Genipin delays corneal stromal enzymatic digestion

Purpose: To evaluate the use of genipin in delaying enzymatic digestion of corneal stroma. Methods: Human corneal stromal tissue was treated with genipin, a known chemical crosslinker, and then along with control tissue was subjected to enzymatic digestion with collagenase. The effects of genipin treatment in retarding stromal digestion were analyzed with phase contrast microscopy, a protein quantification assay, second harmonic generation imaging, and transmission electron microscopy. Results: Genipin increased stromal resistance to enzymatic digestion when compared with untreated stroma. A morphologic analysis and protein quantification showed increased stromal resistance to enzymatic digestion once stromal tissue was treated with genipin. Second harmonic generation imaging revealed persistent fibrillar collagen signaling in genipin-treated tissue in contrast with untreated tissue suggesting that genipin retards collagenolysis. Conclusions: Genipin increases stromal resistance to enzymatic digestion in controlled experiments as demonstrated by protein quantification studies and through morphologic imaging. Translational Relevance: This study explores the novel use of genipin in delaying enzymatic stromal digestion. Delaying stromal melting in the setting of corneal infectious or autoimmune keratitis can potentially decrease clinical morbidity

Evolution of the vertebrate corneal stroma

Although the cornea is the major refractive element of the eye, the mechanisms controlling corneal shape and hence visual acuity remain unknown. To begin to address this question we have used multiphoton, non-linear optical microscopy to image second harmonic generated signals (SHG) from collagen to characterize the evolutionary and structural changes that occur in the collagen architecture of the corneal stroma. Our studies show that there is a progression in complexity of the stromal collagen organization from lower (fish and amphibians) to higher (birds and mammals) vertebrates, leading to increasing tissue stiffness that may control shape. In boney and cartilaginous fish, the cornea is composed of orthogonally arranged, rotating collagen sheets that extend from limbus to limbus with little or no interaction between adjacent sheets, a structural paradigm analogous to 'plywood'. In amphibians and reptiles, these sheets are broken down into broader lamellae that begin to show branching and anastomosing with adjacent lamellae, albeit maintaining their orthogonal, rotational organization. This paradigm is most complex in birds, which show the highest degree of lamellar branching and anastomosing, forming a 'chicken wire' like pattern most prominent in the midstroma. Mammals, on the other hand, diverged from the orthogonal, rotational organization and developed a random lamellar pattern with branching and anastomosing appearing highest in the anterior stroma, associated with higher mechanical stiffness compared to the posterior stroma

Chondroitin sulphate/dermatan sulphate proteoglycans: potential regulators of corneal stem/progenitor cell phenotype in vitro

Chondroitin sulphate (CS) proteoglycans with variable sulphation-motifs along their glycosaminoglycan (GAG) chains are closely associated with the stem cell niche of articular cartilage, where they are believed to influence the characteristics of the resident stem cells. Here, we investigated the immunohistochemical distribution of hybrid CS/dermatan sulphate (DS) GAGs in the periphery of the adult chicken cornea, which is the location of the cornea’s stem cell niche in a number of species, using a monoclonal antibody, 6C3, that recognises a sulphation motif-specific CS/DS GAG epitope. This revealed positive labelling that was restricted to the subepithelial corneal stroma, as well as nearby bony structures within the sclera, called ossicles. When cultivated on cell culture dishes coated with 6C3-rich CS/DS, corneal stromal cells (keratocytes) that had been isolated from embryonic chicken corneas formed circular colonies, which took several days to reach confluency. A flow cytometric analysis of these keratocytes revealed changes in their expression levels of the indicative stem cell markers, Connexin 43 (Cx43), Paired Box 6 (PAX6), B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1), and C-X-C Chemokine Receptor 4 (CXCR4) suggestive of a less-differentiated phenotype compared with expression levels in cells not exposed to CS/DS. These findings support the view that CS/DS promotes the retention of a stem cell phenotype in corneal cells, much as it has been proposed to do in other connective tissues

Neuroprotective properties of levosimendan in an in vitro model of traumatic brain injury

<p>Abstract</p> <p>Background</p> <p>We investigated the neuroprotective properties of levosimendan, a novel inodilator, in an in vitro model of traumatic brain injury.</p> <p>Methods</p> <p>Organotypic hippocampal brain slices from mouse pups were subjected to a focal mechanical trauma. Slices were treated after the injury with three different concentrations of levosimendan (0.001, 0.01 and 0.1 μM) and compared to vehicle-treated slices. After 72 hrs, the trauma was quantified using propidium iodide to mark the injured cells.</p> <p>Results</p> <p>A significant dose-dependent reduction of both total and secondary tissue injury was observed in cells treated with either 0.01 or 0.1 μM levosimendan compared to vehicle-treated slices.</p> <p><b>Conclusion</b></p> <p>Levosimendan represents a promising new pharmacological tool for neuroprotection after brain injury and warrants further investigation in an in vivo model.</p

Chondroitin sulfate as a potential modulator of the stem cell niche in cornea

Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells

Ultrastructural variability of the juxtacanalicular tissue along the inner wall of Schlemm's canal.

Purpose: Increased resistance of aqueous humor drainage from the eye through Schlemm's canal (SC) is the basis for elevated intraocular pressure in glaucoma. Experimental evidence suggests that the bulk of outflow resistance lies in the vicinity of the inner wall endothelial lining of SC and the adjacent juxtacanalicular tissue (JCT). However, there is little understanding of how this resistance is generated, and a detailed understanding of the structure-function relationship of the outflow pathway has not been established yet. In the present study, regional variations in the ultrastructure of the JCT and the inner wall of SC were investigated in three dimensions. Methods: With the use of serial block face scanning electron microscopy (SBF-SEM), the volume occupied by the electron lucent spaces of the JCT compared to that occupied by the cellular and extracellular matrix was investigated and quantified. The distribution of giant vacuoles (GVs) and pores in the inner wall endothelium of SC was further examined. Results: With increasing distance from the inner wall of SC, the volume of the electron lucent spaces increased above 30%. In contrast, the volume of these spaces in immediate contact with the inner wall endothelium was minimal (<10%). Circumferential variability in the type and distribution of GVs was observed, and the percentage of GVs with pores varied between 3% and 27%. Conclusions: These studies provide a detailed quantitative analysis of the ultrastructure of JCT and the distribution of GVs along the circumference of SC in three dimensions, supporting the non-uniform or segmental aqueous outflow

Video-laryngoscopes in the adult airway management: a topical review of the literature

The aim of the present paper is to review the literature regarding video-laryngoscopes (Storz V-Mac and C-Mac, Glidescope, McGrath, Pentax-Airway Scope, Airtraq and Bullard) and discuss their clinical role in airway management. Video-laryngoscopes are new intubation devices, which provide an indirect view of the upper airway. In difficult airway management, they improve Cormack-Lehane grade and achieve the same or a higher intubation success rate in less time, compared with direct laryngoscopes. Despite the very good visualization of the glottis, the insertion and advancement of the endotracheal tube with video-laryngoscopes may occasionally fail. Each particular device’s features may offer advantages or disadvantages, depending on the situation the anaesthesiologist has to deal with. So far, there is inconclusive evidence indicating that video-laryngoscopy should replace direct laryngoscopy in patients with normal or difficult airways