Cells Of Cornea Research Articles

Factors Affecting the Density of Corneal Endothelial Cells Cultured from Donor Corneas

We investigated which specific correlation exists between the endothelial cell density (ECD) of corneal endothelial cell (CEC) cultures and the features of the donor corneas from which they originate. CEC cultures were prepared from one donor cornea or by pooling together cells of more corneas from elderly donors with ECDs lower or higher than 2000 cells/mm2. The ECDs of such primary cultures were evaluated and showed that that ECDs > 2000 cells/mm2 can be obtained only when CECs are isolated from (1) corneas of young donors; (2) at least two elderly donor corneas (if ECD > 2000 cells/mm2), or three elderly donor corneas (if ECD < 2000 cells/mm2). Secondary cultures are all characterized by ECDs < 2000 cells/mm2. Our study highlights the difficulties in obtaining cultures with ECDs > 2000 cells/mm2. Even if achievable with corneas from young donors, this becomes a challenging task when corneas from elderly donors are used (i.e., the overall majority of those collected by eye banks) and particularly when corneas from elderly donors with ECD < 2000 cells/mm2 are used. Pooling more of two corneas to obtain suitable CECs could technically overcome the problem. The above issues should be tackled appropriately before moving into clinical studies.

Mouse Corneal Immune Cell Heterogeneity Revealed by Single-Cell RNA Sequencing.

This study aimed to define the heterogeneity, spatial localization, and functional roles of immune cells in the mouse cornea using single-cell RNA sequencing (scRNA-seq) and immunofluorescent staining. Enriched mouse corneal immune cells (C57BL/6 strain, age 16-20 weeks) underwent single-cell RNA sequencing library preparation, sequencing, and analysis with Seurat, Monocle 3, and CellChat packages in R. Pathway analysis used Qiagen Ingenuity Pathway Analysis software. Immunostaining confirmed cell distribution. We identified 14 distinct immune cell clusters (56% myeloid and 44% lymphoid). Myeloid populations included resident macrophages, conventional dendritic cells (cDC2s), Langerhans cells, neutrophils, monocytes, and mast cells. Additionally, lymphocyte subsets (B, CD8, CD4, γδT, natural killer, natural killer T, and group 2 innate lymphoid cells) were found. We also found three new subtypes of resident macrophages in the cornea. Trajectory analysis suggested a differentiation pathway from monocytes to conventional dendritic cells, resident macrophages, and LCs. Intercellular communication network analysis using cord diagram identified amyloid precursor protein, chemokine (C-C motif) ligands (2, 3, 4, 6, 7, 9, and 12), Cxcl2, Mif, Tnf, Tgfb1, Igf1, and Il10 as prominent ligands involved in these interactions. Sexually dimorphic gene expression patterns were observed, with male myeloid cells expressing genes linked to immune regulation (Egr1, Foxp1, Mrc1, and Il1rn) and females showing higher expression of antigen presentation genes (Clic1, Psmb8, and Psmb9). Finally, immunostaining confirmed the spatial distribution of these cell populations within the cornea. This study unveils a diverse immune landscape in the mouse cornea, with evidence for cell differentiation and sex-based differences. Immunostaining validates the spatial distribution of these populations, furthering our knowledge of corneal immune function.

Suppressive Role of Pigment Epithelium-derived Factor in a Rat Model of Corneal Allograft Rejection.

Immunological rejection is the most common reason for corneal transplantation failure. The importance of T cells in corneal allograft rejection is well demonstrated. Recent studies highlight that pigment epithelium-derived factor (PEDF) plays an immunoregulatory role in ocular diseases by enhancing the suppressive phenotype of regulatory T cells besides its other functions in neurotrophy and antiangiogenesis. The effects of PEDF on immune rejection were examined in rat models of corneal transplantation using slit-lamp microscope observation, immunohistochemistry, flow cytometry, and Western blot. In vitro, we demonstrated PEDF reduced alloreactive T-cell activation using real-time polymerase chain reaction, flow cytometry, and Western blot. Topical administration of PEDF provided corneal transplantation rats with an improved graft survival rate of corneal allografts, reduced hemangiogenesis, and infiltration of immune cells in corneas, in particular, type 17 T helper cells while increased regulatory T cells. Moreover, nerve reinnervation within grafts was promoted in PEDF-treated recipient rats. In vitro, PEDF inhibited alloreactive T-cell activation via the c-Jun N-terminal kinase/c-Jun signaling pathway and upregulated the expressions of interleukin-10 and transforming growth factor-β, emphasizing the suppressive role of PEDF on immune responses. Our results underscore the feasibility of PEDF in alleviating corneal allograft rejection and further illustrate its potential in managing immune-related diseases.

A novel GFP-based strategy to quantitate cellular spatial associations in HSV-1 viral pathogenesis.

Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with in vitro and in vivo assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications.IMPORTANCEThe goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.

Cell Volume Regulation of Endothelial Cells Is Impaired in Keratoconus Cornea

In this work the permeability to water and urea of plasma membranes of endothelial cells of normal corneas and corneas with keratoconus was investigated. The endothelial cells were obtained from surgery material. Measurements of osmotic aqueous permeability (Pf) of endothelial cells in normal and in keratoconus did not reveal significant differences of this parameter in the two studied groups. The control cells and the cells from keratoconus cornea have similar osmotic water permeability (control cells, Pf = 0.53 ± 0.045 cm/s; keratoconus cells, Pf = 0.63 ± 0.041 cm/s; n = 25; p ≥ 0.05). Neither coefficient of urea permeability differed significantly in these groups (control, Pu = 0.049 ± 0.003 cm/s; keratoconus, Pu = 0.056 ± 0.003 cm/s; n = 25; p ≥ 0.05). Analysis of cell volume dynamics based on exponential approximation showed a more pronounced decrease of the cell volume of endothelial cells from keratoconus cornea in hypertonic medium in comparison with the cells from normal cornea. The increase of cell volume caused by isotonic entering of urea into the cells in hypertonic medium also was more pronounced in these cells in comparison with the normal ones. We conclude that there are significant changes in cell volume regulating mechanism in keratoconus cornea endothelial cells.

Corneal endothelial density loss in patients after gonioscopy‑assisted transluminal trabeculotomy.

To compare short-term changes in corneal endothelial cells after gonioscopy-assisted transluminal trabeculotomy(GATT). This retrospective comparative study included 138 patients(138 eyes), and 98 of these patients underwent GATT procedure and 40 underwent SLT procedure as a control group. Changes in the corneal endothelium in patients who underwent GATT and SLT were analyzed retrospectively. Endothelial changes in the central cornea were examined using specular microscopy before and 6 months after the GATT and SLT procedure. Intraocular pressure(IOP), number of glaucoma medications, and side effects were evaluated at visits before and after two methods. One hundred and thirty-eight eyes of 138 patients with a mean age of 62.9±12.7 years in the SLT group and 62.5±11.8 years in the GATT group were included in this study. Pre-procedure mean ± SD IOP was 27.7±3.6 mmHg and 27.4±5.3 mmHg (p=0.173) 2.8±0.5 and 2.9±0.8 (p=0.204) glaucoma drugs are in the SLT and GATT group, respectively. The mean corneal endothelial cell density (CECD) in the SLT group was 2433.1±581.4 cells/mm2 before the procedure and 2435.1±585 cells/mm2 6 months after the procedure, a change of 0.1±0.6% which was not statistically significant (p>0.967).The mean CECD at baseline in the GATT group was 2443.4±508.2 cells/mm2 and decreased to 2290.2±527.7 cells/mm2 6 months after this procedure, representing a cell loss of 6,2±9,1% (p<0.001). GATT caused more CECD damage than SLT at the sixth month after the procedure. Considering the loss of CECD in candidates for GATT, sufficient number of endothelial cells in the central cornea is recommended.

Whole mount immunofluorescence analysis of fresh and stored human donor corneas highlights changes in limbal characteristics during storage

PurposeHuman donor corneas are an essential control tissue for corneal research. We utilized whole mount immunofluorescence (WM-IF) to evaluate how the storage affects the tissue integrity and putative limbal stem cells in human and porcine corneas. Moreover, we compare this information with the marker expression patterns observed in human pluripotent stem cell (hPSC)-derived LSCs. MethodsThe expression of putative LSC markers was analyzed with WM-IF and the fluorescence intensity was quantified in human donor corneas stored for 1–30 days, and in porcine corneas processed 0–6 h after euthanasia. The results were compared with the staining of human and porcine corneal cryosections and with both primary and hPSC-derived LSC cultures. ResultsWM-IF analyses emerged as a more effective method when compared to tissue sections for visualizing the expression of LSC markers within human and porcine corneas. Storage duration was a significant factor influencing the expression of LSC markers, as human tissues stored longer exhibited notable epithelial degeneration and lack of LSC markers. Porcine corneas replicated the expression patterns observed in fresh human tissue. We validated the diverse expression patterns of PAX6 in the limbal-corneal region, which aligned with findings from hPSC-LSC differentiation experiments. ConclusionsWM-IF coupled with quantification of fluorescence intensities proved to be a valuable tool for investigating LSC marker expression in both human and porcine tissues ex vivo. Prolonged storage significantly influences the expression of LSC markers, underscoring the importance of fresh human or substitute control tissue when studying limbal stem cell biology.

Evidence for persistent UV-induced DNA damage and altered DNA damage response in xeroderma pigmentosa patient corneas

Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in the cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts that are repaired by the NER (Nucleotide Excision Repair) pathway. Mutations in the genes coding for NER proteins, as reported in XP patients, would lead to sub-optimal damage repair resulting in clinical signs varying from photo-keratitis to cancerous lesions on the ocular surface. Here, we aimed to provide evidence for the accumulation of DNA damage and activation of DNA repair pathway proteins in the corneal cells of patients with XP. Corneal buttons of patients who underwent penetrating keratoplasty were stained to quantify DNA damage and the presence of activated DNA damage response proteins (DDR) using specific antibodies. Positive staining for pH2A.X and thymidine dimers confirmed the presence of DNA damage in the corneal cells. Positive cells were found in both control corneas and XP samples however, unlike normal tissues, positive cells were found in all cell layers of XP samples indicating that these cells were sensitive to very low levels of UV. pH2A.X-positive cells were significantly more in XP corneas (p < 0.05) indicating the presence of double strand breaks in these tissues. A positive expression of phosphorylated-forms of DDR proteins was noted in XP corneas (unlike controls) such as ataxia telangiectasia mutated/Rad-3 related proteins (ATM/ATR), breast cancer-1 and checkpoint kinases-1 and -2. Nuclear localization of XPA was noted in XP samples which co-localized (calculated using Pearson's correlation) with pATM (0.9 ± 0.007) and pATR (0.6 ± 0.053). The increased presence of these in the nucleus confirms that unresolved DNA damage was accumulating in these cells thereby leading to prolonged activation of the damage response proteins. An increase in pp53 and TUNEL positive cells in the XP corneas indicated cell death likely driven by the p53 pathway. For comparison, cultured normal corneal epithelial cells were exposed to UV-radiation and stained for DDR proteins at 3, 6 and 24 h after irradiation to quantify the time taken by cells with intact DDR pathway to repair damage. These cells, when exposed to UV showed nuclear translocation of DDR proteins at 3 and 6 h which reduced significantly by 24 h confirming that the damaged DNA was being actively repaired leading to cell survival. The persistent presence of the DDR proteins in XP corneas indicates that damage is being actively recognized and DNA replication is stalled, thereby causing accumulation of damaged DNA leading to cell death, which would explain the cancer incidence and cell loss reported in these patients.

Hydrogels based on recombinant spidroin stimulate proliferation and migration of human cornea cells

This article presents the results of studying the impact of recombinant spidroin hydrogel on posterior epithelial cells and human corneal keratocytes in an in vitro experiment. The World Health Organization in its studies has established a high prevalence of corneal injuries among the population of developing countries. In recent years, various technologies have been proposed to restore the damaged surface of the cornea. The use of biodegradable silk-based materials, such as spidroins is one of the main parts of scientific research of corneal regeneration. Spidroinsare well known for their optimal balance of strength and elasticity, which, given their biological compatibility, non-immunogenicity and biodegradability, allows them to be used as a biomaterial for tissue engineering and regenerative medicine. In this reason a detailed assessment of the cytotoxicity of hydrogels based on recombinant rS2/12-RGDS spidroinon the epithelial cells and keratocytes was performed here, taking into attention possible changes of the phenotype and migratory activity of these cells. This study demonstrates the promise and therapeutic potential of hydrogels based on recombinant spidroin.

Solanum nigrum Toxicity and Its Neuroprotective Effect Against Retinal Ganglion Cell Death Through Modulation of Extracellular Matrix in a Glaucoma Rat Model.

Purpose: Glaucoma is a complex degenerative optic neuropathy characterized by loss of retinal ganglion cells (RGCs) leading to irreversible vision loss and blindness. Solanum nigrum has been used for decades in traditional medicine system. However, no extensive studies were reported on its antiglaucoma properties. Therefore, this study was designed to investigate the neuroprotective effects of S. nigrum extract on RGC against glaucoma rat model. Methods: High performance liquid chromatography and liquid chromatography tandem mass spectrometry was used to analyze the phytochemical profile of aqueous extract of S. nigrum (AESN). In vitro, {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) and H2DCFDA assays were used to determine cell viability and reactive oxygen species (ROS) production in Statens Seruminstitut Rabbit Cornea cells. In vivo, AESN was orally administered to carbomer-induced rats for 4 weeks. Intraocular pressure, antioxidant levels, and electrolytes were determined. Histopathological and immunohistochemical analysis was carried out to evaluate the neurodegeneration of RGC. Results: MTT assay showed AESN exhibited greater cell viability and minimal ROS production at 10 μg/mL. Slit lamp and funduscopy confirmed glaucomatous changes in carbomer-induced rats. Administration of AESN showed minimal peripheral corneal vascularization and restored histopathological alterations such as minimal loss of corneal epithelium and moderate narrowing of the iridocorneal angle. Immunohistochemistry analysis showed increased expression of positive BRN3A cells and decreased matrix metalloproteinase (MMP)-9 activation in retina and cornea, whereas western blot analysis revealed downregulation of extracellular matrix proteins (COL-1 and MMP-9) in AESN-treated rats compared with the diseased group rats. Conclusions: AESN protects RGC loss through remodeling of MMPs and, therefore, can be used for the development of novel neurotherapeutics for the treatment of glaucoma.

A pilot study on Langerhans cells in keratoconus patients by in vivo confocal microscopy before and after corneal cross-linking and correlation with eye rubbing

PurposeThe aim of this study was to investigate the corneal microstructure and Langerhans cells using in vivo confocal microscopy in keratoconus patients before and after cross-linking, and to correlate the morphologic findings with clinical and patient-reported outcomes, including eye rubbing (ER) behavior. MethodsPatients with progressive keratoconus undergoing iontophoresis-assisted epithelium-on cross-linking (I-CXL) were consecutively enrolled. In vivo confocal microscopy was performed before and 6 months after treatment. Patients were asked to quantify their ER behavior on a Visual Analogue Scale (VAS) and completed the Keratoconus Outcomes Research Questionnaire and the Ocular Surface Disease Index questionnaires at the same time points. Visual acuity, tear osmolarity, topography, aberrometry, and pachymetry of both eyes were assessed. ResultsThirteen patients were included in this pilot study. Preoperatively, the mean Langerhans cells density was 35,615 cells per mm2, and the median morphology was 3. The mean ER VAS before treatment was 7,077 out of 10. The ER VAS showed significant positive correlations with both Langerhans cells density and morphology of the study eye. After treatment, a statistically significant reduction in ER VAS and in Langerhans cells variables was observed. The mean sub-basal plexus nerve density was comparable to pre-operative values 6 months after I-CXL. ConclusionsBased on this preliminary evidence, the presence of high density of mature Langerhans cells in the central cornea of keratoconus patients and its correlation with eye rubbing support the role of inflammation in keratoconus. The reduction in these markers after treatment may suggest a potential of CXL in moderating immune-related inflammation and eye rubbing in the medium term.

Organ Donation in Syria.

The first living donor kidney transplant in Syria was performed 44 years ago; by the end of 2022, 6265 renal transplants had been performed in Syria. Kidney, bone marrow, cornea, and stem cells are the only organs or tissues that can be transplanted in Syria. Although 3 heart transplants from deceased donors were performed in the late 1980s, cardiac transplant activities have since discontinued. In 2003, national Syrian legislation was enacted authorizing the use of organs from living unrelated and deceased donors. This important law was preceded by another big stride: the acceptance by the higher Islamic religious authorities in Syria in 2001 of the principle of procurement of organs from deceased donors, provided that consent is given by a first- or second-degree relative. After the law was enacted, kidney transplant rates increased from 7 per million population in 2002 to 17 per million population in 2007. Kidney transplants performed abroad for Syrian patients declined from 25% in 2002 to <2% in 2007. Rates plateaued through 2010, before the political crisis started in 2011. Forty-four years after the first successful kidney transplant in Syria, patients needing an organ transplant rely on living donors only. Moreover, 20 years after the law authorizing use of organs from deceased donors, a program is still not in place in Syria. The war, limited resources, and lack of public awareness about the importance of organ donation and transplant appear to be factors inhibiting initiation of a deceased donor program in Syria. A concerted and ongoing education campaign is needed to increase awareness of organ donation, change negative public attitudes, and gain societal acceptance. Every effort must be made to initiate a deceased donor program to lessen the burden on living donors and to enable national self-sufficiency in organs for transplant.

Gene expression in calcium triggered healthy primary limabal epithelial cells, in vitro

Aims/Purpose: Calcium supplementation can induce differentiation of several cell types. The tear film has an important role in maintenance of the ocular surface homeostasis with an ionic calcium concentration of 0.4 mM‐1.0 mM. The effect of calcium concentration on healthy primary limbal epithelial cells (LECs) has not been analysed, yet. Our purpose was to investigate the effect of calcium supplementation on gene expression of primary limbal epithelial cells (LECs) of healthy donor corneas, in vitro.Methods: Primary LECs were isolated from corneoscleral rims of healthy donors (n = 6) and were cultured in serum free low calcium (0.06 mM Ca+2) Keratocyte growth medium 3 (KGM3). The primary LECs were then supplemented by 0.3 mM‐2.0 mM Ca+2 concentrations for 72 h. Control LEC cultures were supplemented by low calcium (0.06 mM Ca+2) KGM3 medium. After cell harvesting, RNA and protein were extracted from treated and control LECs to perform qPCR and western blot analysis.Results: The calcium supplementation had no significant effect on PAX6 mRNA expression after 72 h. The differentiation marker and adhesion protein DSG1 and the cornea specific keratin K3 expression did not change significantly through Ca+2 supplementation (p ≥ 0.5). The retinoic acid signalling component fatty acid binding protein FABP5 expression (p ≥ 0.5) the wound healing and vascularization marker CAV1 gene expression (p ≥ 0.5) did not change significantly through Ca+2 supplementation, after 72 h.Conclusions: Calcium supplementation for 72 h did not change PAX6 expression and did not trigger differentiation of healthy primary limbal epithelial cells. We plan to analyse the effect of longer incubation time in the future in order to trigger a full differentiated status of limbal epithelial cells, using calcium supplementation.

Induced pluripotent stem cells as tool for cornea study in regenerative medicine

Induced Pluripotent Stem Cells technology as tool for cornea study in regenerative medicine.The ocular surface consists of an epithelial surface with specialized regions, including cornea lacrimal gland and conjunctiva. Altogether, these regions play a role in vision maintenance by transmitting light and, as first eye layer, ensure protection for the whole eye.These major roles are subject to defective developments or imbalanced maintenance, due to genetic variants or cellular degeneration, respectively, causing vision‐related problems. Intensive research is performed world‐wide to develop new therapies for such vision defects by using in vitro models, but lack of eye donations or low viability is an obstacle for such assays. At the same time, variability among donors on their genetic or epigenetic background is the main barrier for reproducibility, in addition to advanced age of donor, which is a factor limiting cell expansion or present a high number of senescent cells.A promising approach is to use stem cells to generate ocular surface cells or tissue, by mimicking the embryo development. It exists multiple sources of stem cells able to in vitro models, adult corneal cells isolated from biopsies have the advantage to resemble the organ source, but the collection has the disadvantage to be invasive. An alternative and highly valuable approach to generate organoid is from pluripotent stem cells, carrying the potential to differentiate into most cell types of the body and are subdivided into embryonic stem cells, derived from the inner cell mass of preimplantation embryos, and induced pluripotent stem cells (IPSCs) reprogrammed from adult cells.As IPSCs are one of the most promising approaches to counteract this lack of donations, several assays were performed to select the best conditions to generate cornea cells. The main goal is to characterize the developmental procedure by growth factors modulation and assess the maturation degree for the final product. The most important aspect to generate such final product is the heterogeneity of cell population during differentiation or maturation, as epithelial cornea layer has a different cell origin in comparison to stroma or endothelial cornea.Once differentiation and maturation are achieved, this in vitro model can be used for multiple downstream applications. As therapeutic approaches, organoids can be used as a living biobank or to model corneal diseases, either by using cells from affected patients or by base editing, introducing a causative mutation into the genome. Either patient‐derived or control organoids have the high potential to develop gene therapy, optogenetics, be a source of transplantable cells to replace generating tissue or screen small molecules treatments.For this topic, we will focus on the IPSC technology to derive cornea tissue and its perspective for eye development studies, clinical or preclinical study by comparing the methodology depending of the potential applications.

The protective effect of silver nanoparticles' on epithelial cornea cells against ultraviolet is accompanied by changes in calcium homeostasis and a decrease of the P2X7 and P2Y2 receptors

PurposeThe aim of the study was to evaluate the effect of silver nanoparticles hydrocolloids (AgNPs) on human corneal epithelial cells. Epithelial cells form the outermost and the most vulnerable to environmental stimuli layer of the cornea in the eye. Mechanical stress, UV radiation, and pathogens such as bacteria, viruses, and parasites challenge the fragile homeostasis of the eye. To help combat stress, infection, and inflammation wide portfolio of interventions is available. One of the oldest treatments is colloidal silver. Silver nanoparticle suspension in water is known for its anti-bacterial anti-viral and antiprotozoal action. However, AgNPs interact also with host cells, and the character of the interplay between corneal cells and silver seeks investigation. MethodsThe human epithelial corneal cell line (HCE-2) was cultured in vitro, treated with AgNPs, and subjected to UV. The cell’s viability, migration, calcium concentration, and expression/protein level of selected proteins were investigated by appropriate methods including cytotoxicity tests, “wound healing” assay, Fluo8/Fura2 AM staining, qRT-PCR, and western blot. ResultsIncubation of human corneal cells (HCE-2) with AgNP did not affect cells viability but limited cells migration and resulted in altered calcium homeostasis, decreased the presence of ATP-activated P2X7, P2Y2 receptors, and enhanced the expression of PACAP. Furthermore, AgNPs pretreatment helped restrain some of the deleterious effects of UV irradiation. Interestingly, AgNPs had no impact on the protein level of ACE2, which is important in light of potential SARS-CoV-2 entrance through the cornea. ConclusionsSilver nanoparticles are safe for corneal epithelial cells in vitro.

Fluocinolone Acetonide Loaded Chitosan Nanofiber Scaffolds for Treatment of Ocular Disorders: In Vitro Characterization, Ex-Vivo Corneal and Ex-Vivo Scleral Evaluation

Purpose Drugs administered in the ocular region need to overcome ocular barriers without permanently damaging the ocular tissues. Moreover, ocular disorders of the posterior segment are more difficult to treat due to invasive procedures required to reach the posterior segment. Hence, to treat posterior disorders of the eye an attempt was made to develop nanofiber (NF) scaffolds for effective management of chronic posterior uveitis. Nanofibers (NFs) were formulated using the electrospinning technique. Methods NF scaffolds were formulated using the electrospinning technique. The effect of different concentrations of chitosan on NF production was studied by considering different ratios of chitosan (CS) and polyvinyl alcohol (PVA). Physicochemical characterization of NFs was performed to evaluate developed NFs. Results The optimized NF scaffold had a diameter of 129 ± 3 nm. NF scaffolds were found to have a tensile strength of 0.2882 ± 0.078 N/m2, thickness of 0.16 ± 0.05 mm, and drug entrapment of 95 ± 2.0%. The bioadhesive strength of the NF was found to be 257.3 ± 0.04 g/cm2 indicating high bioadhesion of NFs to the ocular tissues. The in-vitro, ex-vivo corneal and ex-vivo scleral drug release after 12 h was found to be 78.4 ± 1.0%, 65.33 ± 0.2% and 78.41 ± 1.0%, respectively. Ex-vivo whole eye model experiment indicated a concentration of about 40 ± 1.75% of drug permeated from corneal layer to the vitreous humor after 12 h. The Hen’s egg test-chorioallantoic membrane study (HET-CAM) study and in-vitro cytotoxicity study on Statens Seruminstitut Rabbit Cornea (SIRC) cell lines indicated that the developed drug-loaded NF scaffolds were found to be non-toxic as compared to pure drug, thus suggesting cytocompatibility. Conclusion Results of HET-CAM, sterility and ex-vivo studies indicate that the developed formulation is non-toxic, sterile, and effective for the ocular delivery of fluocinolone acetonide to the posterior segment of eye.

ADSC-Exos outperform BMSC-Exos in alleviating hydrostatic pressure-induced injury to retinal ganglion cells by upregulating nerve growth factors.

Mesenchymal stem cells (MSCs) have protective effects on the cornea, lacrimal gland, retina, and photoreceptor cell damage, which may be mediated by exosomes (exos) released by MSCs. To investigate the ameliorating effect of exos derived from different MSCs on retinal ganglion cell (RGC) injury induced by hydrostatic pressure. The RGC injury model was constructed by RGC damage under different hydrostatic pressures (40, 80, 120 mmHg). Then RGCs were cultured with adipose-derived stem cell (ADSC)-Exos and bone marrow-derived stem cell (BMSC)-Exos. Cell Counting Kit-8, transmission electron microscopy, flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction, and western blotting were performed to detect the ameliorating effect of exos on pressure-induced RGC injury. ADSC-Exos and BMSC-Exos were successfully isolated and obtained. The gibbosity of RGCs was lower, the cells were irregularly ellipsoidal under pressure, and the addition of ADSC-Exos and BMSC-Exos significantly restored RGC morphology. Furthermore, the proliferative activity of RGCs was increased and the apoptosis of RGCs was inhibited. Moreover, the levels of lactate dehydrogenase and apoptosis-related proteins were increased, and the concentrations of antiapoptotic proteins and neurotrophic factors were decreased in damaged RGCs. However, the above indicators were significantly improved after ADSC-Exos and BMSC-Exos treatment. These findings indicated that ADSC-Exos and BMSC-Exos could ameliorate RGC injury caused by hydrostatic pressure by inhibiting apoptosis and increasing the secretion of neurotrophic factors.

Myo/Nog Cells Increase in Response to Elevated Intraocular Pressure and Mitigate Ganglion Cell Death in a Mouse Model of Glaucoma

Glaucoma is one of the leading causes of blindness worldwide. Decreased aqueous humor drainage causes an increase in intraocular pressure (IOP), which in turn damages the ganglion cells of the retina and optic nerve. A mouse model of glaucoma was used to examine the behavior of Myo/Nog (M/N) cells, which were previously shown to respond to cataract surgery and retinopathy induced by hypoxia, light damage, and intravitreal injection of human retinal pigment epithelial cells. M/N cells express the skeletal-muscle-specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin, and brain-specific angiogenesis inhibitor 1 (BAI1). Glaucoma was induced by injecting microbeads into the anterior chamber (AC) of the right eye to obstruct the flow of aqueous humor into the trabecular meshwork. IOP was elevated within three days of addition of microbeads. Loss of retinal ganglion cells (RGCs) and thinning of the ganglion cell layer–nerve fiber layer (GCL-NFL) was observed in tissue sections by day 32. The injection of microbeads resulted in an increase in BAI1-positive (+) M/N cells in the trabecular meshwork, ciliary body, canal of Schlemm, cornea, and ganglion cell layer (GCL). M/N cells ingested microbeads. The effect of further increasing the population of M/N cells on IOP and RGC loss was determined by injecting BAI1+ cells isolated from the brain into the AC of both eyes. Exogenous M/N cells prelabeled with CellTracker™ Red were found in the same tissues as the endogenous population of M/N cells in eyes with and without elevated IOP. The addition of M/N cells did not significantly reduce IOP in bead-injected eyes. However, there were significantly more RGCs and the NFL was thicker in glaucomatous eyes with M/N cell supplementation than eyes injected with phosphate-buffered saline. The numbers of RGCs and NFL thickness were similar in glaucomatous and non-glaucomatous eyes after adding M/N cells. These results demonstrate that endogenous M/N cells respond to elevated IOP in the anterior and posterior segments in response to induction of glaucoma. M/N cells’ mitigation of RGC loss may reflect a neuroprotective effect within the retina, as opposed to a significant drop in IOP.

MicroRNA and Protein Cargos of Human Limbal Epithelial Cell-Derived Exosomes and Their Regulatory Roles in Limbal Stromal Cells of Diabetic and Non-Diabetic Corneas.

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos' cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.

Limbal Epithelial Stem Cells in the Diabetic Cornea.

Continuous replenishment of the corneal epithelium is pivotal for maintaining optical transparency and achieving optimal visual perception. This dynamic process is driven by limbal epithelial stem cells (LESCs) located at the junction between the cornea and conjunctiva, which is otherwise known as the limbus. In patients afflicted with diabetes, hyperglycemia-induced impairments in corneal epithelial regeneration results in persistent epithelial and other defects on the ocular surface, termed diabetic keratopathy (DK), which progressively diminish vision and quality of life. Reports of delayed corneal wound healing and the reduced expression of putative stem cell markers in diabetic relative to healthy eyes suggest that the pathogenesis of DK may be associated with the abnormal activity of LESCs. However, the precise role of these cells in diabetic corneal disease is poorly understood and yet to be comprehensively explored. Herein, we review existing literature highlighting aberrant LESC activity in diabetes, focusing on factors that influence their form and function, and emerging therapies to correct these defects. The consequences of malfunctioning or depleted LESC stocks in DK and limbal stem cell deficiency (LSCD) are also discussed. These insights could be exploited to identify novel targets for improving the management of ocular surface complications that manifest in patients with diabetes.