Bone Marrow Mesenchymal Stem Cells (BMSCs)-Derived Exosomes Promotes Proliferation and Differentiation of Retinal Neuron-Like Cells to Repair Corneal Epithelial Damage

Dry eye disease (DED) is a common ocular surface disease. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into various cells, and BMSC-derived exosomes (BMSC-exo) is essential to maintaining BMSCs stemness. This study aimed to elucidate the mechanism underlying BMSCexo in DED. Sixty rats with corneal epithelial injury were treated with BMSCs or BMSC-exo and untreated (each group, n = 20) followed by analysis of the effect of BMSCs and BMSC-exo by evaluating the corneal epithelium damage via measuring the Basso-Beattie-Bresnahan (BBB) score on 1st, 3rd, 7th, 14th, 28th day after treatments. TUNEL staining assessed cell apoptosis, NF200 expression and the number of BrdU-positive cells. There was no significant difference in BBB scores among three groups on the 1st and 3rd day after treatment (p > 0.05) with significant difference on the 7th, 14th, and 28th day (p <0.05); compared with control group, BMSCs group and combination group had significantly higher BBB score (p < 0.05). The amount of apoptotic cells rose on 3rd and then gradually decreased since 7th day. Moreover, BMSCs and BMSC-exo decreased the apoptotic index and increased absorbance of NF200 and BrdU-positive rate (p < 0.05). BMSC-exo alleviates corneal epithelial damage in DED and facilitates wound healing possibly through reducing cell apoptosis and increasing retinal neuron-like cell proliferation protein.

Context-Dependent Effects of Inflammation On Retina Regeneration

Inflammation is required for the proliferation of Müller glia (MG) into multipotent progenitors (MGPCs) in the injured fish and avian retinas. However, its function in retina regeneration has not been fully understood. Here we investigated the role of inflammation in three different retinal regeneration paradigms in zebrafish (stab-injury, NMDA-injury and insulin treatment). We first show that different types of immune cells and levels of inflammatory cytokines were found in the retinas of these paradigms. Though zymosan injection alone was insufficient to induce MG proliferation in the uninjured retina, immune suppression significantly inhibited MGPC formation in all three paradigms. Enhancing inflammation promoted MGPC formation after stab-injury, while exhibiting a context-dependent role in the NMDA or insulin models. Furthermore, proper levels of inflammation promoted MG reprogramming and cell cycle re-entry after stab- or NMDA-injury, but excessive inflammation also suppressed MG proliferation in the latter model. Finally, while inflammation promoted retinal neuron regeneration after stab-injury, immune suppression surprisingly achieved the best regeneration in the NMDA model. Our study reveals the complex and context-dependent role of inflammation during retinal repair in fish, and suggests accurate inflammation management may be crucial for successful retina regeneration in mammals.

Study to elucidate the pharmacological activity of retinalamin in a rat model of ischemic retinopathy

Introduction: Over the past few years, the incidence of retinal ischemic disorders has been increasing, due to a rising prevalence of such socially burdensome diseases as diabetes and hypertension, which ultimately lead to ocular vascular pathology. The identification of new treatment options that would prevent retinal neuron death is a crucial task of modern pharmacology. Materials and methods: The research was carried out on male Wistar rats. Retinopathy was modeled by inducing a 30-min ischemic episode, with a 72-hour period of reperfusion and subsequent administration of Retinalamin and Emoxypine for 10 days. The effectiveness of the drugs was evaluated by electroretinographic, ophthalmoscopic and morphological assessments. Results and discussion: On Day 14 of the experiment, a dose-dependent preservation of the electroretinogram b-wave/a-wave amplitude ratio was observed in the animals treated with Retinalamin depending on a dose (1.39±0.06, 1.46±0.03 and 1.49±0.04 in low (0.214 mg/kg), medium (0.428 mg/kg) and high (0.857 mg/kg) Retinalamin dose groups, respectively). The ophthalmoscopic picture of the fundus oculi also improved following the treatment with Retinalamin (1.42, 1.69 and 1.90 times lower ophthalmoscopic scores compared to placebo-treated animals in low, medium and high dose groups, respectively). The morphologic “coefficient of change” applied to ganglion cell layer was 2.2, 1.7 and 1.6 points in low, medium and high dose Retinalamin groups, respectively. These results are significantly different from both intact and placebo group (p&lt;0.05). Based on the aforementioned experimental findings, we conclude that Retinalamin has a retinoprotective effect and is superior to the drug of comparison (Emoxypine). Conclusion: The greatest neuroprotective effects were shown in the groups receiving Retinalamin. In these groups, the ERG b-wave/a-wave amplitude ratio was preserved, the ophthalmoscopic picture was less pathologic and retinal morphology features were close to those of the intact retina.

Peripheral blood-derived monocytes show neuronal properties and integration in immune-deficient rd1 mouse model upon phenotypic differentiation and induction with retinal growth factors

Background Cell therapy is one of the most promising therapeutic interventions for retinitis pigmentosa. In the current study, we aimed to assess if peripheral blood-derived monocytes which are highly abundant and accessible could be utilized as a potential candidate for phenotypic differentiation into neuron-like cells. Methods The peripheral blood-derived monocytes were reconditioned phenotypically using extrinsic growth factors to induce pluripotency and proliferation. The reconditioned monocytes (RM) were further incubated with a cocktail of growth factors involved in retinal development and growth to induce retinal neuron-like properties. These cells, termed as retinal neuron-like cells (RNLCs) were characterized for their morphological, molecular and functional behaviour in vitro and in vivo. Results The monocytes de-differentiated in vitro and acquired pluripotency with the expression of prominent stem cell markers. Treatment of RM with retinal growth factors led to an upregulation of neuronal and retinal lineage markers and downregulation of myeloid markers. These cells show morphological alterations resembling retinal neuron-like cells and expressed photoreceptor (PR) markers. The induced RNLCs also exhibited relative membrane potential change upon light exposure suggesting that they have gained some neuronal characteristics. Further studies showed that RNLCs could also integrate in an immune-deficient retinitis pigmentosa mouse model NOD.SCID-rd1 upon sub-retinal transplantation. The RNLCs engrafted in the inner nuclear layer (INL) and ganglion cell layer (GCL) of the RP afflicted retina. Mice transplanted with RNLCs showed improvement in depth perception, exploratory behaviour and the optokinetic response. Conclusions This proof-of-concept study demonstrates that reconditioned monocytes can be induced to acquire retinal neuron-like properties through differentiation using a defined growth media and can be a potential candidate for cell therapy-based interventions and disease modelling for ocular diseases.

Different lineage contexts direct common pro-neural factors to specify distinct retinal cell subtypes

How astounding neuronal diversity arises from variable cell lineages in vertebrates remains mostly elusive. By in vivo lineage tracing of ∼1,000 single zebrafish retinal progenitors, we identified a repertoire of subtype-specific stereotyped neurogenic lineages. Remarkably, within these stereotyped lineages, GABAergic amacrine cells were born with photoreceptor cells, whereas glycinergic amacrine cells were born with OFF bipolar cells. More interestingly, post-mitotic differentiation blockage of GABAergic and glycinergic amacrine cells resulted in their respecification into photoreceptor and bipolar cells, respectively, suggesting lineage constraint in cell subtype specification. Using single-cell RNA-seq and ATAC-seq analyses, we further identified lineage-specific progenitors, each defined by specific transcription factors that exhibited characteristic chromatin accessibility dynamics. Finally, single pro-neural factors could specify different neuron types/subtypes in a lineage-dependent manner. Our findings reveal the importance of lineage context in defining neuronal subtypes and provide a demonstration of in vivo lineage-dependent induction of unique retinal neuron subtypes for treatment purposes.

Tau Gene Deletion Does Not Influence Axonal Regeneration and Retinal Neuron Survival in the Injured Mouse Visual System

In the present study, we hypothesized that the microtubule-associated protein Tau may influence retinal neuron survival and axonal regeneration after optic nerve injury. To test this hypothesis, the density of retinal ganglion cells was evaluated by immunostaining retinal flat-mounts for RNA-binding protein with multiple splicing (RBPMS) two weeks after optic nerve micro-crush lesion in Tau-deprived (Tau knock-out (KO)) and wild-type (WT) mice. Axon growth was determined on longitudinal sections of optic nerves after anterograde tracing. Our results showed that the number of surviving retinal ganglion cells and growing axons did not significantly vary between WT and Tau KO animals. Moreover, sustained activation of the neuronal growth program with ciliary neurotrophic factor (CNTF) resulted in a similar increase in surviving neurons and in growing axons in WT and Tau KO mice. Taken together, our data suggest that Tau does not influence axonal regeneration or neuronal survival.

Electrical Stimulation Induces Retinal Müller Cell Proliferation and Their Progenitor Cell Potential

Non-invasive electrical stimulation (ES) is increasingly applied to improve vision in untreatable eye conditions, such as retinitis pigmentosa and age-related macular degeneration. Our previous study suggested that ES promoted retinal function and the proliferation of progenitor-like glial cells in mice with inherited photoreceptor degeneration; however, the underlying mechanism remains obscure. Müller cells (MCs) are thought to be dormant residential progenitor cells that possess a high potential for retinal neuron repair and functional plasticity. Here, we showed that ES with a ramp waveform of 20 Hz and 300 µA of current was effective at inducing mouse MC proliferation and enhancing their expression of progenitor cell markers, such as Crx (cone–rod homeobox) and Wnt7, as well as their production of trophic factors, including ciliary neurotrophic factor. RNA sequencing revealed that calcium signaling pathway activation was a key event, with a false discovery rate of 5.33 × 10−8 (p = 1.78 × 10−10) in ES-mediated gene profiling changes. Moreover, the calcium channel blocker, nifedipine, abolished the observed effects of ES on MC proliferation and progenitor cell gene induction, supporting a central role of ES-induced Ca2+ signaling in the MC changes. Our results suggest that low-current ES may present a convenient tool for manipulating MC behavior toward neuroregeneration and repair.