Retinal Microglial Activation Research Articles

A small peptide antagonist of the Fas receptor inhibits neuroinflammation and prevents axon degeneration and retinal ganglion cell death in an inducible mouse model of glaucoma

BackgroundGlaucoma is a complex, multifactorial disease where apoptosis, microglia activation, and inflammation have been linked to the death of retinal ganglion cells (RGCs) and axon degeneration. We demonstrated previously that FasL-Fas signaling was required for axon degeneration and death of RGCs in chronic and inducible mouse models of glaucoma and that Fas activation triggered RGC apoptosis, glial activation, and inflammation. Here, we investigated whether targeting the Fas receptor with a small peptide antagonist, ONL1204, has anti-inflammatory and neuroprotective effects in a microbead-induced mouse model of glaucoma.MethodsIntracameral injection of microbeads was used to elevate intraocular pressure (IOP) in Fas-deficient (Faslpr) mice and WT C57BL/6J mice that received an intravitreal injection of the Fas inhibitor, ONL1204 (2 μg/1 μl) (or vehicle only), on day 0 or day 7 after microbead injection. The IOP was monitored by rebound tonometry, and at 28 days post-microbead injection, Brn3a-stained RGCs and paraphenylenediamine (PPD)-stained axons were analyzed. The effects of ONL1204 on retinal microglia activation and the expression of inflammatory genes were analyzed by immunostaining of retinal flatmounts and quantitative PCR (qPCR).ResultsRebound tonometry showed equivalent elevation of IOP in all groups of microbead-injected mice. At 28 days post-microbead injection, the RGC and axon counts from microbead-injected Faslpr mice were equivalent to saline-injected (no IOP elevation) controls. Treatment with ONL1204 also significantly reduced RGC death and loss of axons in microbead-injected WT mice when compared to vehicle-treated controls, even when administered after IOP elevation. Confocal analysis of Iba1-stained retinal flatmounts and qPCR demonstrated that ONL1204 also abrogated microglia activation and inhibited the induction of multiple genes implicated in glaucoma, including cytokines and chemokines (GFAP, Caspase-8, TNFα, IL-1β, IL-6, IL-18, MIP-1α, MIP-1β, MIP-2, MCPI, and IP10), components of the complement cascade (C3, C1Q), Toll-like receptor pathway (TLR4), and inflammasome pathway (NLRP3).ConclusionsThese results serve as proof-of-principal that the small peptide inhibitor of the Fas receptor, ONL1204, can provide robust neuroprotection in an inducible mouse model of glaucoma, even when administered after IOP elevation. Moreover, Fas signaling contributes to the pathogenesis of glaucoma through activation of both apoptotic and inflammatory pathways.

Inhibitory effect of Houttuynia cordata Thunb on LPS-induced retinal microglial activation.

To identify the effect of Houttuynia cordata Thunb (HCT) on lipopolysaccharide (LPS)-induced microglial activation and investigate its possible molecular mechanisms. The primary retinal microglial cells were cultured from the retinas of newborn Sprague-Dawley rats and exposed to LPS, and/or HCT with different concentrations. The survival ability of retinal microglia cells was tested by standard MTT method. BrdU cell proliferation assay was used to evaluate the proliferation of retinal microglia. Inflammatory factors in the culture supernatants, including TNF-α, iNOS and IL-1β, were measured using ELISA. Microglia cells' migration was determined with Transwell migration assay. The total p38-MAPK and phosphorylation of p38-MAPK (p-p38-MAPK) were detected with Western blot. Primary retinal microglia in culture exposed to LPS to induce microglia activation. Pretreatment with HCT significantly inhibited the LPS-induced cell proliferation, but not the cell viability. LPS induced inflammatory reaction in microglia and cell migration. HCT significantly reduced LPS-stimulated release of pro-inflammatory factors and decreased the number of migrating cells substantially in a concentration-dependent manner. Moreover, the protein levels of p-p38 MAPK were identified as the up regulation and co-treatment with HCT obviously inhibited the upregulation of p-p38 MAPK, but had no effect on the levels of total p38-MAPK. The data suggest that HCT inhibits LPS-induced retinal microglial activation via suppression of the p-p38-MAPK. HCT may be used for the treatment of ocular diseases characterized by over-activated microglia.

MiR-30a Regulates S100A12-induced Retinal Microglial Activation and Inflammation by Targeting NLRP3

ABSTRACTPurpose: Our previous study has identified that plasma levels of S100A12 are closely associated with presence and severity of diabetic retinopathy (DR). In this work, we explored whether S100A12 can contribute to retinal microglial activation and inflammatory changes of DR via a microRNA-dependent mechanism.Material and Methods: Streptozotocin (STZ)-induced DR model was developed. Retinal microglia of rats were activated through intravitreal injection of S100A12. Differential expression of miRNAs on retinal microglia treated with S100A12 or DMEM/F-12 alone was determined using microarray analysis. Luciferase reporter assays were performed, which explored the regulation of a putative target of miR-30a.Results: S100A12 was increased approximately fivefold in the retina of 16-week diabetic rats compared with nondiabetic retinas. Furthermore, the levels of NLRP3, ASC, caspase-1, IL-1β, and IL-18 were significantly increased in the retina of rats treated with intravitreal injection of S100A12. Moreover, S100A12 induced an increased expression of NLRP3, ASC, caspase-1, IL-1β, and IL-18 in a dose- and time-dependent manner in retinal microglia. S100A12 was a proinflammatory trigger in diabetes-induced retinal microglial activation by activating NLRP3 in vivo and in vitro. In addition, S100A12 induced retinal microglial activation via a miR-30a-dependent mechanism. Mechanistically, S100A12 inhibited miR-30a expression, which was controlled by HDAC, and miR-30a downregulated NLRP3 expression by directly targeting its 3ʹ-UTR.Conclusions: S100A12 plays an important role in the pathogenesis of DR by activating retinal microglia via a miR-30a-dependent mechanism.

Systemic exposure to CpG-ODN elicits low-grade inflammation in the retina

Previous studies have reported that topical exposure to the toll-like receptor (TLR) 9 ligand CpG-ODN causes widespread ocular inflammation, including retinal microglial activation and posterior segment inflammation. Here we sought to determine the effects of systemic exposure to CpG-ODN in the retina and whether this inflammatory response was altered with Cx3cr1 deficiency or hyperglycemia. Male non-diabetic Cx3cr1+/gfp and Cx3cr1gfp/gfp littermates (normoglycemic controls) and Cx3cr1+/gfpIns2Akitaand Cx3cr1gfp/gfpIns2Akita diabetic mice were injected intraperitoneally with 40 μg CpG-ODN. Immunofluorescence staining was performed 1 week later to assess the expression of MHC Class II and glial fibrillary acidic protein (GFAP), as well as to identify morphological changes to microglia and changes in retinal macrophage cell density. Systemic exposure to CpG-ODN induced the upregulated expression of both GFAP on retinal Müller cells and MHC Class II on the retinal vasculature. Additionally, there was an increased accumulation of macrophages in the subretinal space 1 week after exposure to systemic CpG-ODN as well as characteristic morphological changes to microglia indicative of an activated phenotype. These preliminary studies demonstrate that low-grade inflammatory changes were not enhanced in Cx3cr1-deficient or diabetic mice, indicating that the inflammatory response to systemic CpG-ODN in the retina is unaltered in the context of Cx3cr1 deficiency or prolonged hyperglycemia.

Cascade Signals of Papaverine Inhibiting LPS-Induced Retinal Microglial Activation

Studies have shown that papaverine can inhibit lipopolysaccharide (LPS)-induced microglial activation. The retinal primary microglia of newborn SD rats were isolated and purified, and a LPS-induced microglia activation model was established. The protein phosphorylation level of the signaling pathway was detected by western blotting. The transcription and expression of TNF-α, IL-1β, and IL-10 were respectively detected by RT-PCR and ELISA to observe the abnormal activation of primary microglia. The cAMP inhibitor Rp-isomer, PKA inhibitor H89, and MEK inhibitor U0126 were separately added to further investigate the role of MEK/Erk in PAP inhibition of primary microglial activation and the relationship between cAMP/PKA and MEK/Erk. It was found that the level of MEK phosphorylation was upregulated after LPS stimulation, which was blocked by 10 μg/ml of papaverine.10μM U0126 significantly inhibited TNF-α and IL-1β and increased IL-10 transcription and expression in retinal microglia (P < 0.01). Both Rp-isomer and H89 upregulated the phosphorylation levels of MEK and Erk. Papaverine may inhibit inflammatory factors and promote the expression of anti-inflammatory factors through the cAMP/PKA and MEK/Erk pathway, thereby inhibiting LPS-induced activation of primary retinal microglia, and the MEK/Erk pathway may be partially regulated by cAMP/PKA, which can provide theoretical basis and experimental basis for its protection of the central nervous system.

Bilateral activation of retinal microglia: quantitative analysis of the microglia cell number at different time points after laser‐induced ocular hypertension in mice

Bilateral activation of retinal microglia: quantitative analysis of the microglia cell number at different time points after laser‐induced ocular hypertension in mice

Bilateral activation of retinal microglia: quantitative analysis of the area the retina occupied by IBA‐1 + cells in the nerve fiber layer‐ganglion cell layer at different time points after laser‐induced ocular hypertension in mice

Bilateral activation of retinal microglia: quantitative analysis of the area the retina occupied by IBA‐1 + cells in the nerve fiber layer‐ganglion cell layer at different time points after laser‐induced ocular hypertension in mice

Retinal Vascular Abnormalities and Microglia Activation in Mice with Deficiency in Cytochrome P450 46A1–Mediated Cholesterol Removal

CYP46A1 is the cytochrome P450 enzyme that converts cholesterol to 24-hydroxycholesterol, a cholesterol elimination product and a potent liver X receptor (LXR) ligand. We conducted retinal characterizations of Cyp46a1-/- mice that had normal fasting blood glucose levels but up to a 1.8-fold increase in retinal cholesterol. The retina of Cyp46a1-/- mice exhibited venous beading and tortuosity, microglia/macrophage activation, and increased vascular permeability, features commonly associated with diabetic retinopathy. The expression of Lxrα and Lxrβ was increased in both the whole Cyp46a1-/- retina and retinal macroglia/macrophages. The LXR-target genes were affected as well, primarily in activated microglial cells and macrophages. In the latter, the LXR-transactivated genes (Abca1, Abcg1, Apod, Apoe, Mylip, and Arg2) were up-regulated; similarly, there was an up-regulation of the LXR-transrepressed genes (Ccl2, Ptgs2, Cxcl1, Il1b, Il6, Nos2, and Tnfa). For comparison, gene expression was investigated in bone marrow-derived macrophages from Cyp46a1-/- mice as well as retinal and bone marrow-derived macrophages from Cyp27a1-/- and Cyp27a1-/-Cyp46a1-/- mice. CYP46A1 expression was detected in retinal endothelial cells, and this expression was increased in the proinflammatory environment. Retinal Cyp46a1-/- phosphoproteome revealed altered phosphorylation of 30 different proteins, including tight junction protein zonula occludens 1 and aquaporin 4. Collectively, the data obtained establish metabolic and regulatory significance of CYP46A1 for the retina and suggest pharmacologic activation of CYP46A1 as a potential therapeutic approach to dyslipidemia-induced retinal damage.

Transcorneal Electrical Stimulation Inhibits Retinal Microglial Activation and Enhances Retinal Ganglion Cell Survival After Acute Ocular Hypertensive Injury.

PurposeTo investigate the effect of transcorneal electrical stimulation (TcES) on retinal ganglion cell (RGC) function and survival after acute ocular hypertension-related retinal injury in gerbil eyes.MethodsGerbil eyes were subjected to acute ocular hypertensive injury (80 mm Hg for 60 minutes). In the treatment group, TcES was applied to the surgical eye immediately and then twice weekly for a total of 1 month. In the control group, sham TcES was given to the surgical eye at the same time points. Retinal function was assessed and compared between groups using flash electroretinography. For histological analysis, the number of RGC and microglial cells were counted by immunofluorescence staining after the gerbils were sacrificed on day 7 and day 28. Real-time polymerase chain reaction and western blot analysis were conducted to compare expression of interleukin (IL)-10, IL-6, COX-2, tumor necrosis factor (TNF)-α, and NF-κB phosphorylation among groups.ResultsTcES-treated eyes had significantly higher RGC survival at 1 month compared to controls. This was associated with RGC function. Furthermore, TcES-treated eyes were shown to have increased IL-10 expression, with a corresponding reduction in IL-6 and COX-2 expression as well as reduction in NF-κB phosphorylation. This was associated with a suppression in microglial cell activation in TcES-treated eyes.ConclusionsEarly treatment with TcES in gerbils protected the RGC from secondary damage and preserved retinal function in acute ocular hypertensive injury through modulation of the microglial-cell activated local inflammatory response.Translational RelevanceOur study strengthens the argument for translating TcES as a viable treatment in acute glaucoma.

Loss of XBP1 accelerates age-related decline in retinal function and neurodegeneration

BackgroundAging is the strongest risk factor for neurodegenerative diseases and extended age results in neuronal degeneration and functional decline in the visual system. Among many contributing factors to age-related deterioration of neurons is an insufficient activation of the Unfolded Protein Response (UPR) in the endoplasmic reticulum (ER) in response to cellular stress. X-box binding protein 1 (XBP1) is a major component of the UPR and is essential for maintaining protein homeostasis and reducing cellular stresses. Herein, we investigate the role of XBP1 in maintaining morphological and functional integrity in retinal neurons during adulthood and the early stages of aging.MethodsThe basal and induced levels of XBP1 activation in the retina were measured in young adult and aged mice. Conditional knockout (cKO) of XBP1 in retinal neurons was achieved by crossing XBP1 floxed mice with a retina specific Cre-recombinase line (Chx10-Cre). Retinal morphology, neuronal populations including photoreceptors, bipolar cells, and retinal ganglion cells (RGCs), synaptic structure, and microglial activation were examined with immunohistochemistry and staining of retinal sections. Retinal function was evaluated with light-adapted (photopic) and dark adapted (scotopic) electroretinograms. Retinal mitochondrial function and metabolism was assessed by Seahorse XFe24 Extracellular Flux Analyzer.ResultsThe retinas of aged wild type (WT) mice display a significantly reduced basal level of Xbp1s and compromised activation of ER stress response. In XBP1 cKO mice, significant structural degeneration of the retina, evidenced by thinning of retinal layers and a loss of RGCs, and functional defects indicated by diminished photopic and scotopic ERG b-waves are observed at the age of 12–14 months. Furthermore, discontinuous and disorganized synaptic laminae, colocalized with activated microglia, in the inner plexiform layer is found in the XBP1 cKO retinas. In addition, cKO mice demonstrate a significant increase in ectopic synapses between bipolar cells and photoreceptors, which is strikingly similar to WT mice at 20–24 months of age. These changes are associated with defective retinal glycolysis while mitochondrial respiratory function appears normal in the cKO retina.ConclusionsXBP1 cKO mice at 12–14 months of age show significant structural, functional, and metabolic deficits that closely resemble WT mice twice that age. Our findings suggest that the absence of XBP1, a critical component of the UPR, accelerates age-related retinal neurodegeneration.

Activation of retinal and optic nerve microglia following optic nerve crush in rats

Background Traumatic optic neuropathy (TON) is an acute injury of the optic nerve secondary to trauma.As an important immune cell, microglia is involved in a variety of pathologic changes in central nervous system diseases and eye diseases.However, the role of microglia in the lesion development and neural restoration in TON is still unclear. Objective This study was to compare the differences of microglial activation between optic nerve axon and retina after optic nerve crush injury, including the morphological changes, number, and distribution of microglia. Methods Thirty-five SPF female Sprague-Dawley (SD) rats were numbered from 1 to 35 and randomized into normal control group, sham operation group and modeling 6-hour group, modeling 3-day group, modeling 7-day group, modeling 14-day group and modeling 30-day group according to the random number table.Optic nerve injury models were established by crushing optic nerve at retrobulbar 2 mm for 10 seconds.The same operation was carried out but not clamping optic nerve in the sham operation group, and normal control group without any treatment.The morphological changes, number and distribution of microglia in retina and optic nerve axon were assayed and compared by immunofluorescence staining with lectin-FITC fluorescence labeled antibody detection.The use and care of the experimental animals complied with ARVO. Results Microglia were displayed mainly in inner plexiform layer (IPL), and a few of microglia were in inner nuclear layer (INL) and ganglion cell layer (GCL), not any microglia were seen in outer nuclear layer (ONL) and outer plexiform layer (OPL) in the normal control group and sham operation group.Thining synapsis and secondary branches were seen with the dendroid in shape in normal control group.In the model groups, the microglia were more in GCL compared with sham operation group and showed amoeboid microglial cells.The number of microglia in retinas was 6.40±1.52, 7.20±2.05, 12.00±3.54, 14.00±4.06, 18.00±4.36, 18.40±3.13 and 10.80±1.92 in the normal control group, sham operation group, modeling 6-hour group, modeling 3-day group, modeling 7-day group, modeling 14-day group and modeling 30-day group, respectively, and the microglia number were significantly increased in modeling 6-hour group, modeling 3-day group, modeling 7-day group, modeling 14-day group and modeling 30-day group in comparison with the normal control group, and in the modeling 30-day group, the microglial number in retinas was significantly reduced in comparison with the modeling 7-day group and modeling 14-day group (all at P<0.05). The activited microglia in the retinas were nuch more in the modeling 3-day group, modeling 7-day group and modeling 14-day group than that in the sham operation group (P=0.024, 0.009, 0.023). In the optic nerve, the microglial cells were small in size and sparse in arrangement in the normal control group and sham operation group.The cells were enlarged with the amoeboid-like in shape in the model groups and distributed in injury area.The cell number was significantly higher in the modeling 6-hour group, modeling 3-day group, modeling 7-day group and modeling 14-day group than that in the normal control group (P=0.007, 0.001, 0.003, 0.014). The cell number in modeling 30-day group was significnatly reduced in comparison with the modeling 3-day group, modeling 7-day group and modeling 14-day group (all at P<0.05). The activated microglial number were significantly elevated in the modeling 6-hour group, modeling 3-day group, modeling 7-day group compared with the sham operation group (P=0.005, 0.004, 0.030), and the cell number in the modeling 30-day group was evidently lower than that in the modeling 3-day group (P=0.021, 0.004). The cells of activated state in the optic nerve were significantly increased in modeling 6-hour group and modeling 14-day group. Conclusions Microglia are activated and keep increasing number in retina and optic nerve at a certain period after optic nerve injury, and these changes are earlier and more distinct in optic nerve axon than those in retina. Key words: Optic nerve/injury; Retina; Microglia; Activation

Activation of retinal microglia and accumulation of sub‐retinal fluid after systemic challenge with Lipopolysaccharide in mice

PurposeRetinal pathology such as diabetic retinopathy can be exacerbated by systemic viral illness. However, little is known whether systemic inflammation causes activation of retinal microglia. The aim of the present study was to investigate the effect of a systemic inflammatory stimulus with lipopolysaccharide (LPS) on retinal microglia activation.MethodsLPS (1 mg/kg) was administered intravenously for 4 days in Cx3cr1gfp/+ mice, specifically expressing gfp in microglia/macrophages. Retinas were examined with optical coherence tomography, fluorescein angiography and autofluorescence imaging before and after the LPS challenge. A group of mice was fed with the colony stimulating factor‐1 receptor (CSF‐1R) inhibitor PLX5622, which has been shown to deplete retinal microglia after 48 hours, for 7 days and until the end of the experiments. PLX‐fed mice were challenged with LPS as above, imaged and euthanized at day 4 of the LPS challenge.ResultsAccumulation of sub‐retinal fluid and leakage of fluorescein was observed in the retinas of LPS mice, suggesting disruption of the blood retinal barrier (BRB). In vivo imaging and histology showed increased numbers of gfp+ cells in retinas and brains and accumulation around retinal vessels in the LPS group. Increase of gfp+ cell numbers was partially attributed to proliferation, based on Ki67 staining. PLX attenuated the numbers of gfp+ cells and reversed the effects of LPS on sub‐retinal fluid accumulation.ConclusionsOur data suggest that systemic LPS challenge initiates inflammatory responses in the retina and can lead to disruption of the BRB, whose integrity seems to be highly dependent on retinal microglia and/or invading macrophages. Pathology is completely abrogated in microglia depleted mice, suggesting that novel CSF‐1R inhibitors may be a promising target for inflammation mediated retinal diseases.

Impact of minocycline on vascularization and visual function in an immature mouse model of ischemic retinopathy

The role of microglia in the pathophysiology of ischemic retinal diseases has been extensively studied. Retinal microglial activation may be correlated with retinal neovascularization in oxygen-induced retinopathy (OIR), an animal model that has been widely used in retinopathy of prematurity (ROP) research. Minocycline is an antibiotic that decreases microglial activation following hyperoxic and hypoxic-ischemic phases in neonatal rodents. Here, we investigated the effects of minocycline on vascularization and visual function. In our results, we found that after the administration of minocycline, microglial reactivity was reduced in the retina, which was accompanied by an increase in the avascular area at P12, P14 and P17. Although microglial reactivity was reduced at P17, minocycline treatment did not attenuate retinal neovascularization. A changing trend in microglial number was observed, and the apoptosis and proliferation states on different days partly contributed to this change. Further study also revealed that although minocycline downregulated the levels of proinflammatory factors, visual function appeared to be significantly worsened. Collectively, we demonstrated that minocycline disturbed the physiological vascularization of the avascular area and exacerbated visual dysfunction, indicating that minocycline may not be an effective drug and may even be detrimental for the treatment of ischemic retinopathy in immature mammals.

Strong upregulation of inflammatory genes accompanies photoreceptor demise in canine models of retinal degeneration.

We have analyzed the complex pattern of the inflammatory response in early-onset canine models of human retinitis pigmentosa, rcd1, xlpra2 and erd, as well as late-onset xlpra1, in comparative manner. The time course of immune response genes and proteins expression was examined along the timeline of photoreceptors degeneration. Gene expression analysis of the early-onset models prior to and after the peak of photoreceptors death identified the involvement of multiple immune response genes including those encoding constituents of the NLRP3 inflammasome, its substrates, pro-IL1B, pro-IL18, and common components of IL1B, IL18 and TLR4 pathways. Out of two activated caspase-1 cleavage products, IL1B and IL18, only IL1B was detected in rcd1 and xlpra2 while precursor IL18 remained unprocessed in the same protein extract highlighting prominence of IL1B pathway. An overall immune response was most prominent in rcd1 followed by xlpra2 and least prominent in erd. Noticeably, in rcd1 and xlpra2, but not in erd, early induction of the immune response was accompanied by sustained intraretinal migration and activation of retinal microglia. Lastly, delayed activation of the anti-inflammatory factors in all early-onset models was insufficient to counterbalance rapidly progressing inflammation. In contrast to early-onset models, in late-onset xlpra1 retinas a subset of the pro-inflammatory genes was highly upregulated long before any disease-related structural changes occurred, but was counterbalanced by an adequate anti-inflammatory response. Results point out to upregulated immune response accompanying disease progression in animal models of retinal degeneration, and to potential benefits of early anti-inflammatory therapy.

Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration

BackgroundThe bromodomain and extraterminal domain (BET) family proteins (BET2, BET3, and BET4) “read” (bind) histone acetylation marks via two distinct bromodomains (Brom1 and Brom2) facilitating transcriptional activation. These epigenetic “readers” play crucial roles in pathogenic processes such as inflammation. The role of BETs in influencing the degenerative process in the retina is however unknown.MethodsWe employed the rd10 mouse model (Pde6brd10 mutation) of retinitis pigmentosa (RP) to examine the involvement of BET proteins in retinal neurodegeneration.ResultsInhibition of BET activity by intravitreal delivery of JQ1, a BET-specific inhibitor binding both Brom1 and Brom2, ameliorated photoreceptor degeneration and improved electroretinographic function. Rescue effects of JQ1 were related to the suppression of retinal microglial activation in vivo, as determined by decreased immunostaining of activation markers (IBA1, CD68, TSPO) and messenger RNA (mRNA) levels of inflammatory cytokines in microglia purified from rd10 retinas. JQ1 pre-treatment also suppressed microglial activation in vitro, decreasing microglial proliferation, migration, and mRNA expression of inflammatory cytokines (TNFα, MCP-1, IL-1β, IL-6, and RANTES). Expression of BET2, but not BET3 and BET4, was significantly elevated during photoreceptor degeneration at postnatal day (PN)24 in retinas of rd10 mice relative to age-matched wild-type controls. siRNA knockdown of BET2 but not BET4, and the inhibitor of Brom2 (RVX208) but not of Brom1 (Olinone), decreased microglial activation.ConclusionsThese findings indicate that BET inhibition rescues photoreceptor degeneration likely via the suppression of microglial activation and implicates BET interference as a potential therapeutic strategy for the treatment of degenerative retinal diseases.

Elevated Glucose and Interleukin-1β Differentially Affect Retinal Microglial Cell Proliferation.

Diabetic retinopathy is considered a neurovascular disorder, hyperglycemia being considered the main risk factor for this pathology. Diabetic retinopathy also presents features of a low-grade chronic inflammatory disease, including increased levels of cytokines in the retina, such as interleukin-1 beta (IL-1β). However, how high glucose and IL-1β affect the different retinal cell types remains to be clarified. In retinal neural cell cultures, we found that IL-1β and IL-1RI are present in microglia, macroglia, and neurons. Exposure of retinal neural cell cultures to high glucose upregulated both mRNA and protein levels of IL-1β. High glucose decreased microglial and macroglial cell proliferation, whereas IL-1β increased their proliferation. Interestingly, under high glucose condition, although the number of microglial cells decreased, they showed a less ramified morphology, suggesting a more activated state, as supported by the upregulation of the levels of ED-1, a marker of microglia activation. In conclusion, IL-1β might play a key role in diabetic retinopathy, affecting microglial and macroglial cells and ultimately contributing to neural changes observed in diabetic patients. Particularly, since IL-1β has an important role in retinal microglia activation and proliferation under diabetes, limiting IL-1β-triggered inflammatory processes may provide a new therapeutic strategy to prevent the progression of diabetic retinopathy.

Loss of Fractalkine Signaling Exacerbates Axon Transport Dysfunction in a Chronic Model of Glaucoma.

Neurodegeneration in glaucoma results in decline and loss of retinal ganglion cells (RGCs), and is associated with activation of myeloid cells such as microglia and macrophages. The chemokine fractalkine (FKN or Cx3cl1) mediates communication from neurons to myeloid cells. Signaling through its receptor Cx3cr1 has been implicated in multiple neurodegenerative diseases, but the effects on neuronal pathology are variable. Since it is unknown how FKN-mediated crosstalk influences RGC degeneration in glaucoma, we assessed this in a chronic mouse model, DBA/2J. We analyzed a DBA/2J substrain deficient in Cx3cr1, and compared compartmentalized RGC degeneration and myeloid cell responses to those in standard DBA/2J mice. We found that loss of FKN signaling exacerbates axon transport dysfunction, an early event in neurodegeneration, with a significant increase in RGCs with somal accumulation of the axonal protein phosphorylated neurofilament, and reduced retinal expression of genes involved in axon transport, Kif1b, and Atp8a2. There was no change in the loss of Brn3-positive RGCs, and no difference in the extent of damage to the proximal optic nerve, suggesting that the loss of fractalkine signaling primarily affects axon transport. Since Cx3cr1 is specifically expressed in myeloid cells, we assessed changes in retinal microglial number and activation, changes in gene expression, and the extent of macrophage infiltration. We found that loss of fractalkine signaling led to innate immune changes within the retina, including increased infiltration of peripheral macrophages and upregulated nitric oxide synthase-2 (Nos-2) expression in myeloid cells, which contributes to the production of NO and can promote axon transport deficits. In contrast, resident retinal microglia appeared unchanged either in number, morphology, or expression of the myeloid activation marker ionized calcium binding adaptor molecule 1 (Iba1). There was also no significant increase in the proinflammatory gene interleukin 1 beta (Il1β). We conclude that loss of fractalkine signaling causes a selective worsening of axon transport dysfunction in RGCs, which is linked to enhanced Nos-2 expression in myeloid cells. Our findings suggest that distinct mechanisms may contribute to different aspects of RGC decline in glaucoma, with axonal transport selectively altered after loss of Cx3cr1 in microglia and/or macrophages.

Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling.

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue ‘Norgestrel’ is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel’s neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising treatment for RP, with dual actions as a neuroprotective and anti-inflammatory agent in the retina.

Erianin inhibits high glucose-induced retinal angiogenesis via blocking ERK1/2-regulated HIF-1α-VEGF/VEGFR2 signaling pathway

Erianin is a natural compound found in Dendrobium chrysotoxum Lindl. Diabetic retinopathy (DR) is a serious and common microvascular complication of diabetes. This study aims to investigate the inhibitory mechanism of erianin on retinal neoangiogenesis and its contribution to the amelioration of DR. Erianin blocked high glucose (HG)-induced tube formation and migration in choroid-retinal endothelial RF/6A cells. Erianin inhibited HG-induced vascular endothelial growth factor (VEGF) expression, hypoxia-inducible factor 1-alpha (HIF-1α) translocation into nucleus and ERK1/2 activation in RF/6A and microglia BV-2 cells. MEK1/2 inhibitor U0126 blocked HG-induced HIF-1α and ERK1/2 activation in both above two cells. In addition, erianin abrogated VEGF-induced angiogenesis in vitro and in vivo, and also inhibited VEGF-induced activation of VEGF receptor 2 (VEGFR2) and its downstream cRaf-MEK1/2-ERK1/2 and PI3K-AKT signaling pathways in RF/6A cells. Furthermore, erianin reduced the increased retinal vessels, VEGF expression and microglia activation in streptozotocin (STZ)-induced hyperglycemic and oxygen-induced retinopathy (OIR) mice. In conclusion, our results demonstrate that erianin inhibits retinal neoangiogenesis by abrogating HG-induced VEGF expression by blocking ERK1/2-mediated HIF-1α activation in retinal endothelial and microglial cells, and further suppressing VEGF-induced activation of VEGFR2 and its downstream signals in retinal endothelial cells.

Transcorneal electrical stimulation promotes survival of retinal ganglion cells after optic nerve transection in rats accompanied by reduced microglial activation and TNF-α expression

Microglial activation plays a crucial role in the pathological processes of various retinal and optic nerve diseases. TNF-α is a pro-inflammatory cytokine that is rapidly upregulated and promotes retinal ganglion cells (RGCs) death after optic nerve injury. However, the cellular source of TNF-α after optic nerve injury remains unclear. Thus, we aimed to determine the changes of retinal microglial activation in a rat model of optic nerve transection (ONT) after transcorneal electrical stimulation (TES). Furthermore, we assessed TNF-α expression after ONT and evaluated the effects of TES on TNF-α production. Rats were divided into 2 control groups receiving a sham surgery procedure, 2 ONT+Sham TES groups, and 2 ONT+TES groups. The rats were sacrificed on day 7 or 14 after ONT. RGCs were retrogradely labelled by Fluorogold (FG) 7 days before ONT, one TES group and corresponding controls were stimulated on day 0, 4, and the second were stimulated on day 0, 4, 7, 10. Whole-mount immunohistofluorescence, quantification of RGCs and microglia, and western blot analysis were performed on day 7 and 14 after ONT. TES significantly increased RGCs survival on day 7 and 14 after ONT, which was accompanied by reduced microglia on day 7, but not 14. TNF-α was co-localized with ameboid microglia and significantly increased on day 7 and 14 after ONT. TES significantly reduced TNF-α production on day 7 and 14 after ONT. Our study demonstrated that TES promotes RGCs survival after ONT accompanied by reduced microglial activation and microglia-derived TNF-α production.