ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation.

The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.

Dry eye disease in mice activates adaptive corneal epithelial regeneration distinct from constitutive renewal in homeostasis.

Many epithelial compartments undergo constitutive renewal in homeostasis but activate unique regenerative responses following injury. The clear corneal epithelium is crucial for vision and is renewed from limbal stem cells (LSCs). Using single-cell RNA sequencing, we profiled the mouse corneal epithelium in homeostasis, aging, diabetes, and dry eye disease (DED), where tear deficiency predisposes the cornea to recurrent injury. In homeostasis, we capture the transcriptional states that accomplish continuous tissue turnover. We leverage our dataset to identify candidate genes and gene networks that characterize key stages across homeostatic renewal, including markers for LSCs. In aging and diabetes, there were only mild changes with <15 dysregulated genes. The constitutive cell types that accomplish homeostatic renewal were conserved in DED but were associated with activation of cell states that comprise "adaptive regeneration." We provide global markers that distinguish cell types in homeostatic renewal vs. adaptive regeneration and markers that specifically define DED-elicited proliferating and differentiating cell types. We validate that expression of SPARC, a marker of adaptive regeneration, is also induced in corneal epithelial wound healing and accelerates wound closure in a corneal epithelial cell scratch assay. Finally, we propose a classification system for LSC markers based on their expression fidelity in homeostasis and disease. This transcriptional dissection uncovers the dramatically altered transcriptional landscape of the corneal epithelium in DED, providing a framework and atlas for future study of these ocular surface stem cells in health and disease.

Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization.

BACKGROUND: The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization. METHODS: Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium. RESULTS: All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity. CONCLUSION: Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage.

Comparative Analysis of Tear Proteins in Keratoconic Scleral Lens Wearers with Variation in Limbal Clearance.

SIGNIFICANCE: Cytokine and protease analysis revealed relative changes in the post-lens tear film of scleral lenses with low and high limbal clearances. Results from this study indicate that midperipheral lens fit is an important fitting feature that can impact the inflammatory response of a keratoconic eye. PURPOSE: The purpose of this study was to investigate changes in levels of inflammatory mediators in the post-lens tear film of keratoconic scleral lens wearers with varying limbal clearance designs. METHODS: Twenty-two keratoconic eyes were fitted with two sets of scleral lenses that were consistent in lens diameter and central sagittal depth but varied in limbal clearance by approximately 50 µm. Lenses were worn in a randomly assigned order for a 2-week period each. At each follow-up visit, immediately after lens removal, tear samples were collected with a microcapillary tube (10 µL, 0.5 mm in diameter) from the bowl of the inverted scleral lens. Tear cytokine and protease analysis was performed using a multiplex electrochemiluminescent array (Meso Scale Discovery, Rockville, MD) instrument. Levels of interleukins 1, 6, and 8; tumor necrosis factor α; and matrix metalloproteinases 1 and 9 were compared and analyzed. RESULTS: Levels of interleukin 1ß, tumor necrosis factor α, and matrix metalloproteinase 1 increased with high limbal clearance (P = .01, .006, and .02, respectively). No change in interleukins 6 and 8 levels was found (P > .05). A decrease in matrix metalloproteinase 9 was noted in post-lens tear film of scleral lenses with high limbal clearance (P = .10). DISCUSSION: Relative changes in the cytokine and protease levels were found when comparing low and high limbal clearance, indicating that the midperipheral lens fit is an important feature that can impact the inflammatory response of the keratoconic eye.

Opening the Soul Window Manually: Limbal Tissue Scaffolds with Electrospun Polycaprolactone/Gelatin Nanocomposites.

Restricted by the difficulty in fabricating scaffolds suitable for cell proliferation, the use of ex vivo expanded limbal stem cell (LSC) for LSC transplantation, an effective treatment method for patients with limb stem cell deficiency (LSCD), is hard to be widely used in clinical practice. To tackle these challenges, a novel electrospun polycaprolactone (PCL)/gelatin nanocomposite is proposed to make 3D scaffolds for limbal niche cells (LNC) proliferation in vitro, which is a milestone in the treatment of diseases such as LSCD. PCL and gelatin in different weight ratios are dissolved in a mixed solvent, and then electrospinning and cross-linking are performed to prepare a scaffold for cell proliferation. The characterizations of the nanocomposites indicate that the gelatin content has a significant effect on its micro-morphology, thermal properties, crystallinity, degradation temperature, hydrophilicity, and mechanical properties. P8G2-C (PCL: gelatin = 80: 20, cross-linked), with smooth fibers and homogeneous pores, has better hydrophilicity, mechanical properties, and flexibility, so it can support LNC as cell proliferation assays revealed. This detailed investigation presented here demonstrates the feasibility of using PCL/gelatin nanocomposites electrospun fiber membranes as a limbus tissue engineering scaffold, which undoubtedly provide a new perspective for the development of tissue engineering field.

Shape analysis of rectus extraocular muscles with age and axial length using anterior segment optical coherence tomography.

PURPOSE: This study aimed to evaluate the shape of the extraocular muscles (EOMs) in normal subjects using the en-face images of anterior segment optical coherence tomography (AS-OCT). The EOM insertion and the direction of the muscle fibers were investigated. SUBJECTS AND METHODS: A total of 97 healthy normal subjects (194 eyes) at Okayama University Hospital (age, 47.1±21.5 years; range, 8-79 years) participated in the study. A series of 256 tomographic images of the rectus EOMs were captured using the C-scan function of the AS-OCT (CASIA2, TOMEY Co., Japan), and the images were converted to en-face images in multi-TIFF format. The anterior chamber angle to EOM insertion distance (AID) and the angle of the muscle fibers from the insertion site (angle of muscles) were measured from the images. The correlations of AID and angle of muscles with age and axial length were investigated and evaluated. RESULTS: AID and angle of muscles were significantly correlated with age or axial length in some EOMs. The AIDs of medial rectus (MR) (P = 0.000) and superior rectus (SR) (P = 0.005) shortened with age. The AIDs of MR (P = 0.001) and inferior rectus (IR) (P = 0.035) elongated with axial length, whereas lateral rectus (LR) (P = 0.013) shortened. The angles of MR (P = 0.001) and LR (P = 0.000) were found to have a more downward direction toward the posterior in older subjects. CONCLUSION: En-face images can be created by AS-OCT, and the shape of the EOMs in normal subjects using these image measurements was available. With the ability to assess the EOMs, AID and angle of muscles are expected give useful information for treating and diagnosing strabismus-related diseases.

Dynamic Optical Coherence Elastography of the Anterior Eye: Understanding the Biomechanics of the Limbus.

Purpose: Currently, the biomechanical properties of the corneo-scleral limbus when the eye-globe deforms are largely unknown. The purpose of this study is to evaluate changes in elasticity of the cornea, sclera, and limbus when subjected to different intraocular pressures (IOP) using wave-based optical coherence elastography (OCE). Special attention was given to the elasticity changes of the limbal region with respect to the elasticity variations in the neighboring corneal and scleral regions. Methods: Continuous harmonic elastic waves (800 Hz) were mechanically induced in the sclera near the corneo-sclera limbus of in situ porcine eye-globes (n = 8). Wave propagation was imaged using a phase-sensitive optical coherence tomography system (PhS-OCT). The eyes were subjected to five different IOP-levels (10, 15, 20, 30, and 40 mm Hg), and spatially distributed propagation velocities were calculated along corneal, limbal, and scleral regions. Finite element analysis (FEA) of the same regions under the same excitation conditions were conducted for further validation of results. Results: FEA demonstrated that the stiffness of the heterogeneous cornea-limbus-sclera transition can be characterized by phase velocity measurements of the elastic waves produced at 800 Hz in the anterior eye. Experimental results revealed that the wave speed in the limbus (cL = 6.5 m/s) is between the cornea (cc = 2.9 m/s) and sclera (cs = 10.0 m/s) at a physiological IOP level (15 mm Hg) and rapidly increases as the IOP level is increased, even surpassing the wave speed in the sclera. Finally, the change in elastic wave speed in the limbus (ΔcL∼18.5 m/s) was greater than in the cornea (Δcc ∼12.6 m/s) and sclera (Δcs∼8.1 m/s) for the same change in IOP. Conclusions: We demonstrated that wave-based OCE can be utilized to assess limbus biomechanical properties. Moreover, experimental evidence showed that the corneo-scleral limbus is highly nonlinear compared to the cornea and sclera when the eye-globe is deformed by an increase of IOP. This may suggest that the limbus has enough structural flexibility to stabilize anterior eye shape during IOP changes.

Corneal Recovery Following Rabbit Peripheral Blood Mononuclear Cell-Amniotic Membrane Transplantation with Antivascular Endothelial Growth Factor in Limbal Stem Cell Deficiency Rabbits.

Background: Limbal stem cell deficiency (LSCD) is a refractory ocular surface disorder characterized by progressive corneal epithelial degeneration, conjunctivalization, and neovascularization, potentially leading to blindness. There are currently no effective therapeutic options for patients experiencing routine symptomatic treatment failure. Transplantation of amniotic membrane (AM) with adherent stem cells (but not bare AM transplantation alone) has shown promise in preclinical studies for ocular surface restoration. A major limitation, however, is finding a reliable stem cell source. Stem cells can be isolated from the peripheral blood mononuclear cell (PBMC) population, and these PBMC-derived stem cells have numerous advantages over allogeneic and other autologous stem cell types for therapeutic application, including relative ease of acquisition, nonimmunogenicity, and the absence of ethical issues associated with embryonic stem cells. Experiment: We examined the efficacy of autologous PBMC-AM sheet cultures combined with postoperative antiangiogenesis treatment for corneal restoration in LSCD model rabbits. Rabbit PBMCs (rPBMCs) were isolated, labeled with EdU for in vivo tracing, and then cultured on AMs in conditioned medium before transplantation. Rabbits were transplanted with bare AMs (group 1), rPBMC-AM sheets (group 2), or rPBMC-AM sheets plus postoperative treatment with the vascular endothelial growth factor antagonist bevacizumab (group 3). Corneal opacity and neovascularization were monitored by slit-lamp imaging for 8 weeks and corneas were examined histologically at 1 and 2 months. Results: Corneal opacity decreased in all three groups over 8 weeks, but was significantly lower in group 2 and even lower in group 3. Corneal neovascularization was significantly higher in group 1 throughout the observation period, and significantly lower in group 3 than group 1 and 2 by 8 weeks post-transplant. At 4 weeks, the corneal surface completed epithelialization (although thinner than normal) in group 3 but still patchy in groups 1 and 2. By 8 weeks, the epithelium in group 3 was complete and smooth, resembling a normal epithelium. Integrin ß1 as a progenitor marker was also generally higher in groups 2 and 3. Conclusions: Autologous rPBMC-AM sheets with post-transplant topical bevacizumab can effectively facilitate corneal epithelium recovery in a LSCD model, suggesting clinical utility for LSCD-related ocular surface diseases. Impact statement Limbal stem cell deficiency (LSCD) increases corneal opacity and vascularization, resulting in severe visual impairment or even blindness. Traditional surgical limbal transplant is currently the main treatment option for LSCD, but carries the risks of rejection and immunosuppressant side effects. Autologous stem cell-based therapy is a promising alternative approach, but a reliable stem cell source is a major limitation. We report that transplantation of autologous rabbit peripheral blood mononuclear cell-amniotic membrane sheets plus antivascular endothelial growth factor restored avascular transparent cornea in a rabbit LSCD model. These results demonstrate a potentially effective approach for ocular surface reconstruction in bilateral LSCD.

The microanatomy of Bowman's layer in the cornea of the pig: Changes in collagen fibril architecture at the corneoscleral limbus

In most animals, Bowman's layer is a feature of the cornea of the eye, and lies between the sur-face epithelium and the stromal extracellular matrix that makes up the bulk of the cornea. It is comprised of a condensation of disorganised collagen fibrils. However, it has been conjectured that not all species possess Bowman’s layer, and pigs are a species that has classically been stated to lack this anatomical structure, although there is disagreement in the published literature. Here, we studied the porcine cornea using transmission and scanning electron microscopy (TEM and SEM) to ascertain whether Bowman’s layer existed. TEM identified a thin band of disorganised collagen fibrils between the epithelial basement membrane and corneal stroma. SEM images of the central and peripheral corneal surfaces, following removal of the corneal epithelium by cell maceration, revealed a disorganised meshwork of collagen fibrils, with a highly aligned annulus of collagen at the limbus. In between the peripheral cornea and limbus, a "transition zone" is observed where collagenfibrils start to align. Quantification of fibril alignment demonstrates a significant increase in collagen alignment from 0.08 ± 0.04 to 0.33 ± 0.07 (p < 0.001; n = 60; 0 = no alignment, 1 = full alignment) with increasing distance from the corneal centre. These data together lead us to conclude that the porcine cornea does include Bowman's layer, though it is thin (contributing roughly 0.2% of corneal thickness), and thus, reaffirms the porcine cornea's similarity to its human counterpart and usefulness as a model system

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YAP, ΔNp63, and ß-Catenin Signaling Pathways Are Involved in the Modulation of Corneal Epithelial Stem Cell Phenotype Induced by Substrate Stiffness.

Recent studies have established that the phenotype of epithelial stem cells residing in the corneal periphery (the limbus) depends on this niche's distinct biomechanical properties. However, the signaling pathways underlying this dependency are still poorly understood. To address this issue, we investigated the effect of substrate stiffness on the migration, proliferation, and molecular phenotype of human limbal epithelial stem cells (LESCs). Specifically, we demonstrated that cells grown on collagen-based substrates with limbus-like compliance showed higher proliferation and stratification and lower migration capabilities, as well as higher levels of pro-proliferative markers Ki67 and ß-Catenin, and LESC markers ΔNp63, ABCG2, and CK15. In contrast, cells on stiffer substrates lost these stem/progenitor cell markers, but instead expressed the key mechanotransduction factor YAP, as well as elevated levels of BMP4, a promotor of cell differentiation known to be negatively regulated by Wnt/ß-Catenin signaling. This data allowed us to propose a new model that integrates the various molecular pathways involved in LESC response to substrate stiffness. This model will potentially be a useful guide to future research on the mechanisms underlying LESC loss following fibrosis-causing injuries.

The role of corneo-scleral junction in soft contact lens fitting: Case report on nanophthalmos.

BACKGROUND: Nanophthalmos is rare developmental ocular condition characterized by a small eye with short axial length, high hyperopia and high lens to eye volume ratio due to arrested development of eye ball as a result of scleral inelasticity. OBSERVATIONS: A 33 year old woman who presented with a complaint of blurring of distance and near vision in both eyes since childhood came to LV Prasad Eye Institute on July 2017. Best corrected visual acuity was 20/60 using soft contact lens +23.50 diopters for the right and left eyes. Axial lengths of two eyes were markedly shortened along with steep corneal curvatures. Visante anterior segment ocular coherence tomography showed a steep (convex) corneo-scleral junction (CSJ) which might be the reason for ill-fitting with conventionally estimated soft contact lens (SCL) parameters. Finally, selection of the customized hydrogel soft contact lens base curve close to flatter corneal curvature and small diameter showed characteristics of optimal lens fit. CONCLUSION: The corneo-scleral junction profile plays significant role in soft contact lens fitting. An eye with a profile of convex CSJ would require a lens with steeper curvature compared to conventional measurements. Cases of nanophthalmos would require observation of the profile with the help of optical coherence tomography in addition to measurement of corneal curvature prior to fitting soft contact lenses.

Corneal epithelial stem cells for corneal injury.

INTRODUCTION: Ocular surface diseases with limbal insufficiency represent a therapeutic challenge for restoring vision. This corneal deficiency includes both classical ocular diseases (as chemical burns) and rare ocular diseases (as congenital aniridia and ocular cicatricial pemphigoid). AREAS COVERED: Our understanding of limbal epithelial stem cells (LESCs) has increased the potential for treatment options. Pharmacological treatment strategies (as regenerating agent ophthalmic solutions) and especially surgical treatment strategies are available. Isolated LESCs can be produced by limbal primary cultures obtained from explants or cell suspensions. We review the latest cornea surgery techniques. EXPERT OPINION: The adjunction of human limbal mesenchymal cells as a support for limbal stem cell primary cultures appears to be of great interest. Recently, human-induced pluripotent stem cells have allowed the generation of minicorneal organoids. This potential means of creating a three-dimensional cornea with in vitro maturation opens up important research areas for corneal regeneration therapy.

Decellularized corneal lenticule embedded compressed collagen: toward a suturable collagenous construct for limbal reconstruction.

Recently, compressed collagen has attracted much attention as a potential alternative for a limbal epithelial stem cell (LESC) carrier to treat limbal stem cell deficiency (LSCD), in that it can provide mechanically improved collagen fibrillar structures compared to conventional collagen hydrogel. However, its clinical efficacy as an LESC carrier has not yet been studied through in vivo transplantation due to limited mechanical strength that cannot withstand a force induced by surgical suturing and low resistance to enzymatic degradation. This study firstly presents a suturable LESC carrier based on compressed collagen in the form of a biocomposite. The biocomposite was achieved by integrating a decellularized corneal lenticule, which is a decellularized stromal tissue obtained from corneal refractive surgery, inside a compressed collagen to form a sandwich structure. A suture retention test verified that the biocomposite has a much higher suture retention strength (0.56 ± 0.12 N) compared to the compressed collagen (0.02 ± 0.01 N). The biocomposite also exhibited more than 3 times higher resistance to enzymatic degradation, indicating long-term stability after transplantation. In vitro cell culture results revealed that the biocomposite effectively supported the expansion and stratification of the LESCs with expressions of putative stem cell and differentiated corneal epithelial cell markers. Finally, the biocomposite verified its clinical efficacy by stably delivering the LESCs onto an eye of a rabbit model of LSCD and effectively reconstructing the ocular surface.

Influence of the Water-Drinking Test on Intraocular Pressure, Schlemm's Canal, and Autonomic Nervous System Activity.

Purpose: To assess changes in Schlemm's canal (SC), intraocular pressure (IOP), and autonomic nervous system activity in healthy individuals after performing the water-drinking test (WDT). Methods: The SC area (SCAR), trabecular meshwork (TM) thickness, IOP, high frequency (HF) of heart rate variability (HRV), heart rate (HR), and systolic (SBP) and diastolic blood pressure (DBP) were measured in 22 young healthy participants before and after the WDT, which involved drinking a 1-liter water load in 5 minutes.The SC and TM profiles were captured using a Spectralis optical coherence tomography device (anterior segment module). HF was recorded using Kubios HRV Premium software to evaluate parasympathetic nervous system activity. Results: Compared with baseline values, IOP increased significantly (14.9 ± 2.7 mm Hg vs. 18.4 ± 3.3 mm Hg; P < 0.001), whereas HF (1587 ± 930 ms2 vs. 2193 ± 863 ms2; P < 0.001), mean SCAR (6521 ± 1360 µm2 vs. 5180 ± 1455 µm2; P < 0.001), and HR (69 ± 9.7 beats/min vs. 63 ± 8.9 beats/min; P < 0.001) values decreased significantly by 15 minutes after water-loading. Least significant difference pairwise comparison revealed significant fluctuations of all parameters (SCAR, IOP, HF, and HR) at 15 minutes and their recovery at 30-minutes post-WDT. TM thickness, SBP, and DBP post-WDT did not differ significantly. The increase in IOP (r = -0.4047; P = 0.010) and HF (r = -0.386; P = 0.014) correlated significantly with the decrease in SCAR. Conclusions: The WDT may cause parasympathetic nervous system stimulation, leading to the collapse of SC, which leads to increased IOP.

Morphology of the extraocular muscles (m. bulbi) in the pre-hatchling and post-hatchling african black ostriches (struthio camelus domesticus L., 1758) (Aves: Struthioniformes).

The aim of the study was to describe the morphology and the development of the extraocular muscles (EOMs) in the pre-hatchling and post-hatchling African black ostrich. The study involved 50 birds aged between 28 days and 3 years. The EOMs were analyzed morphologically with respect to the location and length of the straight and oblique muscles and the third eyelid muscles, the length and breadth of their tendons as well as the distance and shape of the muscle tendon insertions at the corneal limbus. A histological and histometric analysis were also carried out. The greatest increase in the length of the EOMs was noted in groups III-V. A marked increase in the length of the tendons of the dorsal straight muscle was found in groups II and III, in the tendons of the nasal straight muscle in groups IV and V, in the tendons of the dorsal oblique muscle in groups III to V and in the tendons of the ventral oblique muscle in groups IV and V. There was a significant increase in the breadth of the dorsal straight and ventral oblique muscle tendons in groups IV and V and the tendons of the pyramidal muscle in groups III and V. The distance of the distal insertion of the tendon at the corneal limbus increased steadily with age in all the examined groups. The number of fascicles and muscle fibres, their diameter and length in all the studied EOMs were different in the different groups.

Diurnal Variations in the Morphology of Schlemm's Canal and Intraocular Pressure in Healthy Chinese: An SS-OCT Study.

Purpose: To characterize the diurnal variations in the dimensions of the Schlemm's canal (SC) and its association with intraocular pressure (IOP) using swept-source optical coherence tomography (SS-OCT). Methods: The temporal, nasal, inferior, and superior limbus of 102 eyes of 51 healthy subjects were imaged in vivo by SS-COT at 5 time points of 8 AM, 11 AM, 2 PM, 5 PM, and 8 PM. IOP was measured at the same time by Goldmann applanation tonometry (GAT). The diameter and the cross-sectional area of the SC were measured in ImageJ. The associations between changes in the SC parameters, IOP, and other biometric parameters were determined using a general estimating equations model. The temporal and inferior limbus of 94 eyes of 47 healthy subjects were also imaged before and during the Valsalva maneuver (VM) at 8 PM. Results: Mean IOPs at different time points were 13.37, 12.89, 11.9, 12.02, and 12.36 mm Hg. Of all four quadrants, the detectable rate of SC was highest in the superior quadrant (85.3%) and lowest in the inferior quadrant (75.5%). We found that changes in the SC area and diameter were negatively associated with IOP changes only in the inferior quadrant (P = 0.0046 and P = 0.0332, respectively), after adjusting for age, sex, eye, spherical equivalent, and axial length. The mean SC area and diameter during the VM were significantly higher than prior to the VM (P < 0.001). Conclusions: The changes in the SC parameters were negatively associated with IOP changes only in the inferior quadrant. The VM could expand the SC in healthy subjects. Imaging of the SC may be a useful method to discover the reason why IOP fluctuates, and how SC changes morphologically during the daytime in the future.

The Relationship between the 24-hour Fluctuations in Schlemm's Canal and Intraocular Pressure: An Observational Study using High-Frequency Ultrasound Biomicroscopy.

PURPOSE: To assess 24-hour fluctuations in Schlemm's canal (SC) parameters (cross-sectional area, perimeter) and intraocular pressure (IOP) and the relationship between these fluctuations in healthy individuals. METHODS: SC and IOP were examined in 29 participants at 2:30, 5:30, 11:30, 17:30, and 23:30 within one day. The superior, inferior, nasal, and temporal SC quadrants were evaluated using 80-MHz ultrasound biomicroscopy. RESULTS: SC parameters and IOP fluctuated significantly within 24 hours (all P < 0.05). After age, gender, axial length, and central corneal thickness were adjusted, compared with the baseline (23:30) value, the change in SC cross-sectional area was negatively associated with the change in IOP at 2:30, 5:30, 11:30, and 17:30 (ß = -0.072[-0.094, -0.049], -0.070[-0.102, -0.038], -0.046[-0.079, -0.013], and -0.033[-0.062, -0.004], respectively; P < 0.001, < 0.001, = 0.009, and = 0.028, respectively). The nasal (175.6 ± 36.0 pixels) and inferior (174.8 ± 36.0 pixels) SC cross-sectional areas were significantly larger than the superior area (156.2 ± 27.1 pixels) (P = 0.018 and 0.048, respectively) at 23:30. The observable SC proportion did not change among the quadrants or measurement time points (all P > 0.05). CONCLUSIONS: SC cross-sectional area fluctuated throughout the day and was negatively associated with changes in IOP.

Limbal and corneal epithelial homeostasis.

PURPOSE OF REVIEW: The aim of this review is to describe the underlying mechanisms of corneal epithelial homeostasis in addition to illustrating the vital role of the limbal epithelial stem cells (LESCs) and the limbal niche in epithelial regeneration and wound healing. RECENT FINDINGS: The shedded corneal epithelial cells are constantly replenished by the LESCs which give rise to epithelial cells that proliferate, differentiate, and migrate centripetally. While some recent studies have proposed that epithelial stem cells may also be present in the central cornea, the predominant location for the stem cells is the limbus. The limbal niche is the specialized microenvironment consisting of cells, extracellular matrix, and signaling molecules that are essential for the function of LESCs. Disturbances to limbal niche can result in LESC dysfunction; therefore, limbal stem cell deficiency should also be considered a limbal niche deficiency. Current and in-development therapeutic strategies are aimed at restoring the limbal niche, by medical and/or surgical treatments, administration of trophic factors, and cell based therapies. SUMMARY: The corneal epithelium is constantly replenished by LESCs that are housed within the limbal niche. The limbal niche is the primary determinant of the LESC function and novel therapeutic approaches should be focused on regeneration of this microenvironment.

Ocular Surface Temperature During Scleral Lens Wearing in Patients With Keratoconus.

OBJECTIVE: To evaluate the ocular surface temperature using an infrared thermography camera before and after wearing scleral lens in patients with keratoconus and correlate these results with the tear production and stability. METHODS: A pilot, experimental, short-term study has been performed. Twenty-six patients with keratoconus (36.95±8.95 years) participated voluntarily in the study. The sample was divided into two groups: patients with intrastromal corneal ring (KC-ICRS group) and patients without ICRS (KC group). Schirmer test, tear breakup time (TBUT), and ocular surface temperature in the conjunctiva, limbus, and cornea were evaluated before and after wearing a scleral lens. RESULTS: The patients wore the scleral lenses from 6 to 9 hours with average of 7.59±0.73 hours. No significant changes in Schirmer test and TBUT were found for both groups. No temperature differences were found between the KC-ICRS and the KC groups for all zones evaluated. There was a slight, but statistically significant, increase in the inferior cornea, temporal limbus, and nasal conjunctival temperature for KC-ICRS group and temporal limbus temperature decreasing for the KC group after wearing scleral lens (P<0.05). The conjunctiva and limbus temperature was statistically higher than the central cornea for both groups before and after scleral lenses wearing (P<0.05), but no difference in the peripheral cornea was found. No statistically significant differences in the central corneal temperature were found between the groups after scleral lens wearing (P>0.05). CONCLUSION: Scleral contact lens seems not to modify the ocular surface temperature despite the presence of the tear film stagnation under the lens.

Preservation of Ocular Epithelial Limbal Stem Cells: The New Frontier in Regenerative Medicine.

Significant advances have been made in the field of ocular regenerative medicine. Promising stem cell-based therapeutic strategies have been translated into the clinical practice over the last few decades. These new stem cell-based therapies offer the possibility of permanently restoring corneal epithelium in patients with severe disabling and blinding ocular surface disease. The European Union has already classified stem cell-based therapies as "medicinal products". Therefore, manipulation is strictly regulated according to the defined conditions of good manufacturing practice, with the production of stem cell therapeutics at only accredited production sites authorized by the national regulatory agencies. In this regard, as first medical products are licensed for commercial use in Europe enabling a more widespread access to a stem cell-based therapy, the need for safe, validated and reproducible techniques for ex vivo cultured tissue preservation and distribution are coming to the forefront of research. However, these provide various new challenges for biobanking industry such as the retention of viability, good functionality of stem cells and sterility issues. This chapter provides an overview of the current advances in the field of corneal/limbal epithelial stem cell culture preservation techniques using either hypothermic storage or cryopreservation methods, that were used in different culturing steps (from stem cell isolation to the ex vivo epithelial graft preparation), with the reported impact on the post-thawing product recovery.