PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons

PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis,apoptosis,and nec roptosis,which simultaneously occur during the pathophysiological process of infectious and inflammatory diseases.Although our previous lite rature mining study suggested that PANoptosis might occur in neuronal ischemia/repe rfusion injury,little experimental research has been reported on the existence of PANoptosis.In this study,we used in vivo and in vitro retinal neuronal models of ischemia/repe rfusion injury to investigate whether PAN optosis-like cell death(simultaneous occurrence of pyroptosis,apo ptosis,and necroptosis)exists in retinal neuronal ischemia/repe rfusion injury.Our results showed that ischemia/repe rfusion injury induced changes in morphological features and protein levels that indicate PANoptosis-like cell death in retinal neurons both in vitro and in vivo.Ischemia/repe rfusion inju ry also significantly upregulated caspase-1,caspase-8,and NLRP3 expression,which are important components of the PANoptosome.These results indicate the existence of PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons and provide preliminary experimental evidence for future study of this new type of regulated cell death.

Glycogen synthase kinase-3： a key kinase in retinal neuron apoptosis in early diabetic retinopathy

Background Diabetes-related pathogenic factors can cause retinal ganglion cell （RGC） apoptosis, but the specific mechanism is not very clear. The aim of this study is to investigate the correlation between glycogen synthase kinase-3 （GSK-3） activation and retinal neuron apoptosis. Methods In an in vitro experiment, the number of apoptotic RGC-5 cells differentiated by staurosporine was evaluated via flow cytometry and nuclei staining using Hoechst 33258. GSK-3 phosphorylation and caspase-3 activation in RGC-5 cells after serum deprivation were determined using Western blotting. Mitochondrial membrane potential was detected using the dye 5,5＇,6,6＇-tetrachloro-l,l＇,3,3＇-tetrethyl benzimidalyl carbocyanine iodide, and reactive oxygen species （ROS） levels were measured with dihydroethidium. In an in vivo experiment, the number of apoptotic retinal neurons was evaluated via terminal transferase dUTP nick-end labeling （TUNEL）, and GSK-3 phosphorylation was determined using Western blotting, in the retinal nerve epithelial tissue of rats in which diabetes was induced by intravenous tail-vein injection of streptozotocin for 4 weeks. Results The levels of phosphorylated Ser21/9 in GSK-3α/13 and p-T308/S473-AKT were lower and the cleaved caspase-3 levels were higher in the serum-deprived model （P 〈0.05）. Lithium chloride treatment was associated with a slower rate of apoptosis, increased mitochondrial membrane potential, and decreased ROS levels in differentiated RGC-5 cells （P 〈0.05）. The level of blood glucose and the number of TUNEL-positive cells in the whole-mounted retinas were higher （P 〈0.01）, and the levels of phosphorylated Ser21/9 in GSK-3α/13 and body weight were lower （P 〈0.05）. However, the thickness of the retinal nerve epithelial layer was not significantly less in diabetic rats compared with control group. Lithium chloride intravitreal injection increased the levels of phosphorylated Ser21/9 in GSK-3α/13 and decreased TUNEL-positive cells in the whole-mounted retinas. Conclusion GSK-3 kinase is closely related to retinal neuron apoptosis, and the application of the GSK-3 inhibitor lithium chloride can reduce retinal neuron apoptosis in early diabetic retinopathy.

Neural and Müller glial adaptation of the retina to photoreceptor degeneration

The majority of inherited retinal degenerative diseases and dry age-related macular degeneration are characterized by decay of the outer retina and photoreceptors,which leads to progressive loss of vision.The inner retina,including second-and third-order retinal neurons,also shows aberrant structural changes at all stages of degeneration.Müller glia,the major glial cells maintain retinal homeostasis,activating and rearranging immediately in response to photoreceptor stress.These phenomena are collectively known as retinal remodeling and are anatomically well described,but their impact on visual function is less well characterized.Retinal remodeling has traditionally been considered a detrimental chain of events that decreases visual function.However,emerging evidence from functional assays suggests that remodeling could also be a part of a survival mechanism wherein the inner retina responds plastically to outer retinal degeneration.The visual system’s first synapses between the photoreceptors and bipolar cells undergo rewiring and functionally compensate to maintain normal signal output to the brain.Distinct classes of retinal ganglion cells remain even after the massive loss of photoreceptors.Müller glia possess the regenerative potential for retinal recovery and possibly exert adaptive transcriptional changes in response to neuronal loss.These types of homeostatic changes could potentially explain the well-maintained visual function observed in patients with inherited retinal degenerative diseases who display prominent anatomic retinal pathology.This review will focus on our current understanding of retinal neuronal and Müller glial adaptation for the potential preservation of retinal activity during photoreceptor degeneration.Targeting retinal self-compensatory responses could help generate universal strategies to delay sensory disease progression.

The complexities underlying age-related macular degeneration： could amyloid beta play an important role？

e-related macular degeneration （AMD） causes irreversible loss of central vision for which there is no effective treatment. Incipient pathology is thought to occur in the retina for many years before AMD manifests from midlife onwards to affect a large proportion of the elderly. Although genetic as well as non-genetic/environmental risks are recognized, its complex aetiology makes it difficult to identify susceptibility, or indeed what type of AMD develops or how quickly it progresses in different individuals. Here we summarize the literature describing how the Alzheimer＇s-linked amyloid beta （Aβ） group of misfolding proteins accumulate in the retina. The discovery of this key driver of Alzheimer＇s disease in the senescent retina was unexpected and surprising, enabling an altogether different perspective of AMD. We argue that Aβ fundamentally differs from other substances which accumulate in the ageing retina, and discuss our latest findings from a mouse model in which physiological amounts of Aβ were subretinally-injected to recapitulate salient features of early AMD within a short period. Our discoveries as well as those of others suggest the pattern of Aβ accumulation and pathology in donor aged/AMD tissues are closely reproduced in mice, including late-stage AMD phenotypes, which makes them highly attractive to study dynamic aspects of Aβ-mediated retinopathy. Furthermore, we discuss our findings revealing how Aβ behaves at single-cell resolution, and consider the long-term implications for neuroretinal function. We propose Aβ as a key element in switching to a diseased retinal phenotype, which is now being used as a biomarker for latestage AMD.

Identification of connexin 50 and 57 mRNA in A-type horizontal cells of the rabbit retina

Horizontal cells (HCs) mediate negative feedback to photoreceptors. In the mammalian retina, there are two types of HCs, which are extensively coupled to neighboring cells through homologous gap junctions. The permeability and therefore the strength of feedback can be regulated by light intensity, dopamine and many other factors. However, the component(s) of the most prominent gap junctions, those between A-type HCs in the rabbit retina, is still unknown. In this study, we compared the sequences of many types of mammalian connexins, obtained partial sequences of rabbit connexin 50 and 57. Using specific primers designed against the rabbit sequences, we identified mRNAs of connexin 50 and/or 57 in visually selected single A-type HC using multiplex RT-PCR.

Genetic and epigenetic regulators of retinal Müller glial cell reprogramming

Background:Retinal diseases characterized with irreversible loss of retinal nerve cells,such as optic atrophy and retinal degeneration,are the main causes of blindness.Current treatments for these diseases are very limited.An emerging treatment strategy is to induce the reprogramming of Müller glial cells to generate new retinal nerve cells,which could potentially restore vision.Main text:Müller glial cells are the predominant glial cells in retinae and play multiple roles to maintain retinal homeostasis.In lower vertebrates,such as in zebrafish,Müller glial cells can undergo cell reprogramming to regenerate new retinal neurons in response to various damage factors,while in mammals,this ability is limited.Interestingly,with proper treatments,Müller glial cells can display the potential for regeneration of retinal neurons in mammalian retinae.Recent studies have revealed that dozens of genetic and epigenetic regulators play a vital role in inducing the reprogramming of Müller glial cells in vivo.This review summarizes these critical regulators for Müller glial cell reprogramming and highlights their differences between zebrafish and mammals.Conclusions:A number of factors have been identified as the important regulators in Müller glial cell reprogramming.The early response of Müller glial cells upon acute retinal injury,such as the regulation in the exit from quiescent state,the initiation of reactive gliosis,and the re-entry of cell cycle of Müller glial cells,displays significant difference between mouse and zebrafish,which may be mediated by the diverse regulation of Notch and TGFβ(transforming growth factor-β)isoforms and different chromatin accessibility.

Activation of Parvalbumin-Positive Neurons in Both Retina and Primary Visual Cortex Improves the Feature-Selectivity of Primary Visual Cortex Neurons

Several recent studies using either viral or transgenic mouse models have shown different results on whether the activation of parvalbumin-positive（PV~＋）neurons expressing channelrhodopsin-2（ChR2） in the primary visual cortex（V1） improves the orientation-and direction-selectivity of V1 neurons. Although this discrepancy was thoroughly discussed in a follow-up communication, the issue of using different models to express ChR2 in V1 was not mentioned. We found that ChR2 was expressed in retinal ganglion cells（RGCs） and V1 neurons in ChR2fl/~＋; PV-Cre mice. Our results showed that the activation of PV~＋RGCs using white drifting gratings alone significantly decreased the firing rates of V1 neurons and improved their direction-and orientation-selectivity. Longduration activation of PV~＋interneurons in V1 further enhanced the feature-selectivity of V1 neurons in anesthetized mice, confirming the conclusions from previous findings. These results suggest that the activation of both PV~＋RGCs and V1 neurons improves feature-selectivity in mice.

Validation of glaucoma-like features in the rat episcleral vein cauterization model

Background Glaucoma,an irreversible optic nerve neuropathy,always results in blindness.This study aimed to evaluate glaucoma-like features in the rat episcleral vein cauterization （EVC） model by multiple in vivo and in vitro evidences.Methods Wistar rat was used in this study.The elevated intraocular pressure （IOP） was induced by cauterization of three episcleral veins.lOP was monitored with Tono-Pen XL tonometer.Time-dependent changes to the neuronal retinal layers were quantified by Fourier domain-optical coherence tomography.The function of retina was evaluated by electroretinogram （ERG）.Survival of retinal ganglion cells （RGCs） was quantified by retrograde labeling.Histology study was performed with retinal sections stained with hematoxylin-eosin,glial fibrillary acidic protein,and neuronal nuclear antigen.Retina and aqueous humor protein were extracted and cytotoxic protein tumor necrosis factor alpha （TNF-α） and alpha-2 macroglobulin （α2m） were measured with Western blotting.Results EVC is a relatively facile intervention,with low failure rates （＜5％）.After surgical intervention,chronic mild lOP elevation （about 1.6-fold over normal,P ＜0.05） was induced for at least 6 weeks without requiring a second intervention.High lOP causes chronic and progressive loss of RGCs （averaging about 4％ per week）,progressive thinning of neuronal retinal layers （3-5 μm per week）,and reduction of a-and b-wave in ERG.EVC method can also induce glial cell activation and alterations of inflammation proteins,such as TNF-α and α2m.Conclusion EVC method can establish a robust,reliable,economic and highly reproducible glaucomatous animal model.