Pax6 isoforms shape eye development: Insights from developmental stages and organoid models.

Pax6 is a critical transcription factor involved in the development of the central nervous system. However, in humans, mutations in Pax6 predominantly result in iris deficiency rather than neurological phenotypes. This may be attributed to the distinct functions of Pax6 isoforms, Pax6a and Pax6b. In this study, we investigated the spatial and temporal expression patterns of Pax6 isoforms during different stages of mouse eye development. We observed a strong correlation between Pax6a expression and the neuroretina gene Sox2, while Pax6b showed a high correlation with iris-component genes, including the mesenchymal gene Foxc1. During early patterning from E10.5, Pax6b was expressed in the hinge of the optic cup and neighboring mesenchymal cells, whereas Pax6a was absent in these regions. At E14.5, both Pax6a and Pax6b were expressed in the future iris and ciliary body, coinciding with the integration of mesenchymal cells and Mitf-positive cells in the outer region. From E18.5, Pax6 isoforms exhibited distinct expression patterns as lineage genes became more restricted. To further validate these findings, we utilized ESC-derived eye organoids, which recapitulated the temporal and spatial expression patterns of lineage genes and Pax6 isoforms. Additionally, we found that the spatial expression patterns of Foxc1 and Mitf were impaired in Pax6b-mutant ESC-derived eye organoids. This in vitro eye organoids model suggested the involvement of Pax6b-positive local mesodermal cells in iris development. These results provide valuable insights into the regulatory roles of Pax6 isoforms during iris and neuroretina development and highlight the potential of ESC-derived eye organoids as a tool for studying normal and pathological eye development.

Intrinsic Expression of Coagulation Factors and Protease Activated Receptor 1 (PAR1) in Photoreceptors and Inner Retinal Layers.

The aim of this study was to characterize the distribution of the thrombin receptor, protease activated receptor 1 (PAR1), in the neuroretina. Neuroretina samples of wild-type C57BL/6J and PAR1-/- mice were processed for indirect immunofluorescence and Western blot analysis. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to determine mRNA expression of coagulation Factor X (FX), prothrombin (PT), and PAR1 in the isolated neuroretina. Thrombin activity following KCl depolarization was assessed in mouse neuroretinas ex vivo. PAR1 staining was observed in the retinal ganglion cells, inner nuclear layer cells, and photoreceptors in mouse retinal cross sections by indirect immunofluorescence. PAR1 co-localized with rhodopsin in rod outer segments but was not expressed in cone outer segments. Western blot analysis confirmed PAR1 expression in the neuroretina. Factor X, prothrombin, and PAR1 mRNA expression was detected in isolated neuroretinas. Thrombin activity was elevated by nearly four-fold in mouse neuroretinas following KCl depolarization (0.012 vs. 0.044 mu/mL, p = 0.0497). The intrinsic expression of coagulation factors in the isolated neuroretina together with a functional increase in thrombin activity following KCl depolarization may suggest a role for the PAR1/thrombin pathway in retinal function.

A distal enhancer that directs Otx2 expression in the retinal pigment epithelium and neuroretina.

BACKGROUND: Homeodomain transcription factor Otx2 is essential for embryonic development of multiple head tissues, including retinal pigment epithelium (RPE) and neuroretina. Temporospatial regulation of Otx2 expression is critical for its functions. Molecular dissection of the cis-acting enhancers will help elucidate how Otx2 expression is regulated. RESULTS: We comprehensively characterized distal enhancer hs1150 that was previously identified in a high throughput study. We established multiple transgenic mouse lines in which human hs1150, corresponding mouse hs1150, and two highly conserved sub-fragments in the mouse hs1150 were individually fused to a minimal hsp68 promoter to drive reporter expression. We found that hs1150 enhancer directed reporter expression in the RPE, neuroretina, and brain in a developmentally regulated manner. Human hs1150-directed reporter expression largely recapitulated Otx2 expression in the RPE, in the early neuroretina, and to a lesser degree in the early brain. Mouse hs1150, although shorter than human hs1150, exhibited similar enhancer activity, indicating functional conservation of hs1150 enhancer across species. Both of the highly conserved subfragments in mouse hs1150 enhancer directed reporter expression in the early neuroretina, indicating that the hs1150 enhancer has two functional components. CONCLUSIONS: Our findings provide insight into the molecular mechanisms underlying the regulation of Otx2 retinal expression.

Spare some internal limiting membrane for later: free ILM patch and neurosensory retina graft.

PURPOSE: Editorial to De Giacinto et al case report on free autologous neurosensory retina patch. METHODS: Literature review and experts' opinion RESULTS: In the present issue, De Giacinto et al describe a free autologous neurosensory retina patch to close a chronic macular hole. This new technique was made necessary by an extended internal limiting membrane peeling during the first surgery, that prevented grafting a patch of internal limiting membrane when the hole did not close. We hereby review pros and cons of patching a chronic macular hole with an internal limiting membrane patch, as well as the importance of not over-enlarging a peeling. DISCUSSION: Internal limiting membrane patch can be considered in chronic macular holes. It may not be an option in cases of over-enlargement of a previous peel; free autologous neurosensory retina patch may be a valid alternative in such cases.

Growth hormone protects against kainate excitotoxicity and induces BDNF and NT3 expression in chicken neuroretinal cells.

There is increasing evidence to suggest a beneficial neuroprotective effect of growth hormone (GH) in the nervous system. While our previous studies have largely focused on retinal ganglion cells (RGCs), we have also found conclusive evidence of a pro-survival effect of GH in cells of the inner nuclear layer (INL) as well as a protective effect on the dendritic trees of the inner plexiform layer (IPL) in the retina. The administration of GH in primary neuroretinal cell cultures protected and induced neural outgrowths. Our results, both in vitro (embryo) and in vivo (postnatal), showed neuroprotective actions of GH against kainic acid (KA)-induced excitotoxicity in the chicken neuroretina. Intravitreal injections of GH restored brain derived neurotrophic factor (BDNF) expression in retinas treated with KA. In addition, we demonstrated that GH over-expression and exogenous administration increased BDNF and neurotrophin-3 (NT3) gene expression in embryonic neuroretinal cells. Thus, GH neuroprotective actions in neural tissues may be mediated by a complex cascade of neurotrophins and growth factors which have been classically related to damage prevention and neuroretinal tissue repair.

Track-weighted imaging for neuroretina: Evaluations in healthy volunteers and ischemic optic neuropathy.

BACKGROUND: The use of MRI-tractography to explore the human neuroretina is yet to be reported. Track-weighted imaging (TWI) was recently introduced as a qualitative tractography-based method with high anatomical contrast. PURPOSE: To explore the human retina in healthy volunteers and patients with anterior ischemic optic neuropathy (AION) using TWI reconstructions. STUDY TYPE: Prospective. POPULATION: Twenty AION patients compared with 20 healthy volunteers. FIELD STRENGTH/SEQUENCE: 3.0T MRI diffusion-weighted imaging (DWI) with b-value of 1000 s/mm2 and 60 diffusion-weighting noncollinear directions. ASSESSMENT: We performed constrained spherical deconvolution from the diffusion-weighted signal and volumetric tractography method, whereby 10 million streamlines are initiated from seed points randomly distributed throughout the orbital area. We then reconstructed TWI maps with isotropic voxel size of 300 µm. STATISTICAL TESTS: We tested the effect of the number of diffusion-weighting directions, ocular laterality, and ocular dominance on healthy retinal fascicles distribution. We then performed factorial analysis of variance to test the effects of the presence/absence of the fascicles on the visual field defect in patients. RESULTS: In healthy volunteers, we found more temporal fascicle in right eyes (P = 0.001), more superior fascicles in dominant eyes (P = 0.014), and fewer fascicles with tractography maps based on 30 directions than those based on 45 directions (P = 9 × 10-8 ) and 60 directions (P = 6 × 10-7 ). Eight out of 20 AION patients presented with complete absence of neuroretinal fascicle, side of the disease, which was correlated with visual field mean deviation at the 6-month visit [F(1,17) = 6.97, P = 0.016]. Seven patients presented with a temporal fascicle in the injured eye; this fascicle presence was linked to visual field mean deviation at the 6-month visit [F(1,17) = 8.43, P = 0.009]. DATA CONCLUSION: In AION patients, the presence of the temporal neuroretinal fascicle in the affected eye provides an objective outcome radiological sign correlated with visual performance. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018.

Growth hormone promotes synaptogenesis and protects neuroretinal dendrites against kainic acid (KA) induced damage.

There is increasing evidence that suggests a possible role for GH in retinal development and synaptogenesis. While our previous studies have focused largely on embryonic retinal ganglion cells (RGCs), our current study demonstrates that GH has a synaptogenic effect in retinal primary cell cultures, increasing the abundance of both pre- (SNAP25) and post- (PSD95) synaptic proteins. In the neonatal chick, kainate (KA) treatment was found to damage retinal synapses and abrogate GH expression. In response to damage, an increase in Cy3-GH internalization into RGCs was observed when administered shortly before or after damage. This increase in internalization also correlated with increase in PSD95 expression, suggesting a neuroprotective effect on the dendritic trees of RGCs and the inner plexiform layer (IPL). In addition, we observed the presence of PSD95 positive Müller glia, which may suggest GH is having a neuroregenerative effect in the kainate-damaged retina. This work puts forth further evidence that GH acts as a synaptogenic modulator in the chick retina and opens a new possibility for the use of GH in retinal regeneration research.

Is ellipsoid zone integrity essential for visual recovery in myopic neovascularization after anti-VEGF therapy?

PURPOSE: To evaluate functional prognostic factors and neuroretinal changes after anti-vascular endothelial growth factor (VEGF) treatment in patients with naïve, recent myopic neovascularization (mCNV), as assessed by spectral-domain optical coherence tomography (SD-OCT). METHODS: Specific changes in tomographic features between baseline and final follow-up were retrospectively evaluated by two examiners independently. Imaging was obtained by a multi-modal imaging system which combines fluorescein angiography and SD-OCT. RESULTS: Twenty-two eyes (male, six; female, 16; mean age, 65 ± 14 years) were considered. Mean follow-up was 21.5 ± 14 months. Best-corrected visual acuity (BCVA) improved from 0.38 ± 0.26 to 0.16 ± 0.20 logMAR (p < 0.001). The ellipsoid zone and the external limiting membrane (ELM) were disrupted in 21 (95.5%) and 15 (68.2%) eyes at baseline, and in 16 (72.7%) and nine (40.9%) eyes after therapy respectively. The ellipsoid zone and ELM were typically intact at lesion margins in 13 (59.1%) and 19 eyes (86.5%) respectively at baseline. The inner retina was intact in 20 eyes (91%). Six eyes (27.3%) exhibited complete regression without fibrosis. Absence of hemorrhage and integrity of lesion-adjacent ELM and of lesion-adjacent ellipsoid zone at baseline were factors for better final BCVA (p ≤ 0.05) CONCLUSION: Vision gain might occur despite ellipsoid zone or ELM restoration. Hemorrhage could be considered a negative prognostic factor, integrity of lesion-adjacent ELM and of lesion-adjacent ellipsoid zone as positive prognostic factors. Myopic CNV can also resolve completely without fibrosis.

The Alzheimer's-related amyloid beta peptide is internalised by R28 neuroretinal cells and disrupts the microtubule associated protein 2 (MAP-2).

Age-related Macular Degeneration (AMD) is a common, irreversible blinding condition that leads to the loss of central vision. AMD has a complex aetiology with both genetic as well as environmental risks factors, and share many similarities with Alzheimer's disease. Recent findings have contributed significantly to unravelling its genetic architecture that is yet to be matched by molecular insights. Studies are made more challenging by observations that aged and AMD retinas accumulate the highly pathogenic Alzheimer's-related Amyloid beta (Aß) group of peptides, for which there appears to be no clear genetic basis. Analyses of human donor and animal eyes have identified retinal Aß aggregates in retinal ganglion cells (RGC), the inner nuclear layer, photoreceptors as well as the retinal pigment epithelium. Aß is also a major drusen constituent; found correlated with elevated drusen-load and age, with a propensity to aggregate in retinas of advanced AMD. Despite this evidence, how such a potent driver of neurodegeneration might impair the neuroretina remains incompletely understood, and studies into this important aspect of retinopathy remains limited. In order to address this we exploited R28 rat retinal cells which due to its heterogeneous nature, offers diverse neuroretinal cell-types in which to study the molecular pathology of Aß. R28 cells are also unaffected by problems associated with the commonly used RGC-5 immortalised cell-line, thus providing a well-established model in which to study dynamic Aß effects at single-cell resolution. Our findings show that R28 cells express key neuronal markers calbindin, protein kinase C and the microtubule associated protein-2 (MAP-2) by confocal immunofluorescence which has not been shown before, but also calretinin which has not been reported previously. For the first time, we reveal that retinal neurons rapidly internalised Aß1-42, the most cytotoxic and aggregate-prone amongst the Aß family. Furthermore, exposure to physiological amounts of Aß1-42 for 24 h correlated with impairment to neuronal MAP-2, a cytoskeletal protein which regulates microtubule dynamics in axons and dendrites. Disruption to MAP-2 was transient, and had recovered by 48 h, although internalised Aß persisted as discrete puncta for as long as 72 h. To assess whether Aß could realistically localise to living retinas to mediate such effects, we subretinally injected nanomolar levels of oligomeric Aß1-42 into wildtype mice. Confocal microscopy revealed the presence of focal Aß deposits in RGC, the inner nuclear and the outer plexiform layers 8 days later, recapitulating naturally-occurring patterns of Aß aggregation in aged retinas. Our novel findings describe how retinal neurons internalise Aß to transiently impair MAP-2 in a hitherto unreported manner. MAP-2 dysfunction is reported in AMD retinas, and is thought to be involved in remodelling and plasticity of post-mitotic neurons. Our insights suggest a molecular pathway by which this could occur in the senescent eye leading to complex diseases such as AMD.

Growth hormone reverses excitotoxic damage induced by kainic acid in the green iguana neuroretina.

It is known that growth hormone (GH) is expressed in extrapituitary tissues, including the nervous system and ocular tissues, where it is involved in autocrine/paracrine actions related to cell survival and anti-apoptosis in several vertebrates. Little is known, however, in reptiles, so we analyzed the expression and distribution of GH in the eye of green iguana and its potential neuroprotective role in retinas that were damaged by the intraocular administration of kainic acid (KA). It was found, by Western blotting, that GH-immunoreactivity (GH-IR) was expressed as two isoforms (15 and 26kDa, under reducing conditions) in cornea, vitreous, retina, crystalline, iris and sclera, in varying proportions. Also, two bands for the growth hormone receptor (GHR)-IR were observed (70 and 44kDa, respectively) in the same tissues. By immunofluorescence, GH-IR was found in neurons present in several layers of the neuroretina (inner nuclear [INL], outer nuclear [ONL] and ganglion cell [GCL] layers) as determined by its co-existence with NeuN, but not in glial cells. In addition, GH and GHR co-expression was found in the same cells, suggesting paracrine/autocrine interactions. KA administration induced retinal excitotoxic damage, as determined by a significant reduction of the cell density and an increase in the appearance of apoptotic cells in the INL and GCL. In response to KA injury, both endogenous GH and Insulin-like Growth Factor I (IGF-I) expression were increased by 70±1.8% and 33.3±16%, respectively. The addition of exogenous GH significantly prevented the retinal damage produced by the loss of cytoarchitecture and cell density in the GCL (from 4.9±0.79 in the control, to 1.45±0.2 with KA, to 6.35±0.49cell/mm(2) with KA+GH) and in the INL (19.12±1.6, 10.05±1.9, 21.0±0.8cell/mm(2), respectively) generated by the long-term effect of 1mM KA intraocular administration. The co-incubation with a specific anti-GH antibody, however, blocked the protective effect of GH in GCL (1.4±0.23cell/mm(2)) and INL (11.35±1.06), respectively. Furthermore, added GH induced an increase of 90±14% in the retinal IGF-I concentration and the anti-GH antibody also blocked this effect. These results indicate that GH and GHR are expressed in the iguana eye and may be able to exert, either directly of mediated by IGF-I, a protective mechanism in neuroretinas that suffered damage by the administration of kainic acid.

Internalization and synaptogenic effect of GH in retinal ganglion cells (RGCs).

In the chicken embryo, GH gene expression occurs in the neural retina and retinal GH promotes cell survival and induces axonal growth of retinal ganglion cells. Neuroretinal GH is therefore of functional importance before the appearance of somatotrophs and the onset of pituitary GH secretion to the peripheral plasma (at ED15-17). Endocrine actions of pituitary GH in the development and function of the chicken embryo eye are, however, unknown. This possibility has therefore been investigated in ED15 embryos and using the quail neuroretinal derived cell line (QNR/D). During this research, we studied for the first time, the coexistence of exogenous (endocrine) and local GH (autocrine/paracrine) in retinal ganglion cells (RGCs). In ovo systemic injections of Cy3-labeled GH demonstrated that GH in the embryo bloodstream was translocated into the neural retina and internalized into RGC's. Pituitary GH may therefore be functionally involved in retinal development during late embryogenesis. Cy3-labelled GH was similarly internalized into QNR/D cells after its addition into incubation media. The uptake of exogenous GH was by a receptor-mediated mechanism and maximal after 30-60min. The exogenous (endocrine) GH induced STAT5 phosphorylation and increased growth associated protein 43 (GAP43) and SNAP-25 immunoreactivity. Ex ovo intravitreal injections of Cy3-GH in ED12 embryos resulted in GH internalization and STAT5 activation. Interestingly, the CY3-labeled GH accumulated in perinuclear regions of the QNR/D cells, but was not found in the cytoplasm of neurite outgrowths, in which endogenous retinal GH is located. This suggests that exogenous (endocrine) and local (autocrine/paracrine) GH are both involved in retinal function in late embryogenesis but they co-exist in separate intracellular compartments within retinal ganglion cells.

Retinal debris triggers cytotoxic damage in cocultivated primary porcine RPE cells.

Introduction: One of the most common causes of vision loss in the elderly population worldwide is age-related macular degeneration (AMD). Subsequently, the number of people affected by AMD is estimated to reach approximately 288 million by the year 2040. The aim of this study was to develop an ex vivo model that simulates various aspects of the complex AMD pathogenesis. Methods: For this purpose, primary porcine retinal pigment epithelial cells (ppRPE) were isolated and cultured. One group was exposed to medium containing sodium iodate (NaIO3) to induce degeneration. The others were exposed to different supplemented media, such as bovine serum albumin (BSA), homogenized porcine retinas (HPR), or rod outer segments (ROOS) for eight days to promote retinal deposits. Then, these ppRPE cells were cocultured with porcine neuroretina explants for another eight days. To assess the viability of ppRPE cells, live/dead assay was performed at the end of the study. The positive RPE65 and ZO1 area was evaluated by immunocytochemistry and the expression of RLBP1, RPE65, and TJP1 was analyzed by RT-qPCR. Additionally, drusen (APOE), inflammation (ITGAM, IL6, IL8, NLRP3, TNF), oxidative stress (NFE2L2, SOD1, SOD2), and hypoxia (HIF1A) markers were investigated. The concentration of the inflammatory cytokines IL-6 and IL-8 was determined in medium supernatants from day 16 and 24 via ELISA. Results: Live/dead assay suggests that especially exposure to NaIO3 and HPR induced damage to ppRPE cells, leading in a significant ppRPE cell loss. All supplemented media resulted in decreased RPE-characteristic markers (RPE65; ZO-1) and gene expression like RLBP1 and RPE65 in the cultured ppRPE cells. Besides, some inflammatory, oxidative as well as hypoxic stress markers were altered in ppRPE cells cultivated with NaIO3. The application of HPR induced an enhanced APOE expression. Pre-exposure of the ppRPE cells led to a diminished number of cones in all supplemented media groups compared to controls. Discussion: Overall, this novel coculture model represents an interesting initial approach to incorporating deposits into coculture to mimic AMD pathogenesis. Nevertheless, the effects of the media used need to be investigated in further studies.

Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer's disease.

Introduction: Ergothioneine (Ergo) is a naturally occurring dietary antioxidant. Ergo uptake is dependent on the transporter, organic cation transporter novel-type 1 (OCTN1) distribution. OCTN1 is highly expressed in blood cells (myeloid lineage cells), brain and ocular tissues that are likely predisposed to oxidative stress. Ergo may protect the brain and eye against oxidative damage and inflammation, however, the underlying mechanism remains unclear. Amyloid beta (Aß) clearance is a complex process mediated by various systems and cell types including vascular transport across the blood-brain barrier, glymphatic drainage, and engulfment and degradation by resident microglia and infiltrating innate immune cells. Impaired Aß clearance is a major cause for Alzheimer's disease (AD). Here we investigated neuroretinas to explore the neuroprotective effect of Ergo in a transgenic AD mouse model. Methods: Age-matched groups of Ergo-treated 5XFAD, non-treated 5XFAD, and C57BL/6J wildtype (WT controls) were used to assess Ergo transporter OCTN1 expression and Aß load along with microglia/macrophage (IBA1) and astrocyte (GFAP) markers in wholemount neuroretinas (n = 26) and eye cross-sections (n = 18). Immunoreactivity was quantified by fluorescence or by semi-quantitative assessments. Results and discussion: OCTN1 immunoreactivity was significantly low in the eye cross-sections of Ergo-treated and non-treated 5XFAD vs. WT controls. Strong Aß labeling, detected in the superficial layers in the wholemounts of Ergo-treated 5XFAD vs. non-treated 5XFAD reflects the existence of an effective Aß clearance system. This was supported by imaging of cross-sections where Aß immunoreactivity was significantly low in the neuroretina of Ergo-treated 5XFAD vs. non-treated 5XFAD. Moreover, semi-quantitative analysis in wholemounts identified a significantly reduced number of large Aß deposits or plaques, and a significantly increased number of IBA1(+)ve blood-derived phagocytic macrophages in Ergo-treated 5XFAD vs. non-treated 5XFAD. In sum, enhanced Aß clearance in Ergo-treated 5XFAD suggests that Ergo uptake may promote Aß clearance possibly by blood-derived phagocytic macrophages and via perivascular drainage.

When Sex Matters: Differences in the Central Nervous System as Imaged by OCT through the Retina.

BACKGROUND: Retinal texture has gained momentum as a source of biomarkers of neurodegeneration, as it is sensitive to subtle differences in the central nervous system from texture analysis of the neuroretina. Sex differences in the retina structure, as detected by layer thickness measurements from optical coherence tomography (OCT) data, have been discussed in the literature. However, the effect of sex on retinal interocular differences in healthy adults has been overlooked and remains largely unreported. METHODS: We computed mean value fundus images for the neuroretina layers as imaged by OCT of healthy individuals. Texture metrics were obtained from these images to assess whether women and men have the same retina texture characteristics in both eyes. Texture features were tested for group mean differences between the right and left eye. RESULTS: Corrected texture differences exist only in the female group. CONCLUSIONS: This work illustrates that the differences between the right and left eyes manifest differently in females and males. This further supports the need for tight control and minute analysis in studies where interocular asymmetry may be used as a disease biomarker, and the potential of texture analysis applied to OCT imaging to spot differences in the retina.

Factors influencing mesenchymal stromal cells in in vitro cellular models to study retinal pigment epithelial cell rescue.

Mesenchymal stromal cells (MSC) stop or slow retinal pigment epithelium (RPE) and neuroretina (NR) degeneration by paracrine activity in oxidative stress-induced retinal degenerative diseases. However, it is mandatory to develop adequate in vitro models that allow testing new treatment strategies against oxidative stress before performing in vivo studies. The viable double- and triple-layered setups are composed of separate layers of NR, MSC, and RPE (NR-MSC-RPE, NR-RPE, MSC-RPE) partially mimic in vivo retinal conditions. In this study, the paracrine neuroprotective effect of each setup's microenvironment on hydrogen peroxide (H2O2)-stressed was compared with unstressed RPE cells. RPE cell proliferation viability was assessed on day 1, 3, and 6 using Alamar Blue® (10%), MTT (10%) and a cell viability/cytotoxicity assay kit followed by data analysis. The results showed that RPE cells, highly viable (> 90%) in mixed medium of DMEM and neurobasal A (1:1), lost 50% viability on exposure to 400 µM of H2O2 (P < 0.05). The unexposed groups differed significantly from exposed groups for RPE cell growth (RPE and [Formula: see text]RPE (P < 0.0001), NR-MSC-RPE, and NR-MSC-[Formula: see text]RPE (P < 0.05), NR-RPE and NR-[Formula: see text]RPE (P < 0.01), and MSC-RPE and MSC-[Formula: see text]RPE (P < 0.01). NR-[Formula: see text]RPE and NR-RPE supported RPE cell proliferation viability better than other setups (P < 0.01) and RPE cells proliferated 0.49-fold more in NR-MSC-[Formula: see text]RPE than NR-MSC-RPE. Thus, NR and MSC presence improved significantly each setup's microenvironment for cell rescue, nevertheless, each setup also showed limitations for its use as an in vitro study tool. Health of microenvironment of such setups depends on many factors including cell-secreted trophic factors.

Binary colloidal crystals (BCCs) modulate the retina-related gene expression of hBMSCs - A preliminary study.

Surface topography-induced lineage commitment of human bone marrow stem cells (hBMSCs) has been reported. However, this effect on hBMSC differentiation toward retinal pigment epithelium (RPE)-like cells has not been explored. Herein, a family of cell culture substrates called binary colloidal crystals (BCCs) was used to stimulate hBMSCs into RPE-like cells without induction factors. Two BCCs, named SiPS (silica (Si)/polystyrene (PS)) and SiPSC (Si/carboxylated PS), having similar surface topographies but different surface chemistry was used for cell culture. The result showed that cell proliferation was no difference between the two BCCs and tissue culture polystyrene (TCPS) control. However, the cell attachment, spreading area, and aspect ratio between surfaces were significantly changed. For example, cells displayed more elongated on SiPS (aspect ratio ~7.0) than those on SiPSC and TCPS (~2.0). The size of focal adhesions on SiPSC (~1.6 µm2) was smaller than that on the TCPS (~2.5 µm2). qPCR results showed that hBMSCs expressed higher RPE progenitor genes (i.e., MITF and PAX6) on day 15, and mature RPE genes (i.e., CRALBP and RPE65) on day 30 on SiPS than TCPS. On the other hand, the expression of optical vesicle or neuroretina genes (i.e., MITF and VSX2) was upregulated on day 15 on SiPSC compared to the TCPS. This study reveals that hBMSCs could be modulated into different cell subtypes depending on the BCC combinations. This study shows the potential of BCCs in controlling stem cell differentiation.

Alterations of retinal thickness measured by optical coherence tomography correlate with neurophysiological measures in diabetic polyneuropathy.

AIMS/INTRODUCTION: Diabetic polyneuropathy (DPN) and diabetic retinopathy (DR) are traditionally regarded as microvascular complications. However, these complications may share similar neurodegenerative pathologies. Here we evaluate the correlations in the severity of DPN and changes in the thickness of neuroretinal layers to elucidate whether these complications exist at similar stages of progression. MATERIALS AND METHODS: A total of 43 patients with type 2 diabetes underwent a nerve conduction study (NCS), a macular optical coherence tomography, and a carotid artery ultrasound scan. Diabetic polyneuropathy was classified according to Baba's classification using NCS. The retina was automatically segmented into four layers; ganglion cell complex (GCC), inner nuclear layer/outer plexiform layer (INL/OPL), outer nuclear layer/photoreceptor inner and outer segments, and retinal pigment epithelium (RPE). The thickness of each retinal layer was separately analyzed for the fovea and the parafovea. RESULTS: Fourteen patients were classified as having moderate to severe diabetic polyneuropathy. The thicknesses of the foveal and parafoveal INL/OPL increased in patients with diabetic polyneuropathy compared with patients without. The thickness of the parafoveal retinal pigment epithelium decreased in patients with diabetic polyneuropathy. The thinning of parafoveal ganglion cell complex and foveal and parafoveal retinal pigment epithelium were positively correlated with deterioration of nerve functions in the nerve conduction study, but the thickening of INL/OPL was positively correlated with the nerve function deterioration. The thinning of parafoveal ganglion cell complex and foveal retinal pigment epithelium were positively correlated with the thickening of the carotid intima-media. CONCLUSIONS: Depending on the progression of diabetic polyneuropathy, the ganglion cell complex and retinal pigment epithelium became thinner and the INL/OPL became thicker. These retinal changes might be noteworthy for pathological investigations and for the assessment of diabetic polyneuropathy and diabetic retinopathy.

Ex Vivo Model of Spontaneous Neuroretinal Degeneration for Evaluating Stem Cells' Paracrine Properties.

Ex vivo neuroretina cultures closely resemble in vivo conditions, retaining the complex neuroretina cells dynamics, connections, and functionality, under controlled conditions. Therefore, these models have allowed advancing in the knowledge of retinal physiology and pathobiology over the years. Furthermore, the ex vivo neuroretina models represent an adequate tool for evaluating stem cell therapies over neuroretinal degeneration processes.Here, we describe a physically separated co-culture of neuroretina explants with stem cells to evaluate the effect of stem cells paracrine properties on spontaneous neuroretinal degeneration.

Influence of Chronic Ocular Hypertension on Emmetropia: Refractive, Structural and Functional Study in Two Rat Models.

Chronic ocular hypertension (OHT) influences on refraction in youth and causes glaucoma in adulthood. However, the origin of the responsible mechanism is unclear. This study analyzes the effect of mild-moderate chronic OHT on refraction and neuroretina (structure and function) in young-adult Long-Evans rats using optical coherence tomography and electroretinography over 24 weeks. Data from 260 eyes were retrospectively analyzed in two cohorts: an ocular normotension (ONT) cohort (<20 mmHg) and an OHT cohort (>20 mmHg), in which OHT was induced either by sclerosing the episcleral veins (ES group) or by injecting microspheres into the anterior chamber. A trend toward emmetropia was found in both cohorts over time, though it was more pronounced in the OHT cohort (p < 0.001), especially in the ES group (p = 0.001) and males. IOP and refraction were negatively correlated at week 24 (p = 0.010). The OHT cohort showed early thickening in outer retinal sectors (p < 0.050) and the retinal nerve fiber layer, which later thinned. Electroretinography demonstrated early supranormal amplitudes and faster latencies that later declined. Chronic OHT accelerates emmetropia in Long-Evans rat eyes towards slowly progressive myopia, with an initial increase in structure and function that reversed over time.

Human Mesenchymal Stem Cell Secretome Exhibits a Neuroprotective Effect over In Vitro Retinal Photoreceptor Degeneration.

Retinal photoreceptor degeneration occurs frequently in several neurodegenerative retinal diseases such as age-related macular degeneration, retinitis pigmentosa, or genetic retinal diseases related to the photoreceptors. Despite the impact on daily life and the social and economic consequences, there is no cure for these diseases. Considering this, cell-based therapy may be an optimal therapeutic option. This study evaluated the neuroprotective in vitro potential of a secretome of human bone marrow mesenchymal stem cells (MSCs) for retinal photoreceptors in vitro. We analyzed the photoreceptor morphologic changes and the paracrine factors secreted by human bone marrow MSCs in a physically separated co-culture with degenerated neuroretinas, using organotypic neuroretinal cultures. The results showed that the secretome of human bone marrow MSCs had a neuroprotective effect over the neuroretinal general organization and neuropreserved the photoreceptors from degeneration probably by secretion of neuroprotective proteins. The study of the expression of 1,000 proteins showed increased paracrine factors secreted by MSCs that could be crucial in the neuroprotective effect of the stem cell secretome over in vitro retinal degeneration. The current results reinforce the hypothesis that the paracrine effect of the human bone marrow MSCs may slow photoreceptor neurodegeneration and be a therapeutic option in retinal photoreceptor degenerative diseases.