A Mathematical Model of Interstitial Fluid Flow and Retinal Tissue Deformation in Macular Edema.

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.

Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood-brain barrier integrity.

The neurovascular unit (NVU) is a complex multicellular structure that helps maintain cerebral homeostasis and blood-brain barrier (BBB) integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration, the underlying molecular mechanisms remain unclear. Here, we use zebrafish embryos carrying a mutation in Scavenger Receptor B2, a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells (RGCs), to investigate the interplay among different NVU components. Through live imaging and genetic manipulations, we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling, thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling, leading to severe vascular defects, hemorrhages, and a leaky BBB. Altogether, our findings provide insights into NVU formation and function and offer avenues for investigating diseases involving white matter defects and vascular abnormalities.

Knockdown of thioredoxin interacting protein in Müller cells attenuates photoreceptor apoptosis in streptozotocin-induced diabetic mouse model.

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.

NADPH and NAC synergistically inhibits chronic ocular hypertension-induced neurodegeneration and neuroinflammation through regulating p38/MAPK pathway and peroxidation.

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.

Central neurocytoma exhibits radial glial cell signatures with FGFR3 hypomethylation and overexpression.

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.

Photoreceptor deficits appear at eye opening in Rs1 mutant mouse models of X-linked retinoschisis.

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.

Müller cells under hydrostatic pressure modulate retinal cell survival via TRPV1/PLCγ1 complex-mediated calcium influx in experimental glaucoma.

Glaucoma, an irreversible blinding eye disease, is currently unclear whose pathological mechanism is. This study investigated how transient receptor potential cation channel subfamily V member 1 (TRPV1), 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 (PLCγ1), and P2X purinoceptor 7 (P2X7) modulate the levels of intracellular calcium ions (Ca2+) and adenosine triphosphate (ATP) in Müller cells and retinal ganglion cells (RGCs) under conditions of elevated intraocular pressure (IOP). Müller cells were maintained at hydrostatic pressure (HP). TRPV1- and PLCG1-silenced Müller cells and P2X7-silenced RGCs were constructed by transfection with short interfering RNA (siRNAs). RGCs were cultured with the conditioned media of Müller cells under HP. A mouse model of chronic ocular hypertension (COH) was established and used to investigate the role of TRPV1 in RGCs in vivo. Müller cells and RGCs were analyzed by ATP release assays, intracellular calcium assays, CCK-8 assays, EdU (5-ethynyl-2'-deoxyuridine) staining, TUNEL staining, flow cytometry, and transmission electron microscopy. In vivo changes in inner retinal function were evaluated by hematoxylin and eosin (H&E) staining and TUNEL staining. Western blot analyses were performed to measure the levels of related proteins. Our data showed that HP increased the levels of ATP and Ca2+ influx in Müller cells, and those increases were accompanied by the upregulation of TRPV1 and p-PLCγ1 expression. Suppression of TRPV1 or PLCG1 expression in Müller cells significantly decreased the ATP levels and intracellular Ca2+ accumulation induced by HP. Knockdown of TRPV1, PLCG1, or P2X7 significantly decreased apoptosis and autophagy in RGCs cultured in the conditioned media of HP-treated Müller cells. Moreover, TRPV1 silencing decreased RGC apoptosis and autophagy in the in vivo model of COH. Collectively, inhibition of TRPV1/PLCγ1 and P2X7 expression may be a useful therapeutic strategy for managing RGC death in glaucoma.

Diabetic Müller-Glial-Cell-Specific Il6ra Knockout Mice Exhibit Accelerated Retinal Functional Decline and Thinning of the Inner Nuclear Layer.

Purpose: Interleukin-6 (IL-6) is implicated in the pathology of diabetic retinopathy (DR). IL-6 trans-signaling via soluble IL-6 receptor (IL-6R) is primarily responsible for its pro-inflammatory functions, whereas cis-signaling via membrane-bound IL-6R is anti-inflammatory. Using a Müller-glial-cell-specific Il6ra-/- mouse, we examined how loss of IL-6 cis-signaling in Müller glial cells (MGCs) affected retinal thinning and electroretinography (ERG) response over 9 months of diabetes. Methods: Diabetes was induced in wildtype and knockout mice with streptozotocin (40 mg/kg, daily for 5 days). Spectral domain optical coherence tomography (SD-OCT), ERG, and fundoscopy/fluorescein angiography (FA) were assessed at 2, 6, and 9 months of diabetes. MGCs and bipolar neurons were examined in retinal tissue sections by immunofluorescence. Results: Diabetic MGC Il6ra-/- mice had significantly thinner retinas than diabetic wildtype mice at 2 (-7.6 µm), 6 (-12.0 µm), and 9 months (-5.0 µm) of diabetes, as well as significant thinning of the inner nuclear layer (INL). Diabetic MGC Il6ra-/- mice also showed a reduction in scotopic B-wave amplitude and B-wave/A-wave ratio earlier than wildtype diabetic mice. In retinal sections, we found a decrease in bipolar neuronal marker PKCα only in diabetic MGC Il6ra-/- mice, which was significantly lower than both controls and diabetic wildtype mice. Glutamine synthetase, a Müller cell marker, was reduced in both wildtype and MGC Il6ra-/- diabetic mice compared to their respective controls. Conclusions: IL-6 cis-signaling in MGCs contributes to maintenance of the INL in diabetes, and loss of the IL-6 receptor reduces MGC-mediated neuroprotection of bipolar neurons in the diabetic retina.

The Role of Müller Cells in Diabetic Macular Edema.

Diabetic macular edema (DME) is a common complication of diabetic retinopathy and is the leading cause of vision loss in diabetic patients. Various factors, such as metabolic disorders and inflammation caused by hyperglycemia, are involved in the occurrence and development of DME, but the specific mechanism is still unclear. Müller cells are a type of macroglial cell unique to the fundus, distributed throughout the retina, and they play a unique role in retinal homeostasis. This article reviews the role of Müller cells in the pathological process of DME and the research progress in the treatment of DME by targeting Müller cells through gene therapy.

Mechanism underlying Müller cell pyroptosis and its role in the development of proliferative vitreoretinopathy.

OBJECTIVES: To explore the mechanism underlying Müller Cell Pyroptosis (MCP) and its role in the development of Proliferative Vitreoretinopathy (PVR). METHOD: The expression of pyroptosis-related factors, namely, cysteinyl aspartate-specific proteinase (caspase-1), interleukin (IL)-1ß, IL-18, and Gasdermin D (GSDMD), was detected by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and western blotting at the mRNA and protein levels, respectively, in retinal tissues. Müller and spontaneously Arising Retinal Pigment Epithelia (ARPE)-19 primary cells with GSDMD overexpression or knockdown were cultivated. Western blotting was used to detect the levels of the following pyroptosis-related factors in retinal tissues: caspase-1, IL-1ß, IL-18, and GSDMD. Through Cell Adhesion (CA) experiments, the changes in ARPE-19 CA in each group were observed. The migration and invasion of ARPE-19 cells were measured using the Transwell assay. The proliferation of ARPE-19 cells was measured with a Cell Counting Kit 8 (CCK-8) assay. Finally, the expression of the cytokines IL-1ß and IL-18 in the ARPE-19 cell culture medium was detected using the Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Compared with the surrounding normal tissues, the expression of caspase-1, IL-1ß, IL-18, and GSDMD at the protein and mRNA levels in the retinal proliferative membrane samples of the patients decreased significantly (p < 0.05). MCP significantly enhanced ARPE-19 CA, migration and invasion, proliferation, and cytokine expression (p < 0.05). CONCLUSIONS: MCP can promote the development of PVR lesions.

The Disorganization of Retinal Inner Layers Is Correlated to Müller Cells Impairment in Diabetic Macular Edema: An Imaging and Omics Study.

The disorganization of retinal inner layers (DRIL) is an optical coherence tomography (OCT) biomarker strictly associated with visual outcomes in patients with diabetic macular edema (DME) whose pathophysiology is still unclear. The aim of this study was to characterize in vivo, using retinal imaging and liquid biopsy, DRIL in eyes with DME. This was an observational cross-sectional study. Patients affected by center-involved DME were enrolled. All patients underwent spectral domain optical coherence tomography (SD-OCT) and proteomic analysis of aqueous humor (AH). The presence of DRIL at OCT was analyzed by two masked retinal experts. Fifty-seven biochemical biomarkers were analyzed from AH samples. Nineteen eyes of nineteen DME patients were enrolled. DRIL was present in 10 patients (52.63%). No statistically significant difference was found between DME eyes with and without DRIL, considering the AH concentration of all the analyzed biomarkers except for glial fibrillary acidic protein (GFAP), a biomarker of Müller cells dysfunction (p = 0.02). In conclusion, DRIL, in DME eyes, seems to strictly depend on a major dysfunction of Müller cells, explaining its role not only as imaging biomarker, but also as visual function Müller cells-related parameter.

Insights into CD154-mediated pathways in ocular hypertensive glaucoma: The role of Müller cells and P2X7 in retinal neuroprotection and therapeutic potential.

An elevation of pathologic intraocular pressure (IOP) is the greatest risk factor for glaucoma. CD154 has been reported to bind to CD40 expressed by orbital fibroblasts and be involved in immune and inflammatory responses. However, the function and mechanism of CD154 in ocular hypertensive glaucoma (OHG) are not fully understood. We isolated and characterized Müller cells and subsequently examined the effect of CD154 on ATP release from those cells. After being cocultured with CD154-pretreated Müller cells, retinal ganglion cells (RGCs) were treated with P2X7 siRNAs or a P2X7 inhibitor. Furthermore, mouse models of glaucoma (GC) were injected with P2X7 shRNA. p21, p53, and P2X7 expression were examined, and cellular senescence and apoptosis were detected by ß-Gal and TUNEL staining, retinal pathology was examined by H&E staining, and CD154 and ß-Gal expression were detected by ELISA. CD154 induced ATP release from Müller cells and accelerated the senescence and apoptosis of RGCs that had been cocultured with Müller cells. We also found that treatment with P2X7 could attenuate the senescence and apoptosis of RGCs mediated by Müller cells pretreated with CD154. In vivo studies in GC model mice verified that P2X7 silencing attenuated pathological damage and prevented the senescence and apoptosis of retinal tissue. The study demonstrates how CD154 accelerates the aging and apoptosis of RGCs by co-cultivating Müller cells pretreated with CD154 in OHG. The research implies that CD154 has the potential to become a new therapeutic target for ocular hypertension glaucoma, providing a new research direction for its treatment.

Whorling Sclerosing Ependymoma of the Cervical Spinal Cord Presenting Tanycytic Histopathologic Features: A Rare Case Report.

Tanycytic ependymoma is a neuroectodermal tumor that arises from ependymoglial cells or tanycytes. It is highly uncommon. We reported a 34-year-old man who was diagnosed with intradural-intramedullary tanycytic ependymoma, located at the level of C4-5 who had a 9-months history of neck pain and left arm pain, and numbness on fingers. One month prior to presentation, his left arm numbness and paresthesia deteriorated. The lesion was removed totally by C4, C5 hemilaminoplasty. The histologic pattern of this lesion was consisted of fascicles forming nebula-like whorling structures. Because of these structures, tanycytic ependymoma should be taken into consideration in the differential diagnosis of a whorling-sclerosing variant of meningiomas.

Th22 cells induce Müller cell activation via the Act1/TRAF6 pathway in diabetic retinopathy.

T helper 22 (Th22) cells have been implicated in diabetic retinopathy (DR), but it remains unclear whether Th22 cells involve in the pathogenesis of DR. To investigate the role of Th22 cells in DR mice, the animal models were established by intraperitoneal injection of STZ and confirmed by fundus fluorescein angiography and retinal haematoxylin-eosin staining. IL-22BP was administered by intravitreal injection. IL-22 level was measured by ELISA in vivo and in vitro. The expression of IL-22Rα1 in the retina was assessed by immunofluorescence. We assessed GFAP, VEGF, ICAM-1, inflammatory-associated factors and the integrity of blood-retinal barrier in control, DR, IL-22BP, and sham group. Müller cells were co-cultured with Th22 cells, and the expression of the above proteins was measured by immunoblotting. Plasmid transfection technique was used to silence Act1 gene in Müller cells. Results in vivo and in vitro indicated that Th22 cells infiltrated into the DR retinal and IL-22Rα1 expressed in Müller cells. Th22 cells promoted Müller cells activation and inflammatory factor secretion by secreting IL-22 compared with high-glucose stimulation alone. In addition, IL-22BP ameliorated the pathological alterations of the retina in DR. Inhibition of the inflammatory signalling cascade through Act1 knockdown alleviated DR-like pathology. All in all, the results suggested that Th22 cells infiltrated into the retina and secreted IL-22 in DR, and then IL-22 binding with IL-22Rα1 activated the Act1/TRAF6 signal pathway, and promoted the inflammatory of Müller cells and involved the pathogenesis of DR.

Regulation of inflammation by VEGF/BDNF signaling in mouse retinal Müller glial cells exposed to high glucose.

The inflammatory changes seem to play an important role in the development of diabetic retinopathy (DR). Anti-VEGF therapy has been testified to inhibit inflammation in animal models of diabetes, but the detailed mechanisms during this process are not yet clear. Müller glial cells (MGCs) in the mammalian retina are deeply involved in DR, while the BDNF overexpression reduces inflammation in diabetic mice. In this research, we aimed to explore the relationship between VEGF and BDNF in mouse retinal MGCs during inflammation of diabetes. We examined the expression of glutamine-synthetase (GS), glial fibrillary acidic protein (GFAP), vascular-endothelial growth factor (VEGF), interleukin-1beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α) at different time points after mouse retinal MGCs exposed to high glucose (25 mM). We also explored changes in the expression of brain-derived neurotrophic factor (BDNF), nuclear factor kappa B (NF-κB), IL-1ß, and TNF-α in MGCs after treatments with anti-VEGF, VEGF siRNA, BDNF siRNA, BDNF recombination protein, and NF-κB inhibitor. In mouse retinal MGCs exposed to high glucose, BDNF was increased after treatments with anti-VEGF or VEGF siRNA. BDNF was decreased in MGCs from VEGF overexpressed mice. Moreover, the expressions of NF-κB, IL-1ß, and TNF-α changed with BDNF: NF-κB, IL-1ß, and TNF-α were increased after treatments with BDNF siRNA; NF-κB, IL-1ß, and TNF-α were decreased after treatments with BDNF recombination protein. VEGF may regulate cytokines (IL-1ß and TNF-α) by BDNF/NF-κB signaling pathway. The regulation of the VEGF/BDNF/NF-κB signaling pathway may be a significant therapeutic strategy for DR.

FOVEAL MÜLLER CELL CONE AS A PROGNOSTIC OPTICAL COHERENCE TOMOGRAPHY BIOMARKER FOR INITIAL RESPONSE TO ANTIVASCULAR ENDOTHELIAL GROWTH FACTOR TREATMENT IN CYSTOID DIABETIC MACULAR EDEMA.

PURPOSE: To investigate the effect of the foveal Müller cell cone structure on the anatomical and functional response to intravitreal bevacizumab treatment in patients with diabetic macular edema. METHODS: In 93 treatment-naive eyes with center-involved cystic type diabetic macular edema, spectral-domain optical coherence tomography scans of baseline were retrospectively evaluated to determine the foveal Müller cell cone structure and prognostic features including length of disorganization in the retinal inner layers and ellipsoid zone disruption. The area and circularity of the foveal avascular zone of the superficial and deep capillary plexus 1 month after intravitreal bevacizumab treatment were evaluated using optical coherence tomography angiography. RESULTS: Destruction of the foveal Müller cell cone structure and a large foveal avascular zone in the deep capillary plexus (mm2) correlated strongly with a poor anatomical response (CST > 250 µm) at 1 month after first intravitreal bevacizumab (Exp [B] = 29.444, P = 0.002 and Exp [B] = 12.419, P = 0.013, respectively). A destroyed Müller cell cone structure (P = 0.008) and length of ellipsoid zone disruption (P < 0.001) at baseline were associated with poor visual acuity at 1 month after the first intravitreal bevacizumab. CONCLUSION: The foveal Müller cell cone structure correlates with the response to initial antivascular endothelial growth factor treatment.

Protective Effect of Raf-1 Kinase Inhibitory Protein on Diabetic Retinal Neurodegeneration through P38-MAPK Pathway.

PURPOSE: This study aimed to investigate the effect of Raf-1 kinase inhibitory protein (RKIP) on diabetic retinal neurodegeneration in streptozotocin-treated rat model and high glucose-treated rat Müller cells. METHODS: Control and streptozotocin-treated rats were intravitreally injected with saline, RKIP gene overexpression lentivirus (oeRKIP) or negative control lentivirus (RKIP-vector). Normal or high glucose-treated Müller cells were transfected with saline, RKIP gene overexpression lentivirus or negative control lentivirus. Western blotting and immunofluorescence assay were utilized to evaluate the function of RKIP on the expression of RKIP, p38 mitogen-activated protein kinase (p38-MAPK), glutamate/aspartate transporter (GLAST), glutamine synthetase (GS), glial fibrillar acidic protein (GFAP) and cysteine-aspartic acid protease-3 (caspase-3). A glutamate assay kit was adopted to detect glutamate level in retina samples. Apoptosis of Müller cells was determined by Annexin-V/PI staining and flow cytometry. RESULTS: High glucose-treated Müller cells exhibited promoted apoptosis, while RKIP overexpression in high glucose-treated Müller cells down-regulated the enhanced apoptosis. Compared with rats injected with saline, streptozotocin-treated hyperglycemic rats displayed enhancement in the immunoreactivities of p38-MAPK and GFAP as well as in the protein expression of p38-MAPK and caspase-3. Strikingly, intravitreal injection of RKIP gene overexpression lentivirus in the hyperglycemic rats reversed the augmented immunoreactivities and protein expression mentioned above. Meanwhile, RKIP overexpression in the hyperglycemic rats improved the immunoreactivities and protein expression of RKIP, GS and GLAST. Besides, RKIP down-regulated the increased level of retinal glutamate in the hyperglycemic rats. CONCLUSIONS: Intravitreal injection of RKIP gene overexpression lentivirus functioned in preventing diabetic retinal neurodegeneration in a rat model of diabetes presumably by inhibiting p38-MAPK pathway.

Microglia-derived TNF-α mediates Müller cell activation by activating the TNFR1-NF-κB pathway.

Microglia and its interaction with Müller cells are responsible to retinal surveillance during retinal neurodegeneration, however, the role and mechanism of microglia-derived tumor necrosis factor (TNF)-α in the activation of retinal Müller cells have not been fully elucidated. In the present study, primary microglia and Müller cells were isolated from newborn Sprague-Dawley (SD) rats with purities of 88.2 ± 6.2% and 92.2 ± 2.2%, respectively. By performing immunofluorescence and Western blot analysis, we found that TNF receptor (TNFR)-1 and TNFR2 were expressed in Müller cells. After co-cultured with microglia-conditioned medium (MCM), the elevated mRNA levels of glial fibrillary acidic protein (GFAP), proinflammatory factors (TNF-α, IL-1ß, CXCL-1, CSF-1, NOS2, COX2) and decreased CNTF mRNA levels were found in Müller cells. However, pretreatment with R-7050 (a TNF-α receptor inhibitor) or anti-TNFR1 significantly abrogated the changes. Simultaneously, pretreatment with anti-TNFR2 slightly inhibited the expression of GFAP in MCM-incubated Müller cells. Meanwhile, anti-TNFR1 treatment reversed the increased expression of CSF-1 and IL-1ß induced by TNF-α. Compared to the control groups, the phosphorylation of NF-κB P65, MAPK P38 and ERK1/2 in TNF-α-treated Müller cells was significantly increased. Nevertheless, pretreatment with anti-TNFR1 inhibited the phosphorylation of NF-κB P65 and MAPK p38, especially NF-κB P65. Additionally, pretreatment with Bay117082 (an NF-κB inhibitor) also significantly inhibited NF-κB P65 phosphorylation and GFAP expression. Moreover, anti-TNFR1 and Bay117082 treatment reduced NF-κB P65 phosphorylation of Müller cells induced by MCM. These results suggested that microglia-derived TNF-α served as a vital role in regulating Müller cells activation during retinal neurodegeneration.

Investigation on the Q-markers of Bushen Huoxue Prescriptions for DR treatment based on chemometric methods and spectrum-effect relationship.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetic retinopathy (DR) is a kind of complex complication of late diabetes mellitus with high incidence and risk of blindness. Bushen Huoxue Prescription (BHP), which consists of Rehmanniae radix (RR), Salviae miltiorrhizae radix et rhizoma (SMRR), Ginseng radix et rhizome (GRR) and Puerariae lobatae radix (PLR), has an active effect on the treatment of DR. However, the quality markers (Q-markers) of BHP are not entirely clear. PURPOSE: This study aimed to screen the Q-markers of BHP for DR treatment based on the establishment of spectrum-effect relationship and verified experiment. MATERIALS AND METHODS: In this study, 12 BHP samples (S1-S12) for fingerprint analysis and pharmacological evaluation were prepared according to a four-factor and twelve-level uniform design. High performance liquid chromatography-ultraviolet detector-evaporative light scattering detector (HPLC-UV-ELSD) was employed to analyze the fingerprint on the basis of the characteristics of BHP components. The evaluation of sample similarity was carried out by similarity analysis (SA) and hierarchical cluster analysis (HCA). The pharmacological indicators, including expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in the retina of Sprague Dawley (SD) rats induced by streptozotocin (STZ), were detected by enzyme-linked immunosorbent assay (ELISA). Besides, the spectrum-effect relationship between common peaks of fingerprints and the pharmacological results was investigated by partial least squares regression (PLSR) and canonical correlation analysis (CCA). The results of spectrum-effect relationship were verified by the expression of VEGF and HIF-1α on primary culture retinal Müller cells induced by hyperglycemia and hypoxia. RESULTS: In the HPLC-UV-ELSD fingerprint, 23 common peaks in UV and 14 common peaks in ELSD were identified. The pharmacological results indicated that the expression of VEGF and HIF-1α in the retina of SD rats was inhibited by 12 BHP samples to varying degrees compared with the model group. Based on SA and heatmap of HCA, S4 and S8 were clearly distinguished from other samples. The results of PLSR and CCA revealed that the contents of puerarin, daidzin, salvianolic acid B and ginsenoside Rb1 were inversely correlated with the expression of VEGF and HIF-1α. Hence, the four compounds may be the main active components to prevent and treat DR. The results of intervention on primary culture retinal Müller cells showed that puerarin, daidzin, salvianolic acid B, and ginsenoside Rb1 can significantly inhibit the expression of VEGF and HIF-1α. CONCLUSIONS: The spectrum-effect relationship of BHP was successfully established, and the Q-markers of BHP for the prevention and treatment of DR were preliminarily confirmed. It provides a feasible method for the research of quality control.

Interplay between Müller cells and microglia aggravates retinal inflammatory response in experimental glaucoma.

BACKGROUND: Glaucoma, the leading cause of irreversible blindness, is a retinal neurodegenerative disease, which results from progressive apoptotic death of retinal ganglion cells (RGCs). Although the mechanisms underlying RGC apoptosis in glaucoma are extremely complicated, an abnormal cross-talk between retinal glial cells and RGCs is generally thought to be involved. However, how interaction of Müller cells and microglia, two types of glial cells, contributes to RGC injury is largely unknown. METHODS: A mouse chronic ocular hypertension (COH) experimental glaucoma model was produced. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), transwell co-culture of glial cells, flow cytometry assay, ELISA, Ca2+ image, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the interaction of Müller cells and microglia, and its underlying mechanisms in COH retina. RESULTS: We first showed that Müller cell activation in mice with COH induced microglia activation through the ATP/P2X7 receptor pathway. The activation of microglia resulted in a significant increase in mRNA and protein levels of pro-inflammatory factors, such as tumor necrosis factor-α and interleukin-6. These inflammatory factors in turn caused the up-regulation of mRNA expression of pro-inflammatory factors in Müller cells through a positive feedback manner. CONCLUSIONS: These findings provide robust evidence, for the first time, that retinal inflammatory response may be aggravated by an interplay between activated two types of glial cells. These results also suggest that to reduce the interplay between Müller cells and microglia could be a potential effective strategy for preventing the loss of RGCs in glaucoma.