EBF1 Limits the Numbers of Cochlear Hair and Supporting Cells and Forms the Scala Tympani and Spiral Limbus during Inner Ear Development.

Early B-cell factor 1 (EBF1) is a basic helix-loop-helix transcription factor essential for the differentiation of various tissues. Our single-cell RNA sequencing data suggest that Ebf1 is expressed in the sensory epithelium of the mouse inner ear. Here, we found that the murine Ebf1 gene and its protein are expressed in the prosensory domain of the inner ear, medial region of the cochlear duct floor, otic mesenchyme, and cochleovestibular ganglion. Ebf1 deletion in mice results in incomplete formation of the spiral limbus and scala tympani, increased number of cells in the organ of Corti and Kölliker's organ, and aberrant course of the spiral ganglion axons. Ebf1 deletion in the mouse cochlear epithelia caused the proliferation of SOX2-positive cochlear cells at E13.5, indicating that EBF1 suppresses the proliferation of the prosensory domain and cells of Kölliker's organ to facilitate the development of appropriate numbers of hair and supporting cells. Furthermore, mice with deletion of cochlear epithelium-specific Ebf1 showed poor postnatal hearing function. Our results suggest that Ebf1 is essential for normal auditory function in mammals.

Induction of human ESC-derived and adult primary multipotent limbal stem cells into retinal pigment epithelial cells and corneal stromal stem cells.

Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.

The comparison of corneal higher-order aberration and surgically induced astigmatism between the clear corneal incision and the limbus tunnel incision of posterior chamber implantable collamer lens implantation.

BACKGROUND: This study aimed to compare the corneal high-order aberrations and surgically induced astigmatism between the clear corneal incision and limbus tunnel incision for posterior chamber implantable collamer lens (ICL/TICL) implantation. METHODS: A total of 127 eyes from 73 myopic patients underwent ICL V4c implantation, with 70 eyes receiving clear corneal incisions and 57 eyes receiving limbus tunnel incisions. The anterior and back corneal surfaces were measured and the Root Mean Square of all activated aberrations (TRMS) was calculated, including higher-order aberration (HOA RMS), spherical aberration Z40, coma coefficients (Coma RMS) Z3-1 Z31, and surgically induced astigmatism (SIA). The measurements were taken preoperatively and postoperatively at 1 day, 1 week, and 1, 3, and 6 months. In this study, the corneal higher-order aberration was estimated as the Zernike coefficient calculated up to 5th order. The measurements were taken at a maximum diameter of 6.5 mm using Pentacam. RESULTS: One week after the operation, the corneal back Z31 of the clear corneal incision group was 0.06 ± 0.06, while the limbus tunnel incision group showed a measurement of 0.05 ± 0.06 (p = 0.031). The corneal back Z40 of the clear corneal incision group was -0.02 ± 0.25, compared to -0.04 ± 0.21 in the limbus tunnel incision group (p = 0.01). One month after the operation, the corneal back SIA of the clear corneal incision group was 0.11 ± 0.11, compared to 0.08 ± 0.11of the limbus tunnel incision group (p = 0.013), the corneal total SIA of the clear corneal incision group was 0.33 ± 0.30, compared to 0.15 ± 0.16 in the limbus tunnel incision group (p = 0.004); the clear corneal incision group exhibited higher levels of back astigmatism and total SIA than the limbus tunnel incision in the post-operation one month period. During the 6- month post-operative follow-up period, no significant difference in Z31, Z40, and other HOA RMS data was observed between the two groups. The total SIA of the corneal incision group and the limbus tunnel incision group were 0.24 ± 0.14 and 0.33 ± 0.32, respectively (p = 0.393), showing no significant difference between the two groups 6 months after the operation. CONCLUSION: Our data showed no significant difference in the high-order aberration and SIA between clear corneal incision and limbus tunnel incision up to 6 months after ICL-V4c implantation.

The central and peripheral corneal response to short-term hypoxia.

PURPOSE: To quantify the magnitude and recovery of central and limbal corneal oedema induced by short-term unilateral eyelid closure without contact lens wear. METHODS: The left eye of 10 adults with healthy corneas was patched using a folded eye pad for 30 min. High-resolution optical coherence tomography images (which captured the limbal and central corneal regions simultaneously) were obtained before patching, immediately after eye opening and again at 1, 2, 5, 6, 9, 10, 14 and 15 mins after eyelid opening. Oedema was measured from the limbus (scleral spur) to the central cornea (thinnest corneal location) along the horizontal meridian. RESULTS: A greater amount of limbal oedema was noted (mean [SD] 3.84 [1.79] %) compared to the central cornea (2.48 [0.61] %; p = 0.04) after 30 mins of unilateral eyelid closure. Both central and limbal corneal oedema recovered rapidly following eyelid opening, with no significant differences in the rate of corneal recovery between corneal locations (p = 0.90). CONCLUSIONS: Short-term unilateral eyelid closure resulted in ~55% more relative oedema in the limbal region compared to the central cornea. Rapid recovery of oedema and corneal overshoot (thinning beyond the baseline corneal thickness) was observed within 1-2 min of eyelid opening for both central and peripheral regions.

Factors influencing the therapeutic effect of hyperopic correction on esotropia in patients with partially accommodative esotropia.

OBJECTIVES: To collate data on partially accommodative esotropia (PAET) to better understand this condition's aetiology and to evaluate and predict the therapeutic effect of a hyperopic correction on PAET. METHODS: Eighty-nine consecutive patients diagnosed with PAET with a spherical equivalent (SE) refractive error >+2.50 D were included in this retrospective review. Clinical characteristics, including gender, age, SE, angle of esodeviation, accommodative convergence/accommodation (AC/A) ratio, near-distance disparity (NDD) and anatomical features of the rectus muscles were compared among different PAET subgroups. Multiple linear regression was used to identify independent factors that influenced the therapeutic effect of a hyperopic correction on esotropia. RESULTS: No significant differences were observed for the angle of esodeviation as a function of age in individuals with PAET. The incidence of SE in PAET participants >9 years old was significantly greater than in those <5 and 6-8 years of age. The therapeutic effect of hyperopic correction on esotropia was positively associated with SE both at distance and near. In addition, the limbus insertion distance (LID) of the lateral rectus (LR) muscle was positively associated with NDD at distance, but negatively associated at near. CONCLUSION: A greater incidence of hyperopia was observed in older (>9 years old) PAET patients. A hyperopic correction had a greater effect on esotropia in individuals with a higher SE, larger LID of the LR muscle and a smaller NDD.

Zonular fibre Insertion-to-Limbus Distance (ZLD): normative data to assess lens position and diagnose ectopia lentis.

PURPOSE: Subluxation of the crystalline lens (Ectopia Lentis, EL) can lead to significant visual impairment and serves as a diagnostic criterion for genetic disorders such as the Marfan syndrome. There is no established criterion to diagnose and quantify EL. We prospectively investigated the distance between the zonular fibre insertion and the limbus (ZLD) in healthy subjects as a parameter to assess the position of the lens, quantify EL and provide normative data. METHODS: This prospective, observational, cross-sectional study includes one-hundred-fifty eyes of 150 healthy participants (mean age 28 years, range 4-68). Pupils were dilated with tropicamide 0.5% and phenylephrine 2.5% eyedrops. ZLD was measured in mydriasis at the slit lamp as the distance between the most central visible insertions of the zonular fibres on the lens surface and the corneoscleral limbus. Vertical pupil diameter (PD) and refractive error were recorded. If zonular fibre insertions were not visible, the distance between limbus and the pupillary margin was recorded as ZLD. RESULTS: 145 right and 5 left eyes were examined. 93% of study subjects were Caucasian, 7% were Asian. In eyes with visible zonular fibre insertions (n = 76 eyes), ZLD was 1.30 ± 0.28 mm (mean ± SD, range 0.7-2.1) and PD was 8.79 ± 0.57 mm (7.5-9.8). In the remaining 74 eyes, ZLD was 1.38 ± 0.28 mm (0.7-2.1), and PD was 8.13 ± 0.58 mm (6.7-9.4). For all eyes, ZLD was 1.34 ± 0.29 mm (0.7-2.1), and PD was 8.47 ± 0.66 mm (6.7-9.8). Refractive error and sex did not significantly affect ZLD. Smaller PD and older age were associated with larger ZLD (P < 0.001 and P = 0.036, respectively). CONCLUSION: Average ZLD was 1.34 mm in eyes of healthy subjects. Older age correlated with larger ZLD. These normative data will aid in diagnosing and quantifying EL.

A Chymist Among Beasts: Reading Paracelsus Literally (with a translation of De lunaticis, chapter two).

Paracelsus is an extraordinarily difficult author to interpret, in part because of the seemingly elusive boundary between literal and metaphorical levels of meaning in his work. The present paper argues for a literal reading of Paracelsus, based on comments that he makes in his late Philosophia de divinis operibus & factis & de secretis naturae. The article also includes a translated chapter from one of the treatises in that work, De lunaticis.

Single mRNA detection of Wnt signaling pathway in the human limbus.

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their close microenvironment. It has been shown that Wnt signaling pathway is crucial for LSCs regulation. Previous differential gene profiling studies confirmed the preferential expression of specific Wnt ligands (WNT2, WNT6, WNT11, WNT16) and Wnt inhibitors (DKK1, SFRP5, WIF1, FRZB) in the limbal region compared to the cornea. Among all frizzled receptors, frizzled7 (Fzd7) was found to be preferentially expressed in the basal limbal epithelium. However, the exact localization of Wnt signaling molecules-producing cells in the limbus remains unknown. The current study aims to evaluate the in situ spatial expression of these 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7. Wnt ligands, DKK1, and Fzd7 expression were scattered within the limbal epithelium, at a higher abundance in the basal layer than the superficial layer. SFRP5 expression was diffuse among the limbal epithelium, whereas WIF1 and FRZB expression was clustered at the basal limbal epithelial layer corresponding to the areas of high levels of Fzd7 expression. Quantitation of the fluorescence intensity showed that all 4 Wnt ligands, 3 Wnt inhibitors (WIF1, DKK1, FRZB), and Fzd7 were highly expressed at the basal layer of the limbus, then in a decreasing gradient toward the superficial layer (P < 0.05). The expression levels of all 4 Wnt ligands, FRZB, and Fzd7 in the basal epithelial layer were higher in the limbus than the central cornea (P < 0.05). All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were also highly expressed in the limbal stroma immediately below the epithelium but not in the corneal stroma (P < 0.05). In addition, Fzd7 had a preferential expression in the superior limbus compared to other limbal quadrants (P < 0.05). Taken together, the unique expression patterns of the Wnt molecules in the limbus suggests the involvement of both paracrine and autocrine effects in LSCs regulation, and a fine balance between Wnt activators and inhibitors to govern LSC fate.

Central and peripheral scleral lens-induced corneal oedema.

PURPOSE: To quantify the magnitude of central and peripheral scleral lens-induced corneal oedema for a range of fluid reservoir thicknesses, and to compare these experimental results with theoretical models of corneal oedema both with and without limbal metabolic support (i.e., the lateral transport of metabolites and the influence of the limbal vasculature). METHODS: Ten young healthy participants wore scleral lenses (KATT™, Capricornia Contact Lenses) fitted with low (mean 141 µm), medium (482 µm) and high (718 µm) central fluid reservoir thickness values across three separate study visits. The scleral lens thickness, fluid reservoir thickness and stromal corneal oedema were measured using optical coherence tomography. Oedema was quantified across the central (0-2.5 mm from the corneal apex) and peripheral (1.25-3 mm from the scleral spur) cornea. Experimental data were compared with published theoretical models of central to peripheral corneal oedema. RESULTS: Stromal oedema varied with fluid reservoir thickness (p < 0.001) for both central and peripheral regions. The mean (standard deviation) stromal oedema was greater for the medium (2.08 (1.21)%) and high (2.22 (1.31)%) fluid reservoir thickness conditions compared to the low condition (1.00 (1.01)%) (p ≤ 0.01). Stromal oedema gradually increased from the corneal centre to the periphery by ~0.3% on average (relative increase of 18%), but the change did not reach statistical significance. This trend of increasing, rather than decreasing, oedema towards the limbus is consistent with theoretical modelling of peripheral oedema without metabolic support from the limbus. CONCLUSIONS: The central and peripheral cornea displayed a similar magnitude of oedema, with increasing levels observed for medium and high fluid reservoir thicknesses. The gradual increase in oedema towards the limbus is consistent with a 'without limbal metabolic support' theoretical model.

The influence of soft contact lens material on the corneoscleral profile.

PURPOSE: To objectively quantify changes in corneoscleral profile, as evaluated by the limbus position and corneoscleral junction (CSJ) angle, as a consequence of wearing different soft contact lens (CL) materials. METHODS: Twenty-two healthy participants wore silicone hydrogel (SiHy, MyDay, CooperVision) and hydrogel (Hy, Biomedics 1 day extra, CooperVision) soft CLs for 8 h per lens in their left eye. In each session, corneoscleral topography was captured before and immediately after CL removal with an Eye Surface Profiler. Previously validated automatic and objective algorithms for limbal position and CSJ angle calculation were applied to 360 semi-meridians to investigate the effect of short-term CL wear on corneoscleral topography, globally and by sectors, depending on the soft CL material worn. RESULTS: Short-term soft CL wear significantly impacted limbal position (SiHy: 120 ± 97 µm, Hy: 128 ± 85 µm) and CSJ angle (SiHy: 0.57 ± 0.36°, Hy: 0.55 ± 0.40°); all p < 0.05. A statistically significant difference was found between the sectors with regard to limbus position and CSJ angle before CL wear that remained following lens wear (all pairwise comparisons, p < 0.001). Although individual differences were observed, there was no evidence that one material caused more substantial corneoscleral alterations. CONCLUSION: Corneoscleral profile parameters were altered significantly following 8 h of soft CL wear. The observed changes in limbus position and CSJ angle support the importance of participant-material biocompatibility.

The Role of Sensory Innervation in Homeostatic and Injury-Induced Corneal Epithelial Renewal.

The cornea is the window through which we see the world. Corneal clarity is required for vision, and blindness occurs when the cornea becomes opaque. The cornea is covered by unique transparent epithelial cells that serve as an outermost cellular barrier bordering between the cornea and the external environment. Corneal sensory nerves protect the cornea from injury by triggering tearing and blink reflexes, and are also thought to regulate corneal epithelial renewal via unknown mechanism(s). When protective corneal sensory innervation is absent due to infection, trauma, intracranial tumors, surgery, or congenital causes, permanent blindness results from repetitive epithelial microtraumas and failure to heal. The condition is termed neurotrophic keratopathy (NK), with an incidence of 5:10,000 people worldwide. In this report, we review the currently available therapeutic solutions for NK and discuss the progress in our understanding of how the sensory nerves induce corneal epithelial renewal.

Ocular surface squamous neoplasia with 360° limbal involvement: a study of 130 patients.

PURPOSE: To describe the risk factors, clinical features and management outcomes of ocular surface squamous neoplasia (OSSN) with 360° of limbal involvement (360-OSSN) and compare with segmental limbal involvement (SL-OSSN). METHODS: Retrospective comparative study of 360-OSSN vs SL-OSSN. All 360-OSSN and every 10th patient with SL-OSSN during the study period (2012-2020) were included. Lesions with uncertain diagnosis were excluded. RESULTS: Of 1250 patients diagnosed with OSSN during the study period, 30 (2%) had 360-OSSN. A total of 100 patients of OSSN with SL-OSSN were included for comparison. 360-OSSN patients more often had longer duration of symptoms (mean, 17 vs 8 months; p, 0.003), prior misdiagnosis (17% vs 6%, p, 0.13) and prior intervention (47% vs 13%; p, 0.0002) than patients with SL-OSSN. 360-OSSN had higher incidence of scleral fixity (57% vs 16%; p < 0.0001), corneal/scleral melt (17% vs 0%; p, 0.0005), intraocular tumor extension (17% vs 0%; p, 0.003), orbital tumor extension (33% vs 1%; p < 0.0001), and advanced T stage at presentation (Tis: 37% vs 76%, T1: 0% vs 15%; T2: 7% vs 4%; T3: 27% vs 4%; T4: 30% vs 1%; p < 0.001). Over a mean follow-up of 14 months, lymph node metastasis (8% vs 0%; p, 0.05) and distant metastasis (4% vs 0%; p, 0.23) were more common in 360-OSSN group compared to SL-OSSN group. CONCLUSION: Risk factors of 360-OSSN include prolonged symptoms, prior misdiagnosis and prior intervention. It represents an advanced form of disease with propensity for corneo-scleral melt and invasive disease which requires aggressive management.

Analysis of different conditioned media secreted by limbal progenitor cells in the modulation of corneal healing.

Ex vivo cultivation and transplantation of limbal epithelial cells has been reported as an alternative source for ocular surface reconstruction. However, until now, the functional improvement of these patients is limited due to the low survival rate of the transplanted cells. Consequently, the clinical benefits of this therapeutic strategy are only temporary and can assign them to paracrine effects associated with the transplanted cells. With this background in mind, we aimed to analyze the effect of different conditioned media containing growth factors secreted by limbal progenitor cells on corneal epithelial healing, both in vitro and in vivo. Limbal tissue was used to obtain different conditioned media (CM). For the in vitro assay, corneal epithelial cells were treated with CM and the epithelial migration was analyzed. Growth factors in the CM were identified with ELISA and multiplex. For the in vivo assay in rats, total limbal stem cell deficiency (LSCD) was induced with an abrasive injury to the ocular surface, and the animals were treated with different CM. Clinical and histological analyses were performed. In the in vitro assay, treatment with limbal fibroblast (LF CM) was more effective compared to the other CM, and analysis revealed high concentrations of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF). In the in vivo assay, animals treated with LF CM showed epithelial defect improvement, maintenance of thickness, and decreased opacity and neovascularization. This treatment also allowed better ocular surface tissue organization when compared to the other treatments. The in vitro and in vivo experiments showed better outcomes in the corneal wound healing for the LF CM treatment. The high concentrations of KGF and HGF, linked to epithelial cell migration and proliferation, may correlate to the best results found in this treatment.

Impact of the transcription factor IRF8 on limbal epithelial progenitor cells in a mouse model.

The limbus of the eye is the location of the corneal epithelial stem cell niche. These cells are necessary for continuous renewal of the corneal epithelium. In the case of limbal stem cell deficiency, these cells are damaged, and the whole cornea becomes opaque. It is important to be able to identify stem cells that could be applied for new therapeutic strategies. There are various known markers to characterize these cells, including p63, Nanog, oct4 and FGFR2. However, none of these markers are exclusively expressed in these stem cells (they are also expressed in transient amplified cells). It seems likely that a combination of stem cell markers will be necessary for corneal stem cell identification. The aim of this study was to detect IRF8 in limbal epithelial stem cells and to determine its function. In a mouse model, IRF8 could be detected in limbal and basal epithelial cells of the cornea by histological and immunohistological staining of wild-type mouse eyes. Furthermore, the limbus of the eye was significantly smaller in IRF8-knockout mice than in wild-type mice, and the expression of Nanog was lower in IRF8-knockout mice. This suggests that IRF8 has an influence on the maintenance of stem cell properties in the limbus, possibly by affecting the expression of Nanog. Furthermore, IRF8 has an impact on E-cadherin and N-cadherin expression in the mouse eye.

Human ocular surface microcirculation quantified by in vivo computer assisted video microscopy and diffuse reflectance spectroscopy.

Non-invasive imaging techniques are increasingly used to objectively quantify anterior segment structures of the eye. In this study, we apply the novel oxygen delivery index (ODIN) concept that, quantifies microvascular capacity for oxygen delivery, to the ocular surface in healthy humans. The purpose of the study was to test the applicability of the technologies used for data acquisition from the human ocular surface. We also validated whether the ODIN concept has sufficient sensitivity to detect and differentiate between microvascular structure and function in limbal and bulbar conjunctiva. Multiple ocular surface measurements using computer-assisted video microscopy (field of view: 1.6 mm × 0.9 mm) and diffuse reflectance spectroscopy (measuring volume: ∼0.1 mm3) were obtained from limbal and bulbar conjunctiva in 20 healthy volunteers. Three parameters were extracted during analyses: Functional capillary density, capillary flow velocity, and microvascular oxygen saturation. Functional capillary density was higher at limbus than in bulbar conjunctiva (11.2 ± 1.8 c/mm versus 5.2 ± 1.2 c/mm, p < 0.01), and microvascular oxygen saturation was lower at limbus (77 ± 8%) as compared to bulbar conjunctiva (89 ± 6%), p < 0.01. More than 80% of scored capillaries had continuous blood flow and no difference was seen between the recording sites (p = 0.68). In conclusion, the ODIN concept is applicable for the assessment of human ocular surface microvascular function and has sufficient sensitivity to detect increased capillary density and oxygen extraction at limbus as compared with bulbar conjunctiva.

Ocular surface microcirculation is better preserved with pulsatile versus continuous flow during cardiopulmonary bypass-An experimental pilot.

BACKGROUND: Non-pulsatile cardiopulmonary bypass (CPB) may induce microvascular dysregulation. In piglets, we compared ocular surface microcirculation during pulsatile versus continuous flow (CF) bypass. METHODS: Ocular surface microcirculation in small tissue volumes (~0.1 mm3 ) at limbus (high metabolic rate) and bulbar conjunctiva (low metabolic rate) was examined in a porcine model using computer assisted video microscopy and diffuse reflectance spectroscopy, before and after 3 and 6 h of pulsatile (n = 5 piglets) or CF (n = 3 piglets) CPB. Functional capillary density, capillary flow velocity and microvascular oxygen saturation were quantified. RESULTS: At limbus, velocities improved with pulsatility (p < 0.01) and deteriorated with CF (p < 0.01). In bulbar conjunctiva, velocities were severely reduced with CF (p < 0.01), accompanied by an increase in capillary density (p < 0.01). Microvascular oxygen saturation decreased in both groups. CONCLUSION: Ocular surface capillary densities and flow patterns are better preserved with pulsatile versus CF during 6 h of CPB in sleeping piglets.

Vernal keratoconjunctivitis with a limbal mass lesion developing independently of severe papillae formation at the tarsal conjunctiva: a case report.

BACKGROUND: A hypertrophic limbal mass lesion is an uncommon finding of vernal keratoconjunctivitis; it normally occurs in eyes with severe papillae formation in the tarsal conjunctiva. We present a case with a limbal mass lesion in a patient with relatively mild allergic findings in the tarsal conjunctiva. CASE PRESENTATION: A 12-year-old Japanese boy displaying allergic conjunctivitis presented with a mass lesion at the inferior limbus in the left eye. Relatively mild papillae formation was found on the tarsal conjunctiva in both eyes. We diagnosed that the mass lesion resulted from limbal vernal keratoconjunctivitis and resected it for therapeutic purposes. Histopathological examination showed that eosinophils, lymphocytes, and fibroblasts were present in the subepithelial lesion and the substantia propria of the mass lesion. Immunohistochemical staining detected diffuse and rich infiltration of CD3-positive T lymphocytes and a relatively small number of CD20-positive B lymphocytes and CD138-positive plasma cells that tended to aggregate. The histopathologic features suggested that the limbal mass lesion had similar structures to the papillae at the tarsal conjunctiva of vernal keratoconjunctivitis. CONCLUSION: The limbal mass lesion as a finding of vernal keratoconjunctivitis can occur even if the papillae formation at the patient's tarsal conjunctiva is mild.

Transcriptomic Profiling of Human Limbus-Derived Stromal/Mesenchymal Stem Cells-Novel Mechanistic Insights into the Pathways Involved in Corneal Wound Healing.

Limbus-derived stromal/mesenchymal stem cells (LMSCs) are vital for corneal homeostasis and wound healing. However, despite multiple pre-clinical and clinical studies reporting the potency of LMSCs in avoiding inflammation and scarring during corneal wound healing, the molecular basis for the ability of LMSCs remains unknown. This study aimed to uncover the factors and pathways involved in LMSC-mediated corneal wound healing by employing RNA-Sequencing (RNA-Seq) in human LMSCs for the first time. We characterized the cultured LMSCs at the stages of initiation (LMSC-P0) and pure population (LMSC-P3) and subjected them to RNA-Seq to identify the differentially expressed genes (DEGs) in comparison to native limbus and cornea, and scleral tissues. Of the 28,000 genes detected, 7800 DEGs were subjected to pathway-specific enrichment Gene Ontology (GO) analysis. These DEGs were involved in Wnt, TGF-ß signaling pathways, and 16 other biological processes, including apoptosis, cell motility, tissue remodeling, and stem cell maintenance, etc. Two hundred fifty-four genes were related to wound healing pathways. COL5A1 (11.81 ± 0.48) and TIMP1 (20.44 ± 0.94) genes were exclusively up-regulated in LMSC-P3. Our findings provide new insights involved in LMSC-mediated corneal wound healing.

Limbal stem cell diseases.

The function of limbal stem/progenitor cells (LSCs) is critical to maintain corneal epithelial homeostasis. Many external insults and intrinsic defects can be deleterious to LSCs and their niche microenvironment, resulting in limbal stem cell dysfunction or deficiency (LSCD). Ocular comorbidities, frequent in eyes with LSCD, can exacerbate the dysfunction of residual LSCs, and limit the survival of transplanted LSCs. Clinical presentation and disease evolution vary among different etiologies of LSCD. New ocular imaging modalities and molecular markers are now available to standardize the diagnosis criteria and stage the severity of the disease. Medical therapies may be sufficient to reverse the disease if residual LSCs are present. A stepwise approach should be followed to optimize the ocular surface, eliminate the causative factors and treat comorbid conditions, before considering surgical interventions. Furthermore, surgical options are selected depending on the severity and laterality of the disease. The standardized diagnostic criteria to stage the disease is necessary to objectively evaluate and compare the efficacy of the emerging customized therapies.

Outcomes of surgical interventions for the treatment of limbal stem cell deficiency.

Background & objectives: In the current scenario, with availability of different surgical procedures for limbal stem cell deficiency (LSCD), there exists no common consensus as to the standardization of the management protocol for the same. In addition, there also exists diversity in the views about the clinical diagnosis, ancillary investigations and clinical parameters. The objective of the present study was to evaluate the reported outcomes of surgical interventions for the management of LSCD. Methods: A systematic review of published literature on limbal stem cell transplantation (LSCT) was performed using Ovid Medline, Embase and PubMed for a duration of 2009 to 2019. Original studies including prospective, retrospective case series and randomized controlled trials, articles in English language, articles with access to full text and studies with more than or at least 10 patients were included in this review. Data related to clinical and visual outcomes were evaluated, and pool estimates of different surgeries were calculated using random-effects model and individually using Pearson's Chi-square test. Results: A total of 1133 abstracts were evaluated. Finally, 17 studies were included for the analysis. Among these 17 studies, direct limbal lenticule transplantation was performed in five studies, of which autologous tissue from the fellow eye [conjunctival limbal autograft (CLAU)], allograft from a cadaver/live donor [keratolimbal allograft (KLAL)/conjunctival limbal allograft (CLAL)] and combination of CLAU plus KLAL were done in one, three and one studies, respectively. The ex vivo expanded cultivated limbal epithelial transplantation (CLET) was reported in six studies and simple limbal epithelial transplantation (SLET) in four studies. Two were comparative studies comparing CLET and CLAL (living-related CLAL) with cadaveric KLAL, respectively. Outcome analysis of the included studies showed significant heterogeneity. Calculated pool rate for various types of surgeries was calculated. The pool estimate for CLAL was 67.56 per cent [95% confidence interval (CI), 41.75-93.36; I2=83.5%, P=0.002]. For KLAL, this value was 63.65 per cent (95% CI, 31.38-95.91; I2=92.4%, P=0.000). Pool estimate for CLET was 78.90 per cent (95% CI, 70.51-87.28; I2=73.6%, P=0.001). Corresponding values for SLET were 79.08 per cent (95% CI, 74.10-84.07; I2=0.0%, P=0.619). CLAU and combination of CLAU plus KLAL were done in one study each; hence, statistical analysis could not be done. The functional outcome in terms of gain in visual acuity post-operatively was better in KLAL (P<0.005) and SLET group as compared to CLET group. Interpretation & conclusions: The present analysis suggests that though the anatomical success rates were almost identical between SLET, CLET, CLAL, and KLAL procedures, the functional success rates were better following KLAL and SLET procedures as compared to CLET. Decision for LSCT for cases of ocular burns based on either clinical judgement of the surgeon or individual diagnosis remains a suitable option.