Redox state sustained by reactive oxygen species (ROS) is crucial for regeneration; however, the interplay between oxygen (O₂), ROS and hypoxia-inducible factors (HIF) remains elusive. Here we observe, using an optic-based probe (optrode), an elevated and steady O₂ influx immediately upon amputation. The spatiotemporal O₂ influx profile correlates with the regeneration of Xenopus laevis tadpole tails. Inhibition of ROS production but not ROS scavenging decreases O₂ influx. Inhibition of HIF-1α impairs regeneration and stabilization of HIF-1α induces regeneration in the refractory period. In the regeneration bud, hypoxia correlates with O₂ influx, ROS production, and HIF-1α stabilization that modulate regeneration. Further analyses reveal that heat shock protein 90 is a putative downstream target of HIF-1α while electric current reversal is a de facto downstream target of HIF-1α. Collectively, the results show a mechanism for regeneration via the orchestration of O₂ influx, ROS production, and HIF-1α stabilization.

Reactive molecular oxygen (O₂) plays important roles in bioenergetics and metabolism and is implicated in biochemical pathways underlying angiogenesis, fertilization, wound healing and regeneration. Here we describe how to use the scanning micro-optrode technique (SMOT) to measure extracellular fluxes of dissolved O₂. The self-referencing O₂-specific micro-optrode (also termed micro-optode and optical fiber microsensor) is a tapered optical fiber with an O₂-sensitive fluorophore coated onto the tip. The O₂ concentration is quantified by fluorescence quenching of the fluorophore emission upon excitation with blue-green light. The micro-optrode presents high spatial and temporal resolutions with improved signal-to-noise ratio (in the picomole range). In this protocol, we provide step-by-step instructions for micro-optrode calibration, validation, example applications and data analysis. We describe how to use the technique for cells (Xenopus oocyte), tissues (Xenopus epithelium and rat cornea), organs (Xenopus gills and mouse skin) and appendages (Xenopus tail), and provide recommendations on how to adapt the approach to different model systems. The basic, user-friendly system presented here can be readily installed to reliably and accurately measure physiological O₂ fluxes in a wide spectrum of biological models and physiological responses. The full protocol can be performed in ~4 h.

Reactive molecular oxygen (O₂) plays important roles in bioenergetics and metabolism and is implicated in biochemical pathways underlying angiogenesis, fertilization, wound healing and regeneration. Here we describe how to use the scanning micro-optrode technique (SMOT) to measure extracellular fluxes of dissolved O₂. The self-referencing O₂-specific micro-optrode (also termed micro-optode and optical fiber microsensor) is a tapered optical fiber with an O₂-sensitive fluorophore coated onto the tip. The O₂ concentration is quantified by fluorescence quenching of the fluorophore emission upon excitation with blue-green light. The micro-optrode presents high spatial and temporal resolutions with improved signal-to-noise ratio (in the picomole range). In this protocol, we provide step-by-step instructions for micro-optrode calibration, validation, example applications and data analysis. We describe how to use the technique for cells (Xenopus oocyte), tissues (Xenopus epithelium and rat cornea), organs (Xenopus gills and mouse skin) and appendages (Xenopus tail), and provide recommendations on how to adapt the approach to different model systems. The basic, user-friendly system presented here can be readily installed to reliably and accurately measure physiological O₂ fluxes in a wide spectrum of biological models and physiological responses. The full protocol can be performed in ~4 h.

The posterior face of the cornea consists of the corneal endothelium, a monolayer of cuboidal cells that secrete and attach to Descemet's membrane, an exaggerated basement membrane. Dysfunction of the endothelium compromises the barrier and pump functions of this layer that maintain corneal deturgesence. A large number of corneal endothelial dystrophies feature irregularities in Descemet's membrane, suggesting that cells create and respond to the biophysical signals offered by their underlying matrix. This review provides an overview of the bidirectional relationship between Descemet's membrane and the corneal endothelium. Several experimental methods have characterized a richly topographic and compliant biophysical microenvironment presented by the posterior surface of Descemet's membrane, as well as the ultrastructure and composition of the membrane as it builds during a lifetime. We highlight the signaling pathways involved in the mechanotransduction of biophysical cues that influence cell behavior. We present the specific example of Fuchs' corneal endothelial dystrophy as a condition in which a dysregulated Descemet's membrane may influence the progression of disease. Finally, we discuss some disease models and regenerative strategies that may facilitate improved treatments for corneal dystrophies.

Purpose

Fuchs endothelial corneal dystrophy (FECD) is a progressive corneal disease that impacts the structure and stiffness of the Descemet's membrane (DM), the substratum for corneal endothelial cells (CECs). These structural alterations of the DM could contribute to the loss of the CECs resulting in corneal edema and blindness. Oxidative stress and transforming growth factor-β (TGF-β) pathways have been implicated in endothelial cell loss and endothelial to mesenchymal transition of CECs in FECD. Ascorbic acid (AA) is found at high concentrations in FECD and its impact on CEC survival has been investigated. However, how TGF-β and AA effect the composition and rigidity of the CEC's matrix remains unknown.

Methods

In this study, we investigated the effect of AA, TGF-β1 and TGF-β3 on the deposition, ultrastructure, stiffness, and composition of the extracellular matrix (ECM) secreted by primary bovine corneal endothelial cells (BCECs).

Results

Immunofluorescence and electron microscopy post-decellularization demonstrated a robust deposition and distinct structure of ECM in response to treatments. AFM measurements showed that the modulus of the matrix in BCECs treated with TGF-β1 and TGF-β3 was significantly lower than the controls. There was no difference in the stiffness of the matrix between the AA-treated cell and controls. Gene Ontology analysis of the proteomics results revealed that AA modulates the oxidative stress pathway in the matrix while TGF-β induces the expression of matrix proteins collagen IV, laminin, and lysyl oxidase homolog 1.

Conclusions

Molecular pathways identified in this study demonstrate the differential role of soluble factors in the pathogenesis of FECD.

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFβ) signaling. Initiation of the TGFβ signaling cascade results from binding of TGFβ to the labile type I TGFβ receptor (TGFβRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFβ signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence. In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFβ1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed. In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFβ1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls. Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.

Laser-induced experimental glaucoma (ExGl) in non-human primates (NHPs) is a common animal model for ocular drug development. While many features of human hypertensive glaucoma are replicated in this model, structural and functional changes in the unlasered portions of trabecular meshwork (TM) of laser-treated primate eyes are understudied. We studied NHPs with ExGl of several years duration. As expected, ExGl eyes exhibited selective reductions of the retinal nerve fiber layer that correlate with electrophysiologic measures documenting a link between morphologic and elctrophysiologic endpoints. Softening of unlasered TM in ExGl eyes compared to untreated controls was observed. The degree of TM softening was consistent, regardless of pre-mortem clinical findings including severity of IOP elevation, retinal nerve fiber layer thinning, or electrodiagnostic findings. Importantly, this softening is contrary to TM stiffening reported in glaucomatous human eyes. Furthermore, microscopic analysis of unlasered TM from eyes with ExGl demonstrated TM thinning with collapse of Schlemm's canal; and proteomic analysis confirmed downregulation of metabolic and structural proteins. These data demonstrate unexpected and compensatory changes involving the TM in the NHP model of ExGl. The data suggest that compensatory mechanisms exist in normal animals and respond to elevated IOP through softening of the meshwork to increase outflow.

Re-epithelialization of wounds is a critical element of wound closure. Growth factors have been used in combination with conventional wound management to promote closure, but the method of delivery has been limited to the topical application of ointment formulations. Cytoactive factors delivered in this way have short resident times in wounds and have met with limited success. Here, we demonstrate that methods used to covalently immobilize proteins on synthetic materials can be extended to immobilize cytoactive factors such as the epidermal growth factor (EGF) onto the wound beds of genetically diabetic mice that exhibit impaired healing. Full-thickness splinted excisional wounds were created in diabetic (db/db) mice with a well-defined silicone splint to limit wound contracture. Wound surfaces were treated with a reducing agent to expose sulfhydryl groups and subsequently treated with EGF modified with a heterobifunctional crosslinker. This allowed for the covalent immobilization of the EGF to the wound surface. The conjugation chemistry was validated in vitro and in vivo. In a separate group of mice, wounds were topically treated twice daily with soluble EGF. The mice were evaluated over 11 days for wound closure. This covalent immobilization strategy resulted in EGF being retained on the wound surface for 2 days and significantly increased epithelial wound closure by 20% compared to wounds treated with topical EGF or topical vehicle. Covalent immobilization was not only therapeutically effective but also delivered a markedly reduced load of growth factor to the wound surface compared to topical application (when only 180 ng of EGF was immobilized onto the wound surface in comparison with 7200 ng of topically applied EGF over a period of 11 days). No adverse effects were observed in treated wounds. Results obtained provide proof of concept for the effectiveness of covalent immobilization in the treatment of dysregulated wounds. The covalent immobilization of cytoactive factors represents a potentially transformative approach to the management of difficult chronic wounds.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.