the rat model, where either NADPH or NAC showed crossover effects on inhibiting peroxidation and p38/MAPK pathway activation. Moreover, the combination of NADPH and NAC ameliorated RGC electrophysiological function and prevented Müller cell gliosis to the greatest extent. These data illustrated conjoined mechanisms in glaucomatous RGC injury and Müller cell gliosis and suggested that NADPH and NAC collaborate as a neuroprotective and anti-inflammatory combination treatment for glaucoma and other underlying human neurodegenerative diseases.

Asunto(s)

Acetilcisteína , NADP , Hipertensión Ocular , Ratas Sprague-Dawley , Células Ganglionares de la Retina , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , NADP/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Hipertensión Ocular/metabolismo , Hipertensión Ocular/tratamiento farmacológico , Hipertensión Ocular/patología , Acetilcisteína/farmacología , Ratas , Masculino , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Glaucoma/metabolismo , Glaucoma/patología , Glaucoma/tratamiento farmacológico , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Humanos , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Modelos Animales de Enfermedad , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Enfermedad Crónica , Fármacos Neuroprotectores/farmacología , Células Cultivadas , Peroxidación de Lípido/efectos de los fármacos

RESUMEN

Müller glia and microglia are capable of phagocytosing fragments of retinal cells in response to retinal injury or degeneration. However, the direct evidence for their mutual interactions between Müller glia and microglia in the progression of retinal degeneration (RD) remains largely unclear. This study aims to construct a progressive RD mouse model and investigate the activated pattern of Müller glia and the interplay between Müller glia and microglia in the early stage or progression of RD. A Prohibitin 2 (Phb2) photoreceptor-specific knockout (RKO) mouse model was generated by crossing Phb2flox/flox mice with Rhodopsin-Cre mice. Optical Coherence Tomography (OCT), histological staining, and Electroretinography (ERG) assessed retinal structure and function, and RKO mice exhibited progressive RD from six weeks of age. In detail, six-week-old RKO mice showed no significant retinal impairment, but severe vision dysfunction and retina thinning were shown in ten-week-old RKO mice. Furthermore, RKO mice were sensitive to Light Damage (LD) and showed severe RD at an early age after light exposure. Bulk retina RNA-seq analysis from six-week-old control (Ctrl) and RKO mice showed reactive retinal glia in RKO mice. The activated pattern of Müller glia and the interplay between Müller glia and microglia was visualized by immunohistology and 3D reconstruction. In six-week-old RKO mice or light-exposed Ctrl mice, Müller glia were initially activated at the edge of the retina. Moreover, in ten-week-old RKO mice or light-exposed six-week-old RKO mice with severe photoreceptor degeneration, abundant Müller glia were activated across the whole retinas. With the progression of RD, phagocytosis of microglia debris by activated Müller glia were remarkably increased. Altogether, our study establishes a Phb2 photoreceptor-specific knockout mouse model, which is a novel mouse model of RD and can well demonstrate the phenotype of progressive RD. We also report that Müller glia in the peripheral retina is more sensitive to the early damage of photoreceptors. Our study provides more direct evidence for Müller glia engulfing microglia debris in the progression of RD due to photoreceptor Phb2 deficiency.

Asunto(s)

Células Ependimogliales , Microglía , Células Fotorreceptoras de Vertebrados , Prohibitinas , Degeneración Retiniana , Animales , Ratones , Modelos Animales de Enfermedad , Electrorretinografía , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Microglía/patología , Fagocitosis/fisiología , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/deficiencia , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Tomografía de Coherencia Óptica

RESUMEN

We explored the effect of inhibition of thioredoxin interacting protein (Txnip) on neuroprotection in Müller cells under high glucose. Wild-type (WT) and Txnip knockout (Txnip-/-) mice were used to establish a streptozotocin (STZ)-induced diabetes model and a Müller cells high glucose model. We detected BDNF expression and PI3K/AKT/CREB pathway activation levels in the retina and Müller cells of each group in vivo and in vitro experiments. The Txnip-/- STZ group showed higher expression of BDNF and phosphorylation of PI3K/AKT/CREB in retina, and less retinal photoreceptor apoptosis was observed in Txnip-/- diabetic group than in WT. After using an inhibitor of PI3K signaling pathway, BDNF expression was reduced; In vitro co-cultured with Müller cells in different groups, 661 W cells showed different situations, Txnip-/- Müller cells maximum downregulated Cleaved-caspase 3 expression in 661 W, accompanied by an increase in Bcl-2/Bax ratio. These findings indicate that inhibiting endogenous Txnip in mouse Müller cells can promote their expression and secretion of BDNF, thereby reducing HG induced photoreceptor apoptosis and having important neuroprotective effects on DR. The regulation of BDNF expression by Txnip may be achieved by activating the PI3K/AKT/CREB pathway. This study suggests that regulating Txnip may be a potential target for DR treatment.

Asunto(s)

Apoptosis , Proteínas Portadoras , Diabetes Mellitus Experimental , Células Ependimogliales , Fosfatidilinositol 3-Quinasas , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Células Ependimogliales/metabolismo , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/patología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Ratones , Transducción de Señal/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratones Noqueados , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/patología , Técnicas de Silenciamiento del Gen , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Masculino , Retinopatía Diabética/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Modelos Animales de Enfermedad

RESUMEN

PURPOSE: FTY720 is an agonist of the Sphingosine-1-phosphate (S1P) receptor 1, 3, 4, and 5 and a functional antagonist of the S1P1 receptor; it can inhibit the activation of mTOR/NF-κB and has therapeutic potential in inflammatory disease. This study was designed to determine the role of the inflammatory process in diabetic retinopathy and investigate the effect of FTY720 on high glucose (HG)-induced rat retinal Müller cells (rMC-1 cells). METHODS: In the present study, the role of FTY720 in inhibiting inflammation and its underlying mechanism were investigated. rMC-1 cells were treated without or with HG, FTY720, CQ, or RAP. Cell viability was examined by CCK-8 assay; cell activation was assessed by western blot analysis and IF staining; and cell migration was evaluated by a scratch wound healing assay. The expression of inflammation-associated proteins and autophagy-related proteins was evaluated by transmission electron microscopy, AO staining, MDC-labeled autophagic vacuoles, western blot analysis and ELISA. RESULTS: Western blot analysis and IF staining showed that the level of the rMC-1 cell marker GFAP was decreased, while GS was increased in FTY720 groups compared to that in the HG group. The healing assay results showed that compared with HG treatment, FTY720 treatment significantly reduced cell migration. Western blot analysis, ELISA and IF staining showed that compared with HG, FTY720 reduced proinflammatory proteins by inhibiting the mechanistic target of the mTOR/NF-κB signaling pathway and regulating autophagy. CONCLUSIONS: This study suggests that in an HG-induced rMC-1 cell model, FTY720 significantly inhibited the production of inflammatory cytokines by inhibiting mTOR/NF-κB signaling and regulating autophagy. These findings were associated with a decrease in rMC-1 cell injury, suggesting that FTY720 or related compounds may be valuable modulators of HG-induced retinal injury.

Asunto(s)

Autofagia , Western Blotting , Movimiento Celular , Supervivencia Celular , Retinopatía Diabética , Células Ependimogliales , Clorhidrato de Fingolimod , FN-kappa B , Transducción de Señal , Serina-Treonina Quinasas TOR , Clorhidrato de Fingolimod/farmacología , Animales , Ratas , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Serina-Treonina Quinasas TOR/metabolismo , FN-kappa B/metabolismo , Autofagia/efectos de los fármacos , Retinopatía Diabética/metabolismo , Retinopatía Diabética/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Moduladores de los Receptores de fosfatos y esfingosina 1/farmacología , Células Cultivadas , Inmunosupresores/farmacología , Microscopía Electrónica de Transmisión , Progresión de la Enfermedad

RESUMEN

Diabetic retinopathy (DR) is a significant complication of diabetes that often leads to blindness, impacting Müller cells, the primary retinal macroglia involved in DR pathogenesis. Reactive oxygen species (ROS) play a crucial role in the development of DR. The objective of this study was to investigate the involvement of sestrin2 in DR using a high-glucose (HG)-induced Müller cell model and assessing cell proliferation with 5-ethynyl-2-deoxyuridine (EdU) labeling. Following this, sestrin2 was upregulated in Müller cells to investigate its effects on ROS, tube formation, and inflammation both in vitro and in vivo, as well as its interaction with the nuclear factor erythroid2-related factor 2 (Nrf2) signaling pathway. The findings demonstrated a gradual increase in the number of EdU-positive cells over time, with a subsequent decrease after 72 h of exposure to high glucose levels. Additionally, the expression of sestrin2 exhibited a progressive increase over time, followed by a decrease at 72 h. The rh-sestrin2 treatment suppressed the injury of Müller cells, decreased ROS level, and inhibited the tube formation. Rh-sestrin2 treatment enhanced the expression of sestrin2, Nrf2, heme oxygenase-1 (HO-1), and glutamine synthetase (GS); however, the ML385 treatment reversed the protective effect of rh-sestrin2. Finally, we evaluated the effect of sestrin2 in a DR rat model. Sestrin2 overexpression treatment improved the pathological injury of retina and attenuated the oxidative damage and inflammatory reaction. Our results highlighted the inhibitory effect of sestrin2 in the damage of retina, thus presenting a novel therapeutic sight for DR.

Asunto(s)

Retinopatía Diabética , Especies Reactivas de Oxígeno , Sestrinas , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Animales , Especies Reactivas de Oxígeno/metabolismo , Ratas , Masculino , Ratas Sprague-Dawley , Factor 2 Relacionado con NF-E2/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Glucosa/metabolismo , Proliferación Celular/efectos de los fármacos , Células Ependimogliales/metabolismo , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/patología , Transducción de Señal/efectos de los fármacos , Peroxidasas/metabolismo , Células Cultivadas

RESUMEN

We explored the genomic events underlying central neurocytoma (CN), a rare neoplasm of the central nervous system, via multiomics approaches, including whole-exome sequencing, bulk and single-nuclei RNA sequencing, and methylation sequencing. We identified FGFR3 hypomethylation leading to FGFR3 overexpression as a major event in the ontogeny of CN that affects crucial downstream events, such as aberrant PI3K-AKT activity and neuronal development pathways. Furthermore, we found similarities between CN and radial glial cells based on analyses of gene markers and CN tumor cells and postulate that CN tumorigenesis is due to dysregulation of radial glial cell differentiation into neurons. Our data demonstrate the potential role of FGFR3 as one of the leading drivers of tumorigenesis in CN.

Asunto(s)

Metilación de ADN , Células Ependimogliales , Neurocitoma , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Humanos , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Neurocitoma/genética , Neurocitoma/patología , Neurocitoma/metabolismo , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Regulación Neoplásica de la Expresión Génica

RESUMEN

Phosphodiesterase (PDE) inhibitors - such as vardenafil - are used primarily for treating erectile dysfunction via increasing cyclic guanosine monophosphate (cGMP) levels. Recent studies have also demonstrated their significant cardioprotective effects in several diseases, including diabetes, upon long-term, continuous application. However, PDE inhibitors are not specific for PDE5 and also inhibit the retinal isoform. A sustained rise in cGMP in photoreceptors is known to be toxic; therefore, we hypothesized that long-term vardenafil treatment might result in retinotoxicity. The hypothesis was tested in a clinically relevant animal model of type 2 diabetes mellitus. Histological experiments were performed on lean and diabetic Zucker Diabetic Fatty rats. Half of the animals were treated with vardenafil for six months, and the retinal effects were evaluated. Vardenafil treatment alleviated rod outer segment degeneration but decreased rod numbers in some positions and induced changes in the interphotoreceptor matrix, even in control animals. Vardenafil treatment decreased total retinal thickness in the control and diabetic groups and reduced the number of nuclei in the outer nuclear layer. Müller cell activation was detectable even in the vardenafil-treated control animals, and vardenafil did not improve gliosis in the diabetic group. Vardenafil-treated animals showed complex retinal alterations with improvements in some parameters while deterioration in others. Our results point towards the retinotoxicity of vardenafil, even without diabetes, which raises doubts about the retinal safety of long-term continuous vardenafil administration. This effect needs to be considered when approving PDE inhibitors for alternative indications.

Asunto(s)

Diabetes Mellitus Experimental , Inhibidores de Fosfodiesterasa 5 , Ratas Zucker , Diclorhidrato de Vardenafil , Diclorhidrato de Vardenafil/farmacología , Diclorhidrato de Vardenafil/toxicidad , Animales , Ratas , Inhibidores de Fosfodiesterasa 5/farmacología , Masculino , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/patología , Retina/efectos de los fármacos , Retina/patología , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/patología , Células Ependimogliales/metabolismo

RESUMEN

Glaucoma is considered a neurodegenerative disease characterized by progressive visual field defects that may lead to blindness. Although controlling intraocular pressure (IOP) is the mainstay of glaucoma treatment, some glaucoma patients have unmet needs due to unclear pathogenic mechanisms. Recently, there has been growing evidence that neuroinflammation is a potential target for the development of novel antiglaucoma agents. In this study, we investigated the protective effects and cellular mechanisms of H7E, a novel small molecule inhibits HDAC8, using in vitro and in vivo glaucoma-like models. Importantly, H7E mitigated extracellular MMP-9 activity and MCP-1 levels in glutamate- or S100B-stimulated reactive Müller glia. In addition, H7E inhibited the upregulation of inflammation- and proliferation-related signaling pathways, particularly the ERK and JNK MAPK pathways. Under conditions of oxidative damage, H7E prevents retinal cell death and reduces extracellular glutamate released from stressed Müller glia. In a mouse model of NMDA-induced retinal degeneration, H7E alleviated functional and structural defects within the inner retina as assessed by electroretinography and optical coherence tomography. Our results demonstrated that the newly identified compound H7E protects against glaucoma damage by specifically targeting HDAC8 activity in the retina. This protective effect is attributed to the inhibition of Müller glial activation and the prevention of retinal cell death caused by oxidative stress.

Asunto(s)

Células Ependimogliales , Glaucoma , Inhibidores de Histona Desacetilasas , Histona Desacetilasas , Ratones Endogámicos C57BL , Estrés Oxidativo , Animales , Estrés Oxidativo/efectos de los fármacos , Glaucoma/tratamiento farmacológico , Glaucoma/metabolismo , Glaucoma/patología , Inhibidores de Histona Desacetilasas/farmacología , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Ratones , Histona Desacetilasas/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Modelos Animales de Enfermedad , Fármacos Neuroprotectores/farmacología , Masculino , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/prevención & control

RESUMEN

X-linked retinoschisis (XLRS) is an early onset degenerative retinal disease characterized by cystic lesions in the middle layers of the retina. These structural changes are accompanied by a loss of visual acuity and decreased contrast sensitivity. XLRS is caused by mutations in the gene Rs1 which encodes the secreted protein Retinoschisin 1. Young Rs1-mutant mouse models develop key hallmarks of XLRS including intraretinal schisis and abnormal electroretinograms. The electroretinogram (ERG) comprises activity of multiple cellular generators, and it is not known how and when each of these is impacted in Rs1 mutant mice. Here we use an ex vivo ERG system and pharmacological blockade to determine how ERG components generated by photoreceptors, ON-bipolar, and Müller glial cells are impacted in Rs1 mutants and to determine the time course of these changes. We report that ERG abnormalities begin near eye-opening and that all ERG components are involved.

Asunto(s)

Moléculas de Adhesión Celular , Modelos Animales de Enfermedad , Electrorretinografía , Proteínas del Ojo , Retinosquisis , Animales , Retinosquisis/genética , Retinosquisis/fisiopatología , Ratones , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Ratones Endogámicos C57BL , Mutación , Células Ependimogliales/patología , Células Ependimogliales/metabolismo , Masculino , Células Bipolares de la Retina/patología , Células Bipolares de la Retina/metabolismo