Endothelial Pump Research Articles

NH3/NH4 + allosterically activates SLC4A11 by causing an acidic shift in the intracellular pK that governs H+(OH-) conductance.

SLC4A11 is the most abundant membrane transport protein in corneal endothelial cells. Its functional presence is necessary to support the endothelial fluid pump that draws fluid from the corneal stroma, preventing corneal edema. Several molecular actions have been proposed for SLC4A11 including H2O transport and cell adhesion. One of the most reproduced actions that SLC4A11 mediates is a H+ (or OH-) conductance that is enhanced in the presence of NH4Cl. The mechanism by which this occurs is controversial with some providing evidence in favor of NH3-H+ cotransport and others providing evidence for uncoupled H+ transport that is indirectly stimulated by the effects of NH4Cl upon intracellular pH and membrane potential. In the present study we provide new evidence and revisit previous studies, to support a model in which NH4Cl causes direct allosteric activation of SLC4A11 by means of an acidic shift in the intracellular pK (pKi) that governs the relationship between intracellular pH (pHi) and SLC4A11 H+-conductance. These findings have important implications for the assignment of a physiological role for SLC4A11.

Characterization of a Novel Mouse Model for Fuchs Endothelial Corneal Dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a progressive blinding disorder, characterized by increased corneal endothelial excrescences (guttae), corneal endothelial cell loss, and edema. These symptoms are hypothesized to be caused by changes in the extracellular matrix (ECM) and mitochondrial dysfunction in the corneal endothelium. Despite this clinical and biological relevance, a comprehensive animal model that recapitulates all the major disease characteristics is currently unavailable. In this study, we develop such a model to improve our understanding of the signaling pathways involved in the FECD progression and develop strategies for early intervention. To generate a comprehensive FECD model, we generated a double mutant mouse bearing tamoxifen-inducible knockdown of Slc4a11 and the Col8a2 (Q455K) mutation. We performed optical coherence tomography (OCT) and in vivo confocal microscopy using the Heidelberg Retinal Tomography 3 - Rostock Cornea module (HRT3-RCM) on the mice at 5 weeks of age before tamoxifen feeding to establish baseline values for corneal thickness, endothelial cell density, and test for the presence of guttae. We measured these parameters again post-tamoxifen treatment at 16 weeks of age. We collected corneas at 16 weeks to perform histopathology, immunofluorescence staining for tight junctions, adherens junctions, and oxidative stress. We evaluated endothelial pump function using a lactate assay. The double mutant tamoxifen-fed animals showed the presence of guttae, and displayed increased corneal thickness and decreased endothelial cell density. Endothelial cells showed altered morphology with disrupted adherens junctions and elevated reactive oxygen species (ROS). Finally, we found that stromal lactate concentrations were elevated in the double mutant mice, indicative of compromised endothelial pump function. Overall, this mouse model recapitulates all the important phenotypic features associated with FECD.

Topical application of calcitonin gene-related peptide as a regenerative, antifibrotic, and immunomodulatory therapy for corneal injury

Calcitonin gene-related peptide (CGRP) is a multifunctional neuropeptide abundantly expressed by corneal nerves. Using a murine model of corneal mechanical injury, we found CGRP levels in the cornea significantly reduced after injury. Topical application of CGRP as an eye drop accelerates corneal epithelial wound closure, reduces corneal opacification, and prevents corneal edema after injury in vivo. CGRP promotes corneal epithelial cell migration, proliferation, and the secretion of laminin. It reduces TGF-β1 signaling and prevents TGF-β1-mediated stromal fibroblast activation and tissue fibrosis. CGRP preserves corneal endothelial cell density, morphology, and pump function, thus reducing corneal edema. Lastly, CGRP reduces neutrophil infiltration, macrophage maturation, and the production of inflammatory cytokines in the cornea. Taken together, our results show that corneal nerve-derived CGRP plays a cytoprotective, pro-regenerative, anti-fibrotic, and anti-inflammatory role in corneal wound healing. In addition, our results highlight the critical role of sensory nerves in ocular surface homeostasis and injury repair.

Comparison of the Central Corneal Thickness between a Scheimpflug Imaging-Based Device and an Optical Measurement-Based Device

Background Central corneal thickness is a critical parameter in corneal evaluation for a wide range of purposes in ophthalmology such as refractive surgeries including LASIK, cataract surgeries to assess endothelial pump function preoperatively to preserve the corneal transparency, and glaucoma in terms of diagnosis, management and follow up to avoid over/underestimation of intraocular pressure due to variations in CCT. Aim of the Work The study will investigate the discrepancy in measuring the CCT between Scheimpflug imaging using the Nidek AL-Scan and optical pachymetry using the Nidek Specular Microscope CEM-530. Patients and Methods In our study, we compared CCT measurements of both devices among 70 eyes of 35 normal subjects with a nonsignificant past ocular history. All subjects attended Ophthalmology Department at Ain Shams University Hospitals and all measurements were taken by the same examiner at the same time of the day. Our study is an observational comparative crosssectional study for six months in Ain Shams University Hospitals, Cairo, Egypt after approval of the research ethical committee on normal people without any history of ocular pathology except for refractive errors. Results The mean age of the study subjects was 29.49 ± 7.98 years (range 20-48 years). Regarding age, sex, and refraction, there was no statistically significant difference between CCT measurements by each individual device. However, there was a statistically significant difference between CCT measurements when compared by both devices. The difference between the mean ± SD of both devices was 7.17 ± 0.98 µm. Conclusion There is a statistically significant difference in central corneal thickness measurement between Nidek AL-Scan and Nidek Specular Microscope CEM-530. Several studies are needed to further correlate CCT with other parameters measured by both devices, namely anterior chamber depth and axial length.

Perspiratio Insensibilis of the Cornea

Introduction: The deswelling of the cornea via the natural pathways of perspiratio insensibilis and endothelial pump has been known for a long time. However, reliable data on the amount of perspiratio insensibilis, especially in edematous corneas, have been lacking until now. Material and Methods: In the Ex Vivo Eye Irritation Test (EVEIT), corneas can be observed purely physically under defined and stable biochemical conditions. The thickness and temperature of the corneas is measured by infrared thermography and Optical Coherence Tomography (OCT). Three corneas in stable MEM EVEIT culture and three corneas with induced and reversible edema in an EVEIT osmolar deficiency culture were placed in at 20°C and 60% humidity while tracking thickness and temperature for 30 minutes. Results: Over a period of 30 minutes, the temperature of both, healthy and edematous EVEIT corneas decreased from an initial 28°C to 21°C. The corneal thickness decreased by 51 ±12 μm in the healthy corneas and by 131 ±16 μm in the edematouscorneas. Conclusion: The considerable thickness differences between healthy and swollen corneas might give a physical and physiological explanation of some observable clinical effects in Fuchs’s endothelial dystrophy. The clinically observed effect of clearing of the cornea during the waking period. Furthermore, some phenomena of Dry Eye Syndrome (DES) can be explained by this difference of increased water release from the eye.

Comparison of central corneal thickness using anterior segment optical coherence tomography, ultrasound pachymetry, and orbscan

Background The thickness of the cornea is a sensitive and important indicator for the health of the cornea. It is beneficial for evaluating endothelial pump function and corneal barrier, monitoring the disorders of the cornea such as keratoconus and corneal edema, and choosing patients for refractive surgery. Aim The aim of this work was to compare central corneal thickness (CCT) using orbscan, ultrasound pachymetry (USP), and anterior segment optical coherence tomography (AS-OCT). Patients and methods This prospective study was carried out on 60 eyes of 30 individuals aged from 18 to 40 years old and with clinically normal cornea who attended the outpatient clinic of Tanta University Hospital and Nour El Ein Hospital from April 2018 till March 2019. Results There was no statistically significant difference in mean minimal measurement of the thickness of the central cornea as measured by the three devices. There was a statistically significant difference in the mean maximal measurement of the CCT as detected by the three devices. The average CCT was statistically significantly different across the three devices. Conclusions OCT, USP, and orbscan were reliable for CCT measurement. Orbscan and USP were more reproducible, but OCT and ultrasound were more technically dependent. The average CCT was the highest by orbscan.

Again about carbonic anhydrase inhibitors: modern aspects of the use of ICA in ophthalmology

For more than 70 years, the inhibitors of carbonic anhydrase (ICA) have been used in the treatment of glaucoma. Since 1995, topical forms of ICA have appeared among antiglaucoma drugs — dorzolamide, and since 1999 — brinzolamide. The hypotensive efficacy and safety of topical ICA gave rise to the widespread use of these drugs in the treatment of various forms of glaucoma. The peculiarities of the mechanism of action made it possible to create on their basis effective fixed combinations, which allow reducing the intraocular pressure by up to 40 % in patients with glaucoma. The attractiveness of the drug has expanded due to the effect of ICA on various tissues of the organ of vision. Thus, ICAs effectively improve the hemodynamics of the eye and optic nerve. The antioxidant activity of the drug makes it possible to expand the indications for its use in patients with vascular patho-logy of the retina and optic nerve. The biomechanical characte-ristics of the drug allowed its effective and safe use in the complex treatment of macular edema of various etiologies. The adjuvant use of topical ICAs expands the possibilities of anti-VEGF therapy in the treatment of macular edema of various etiologies. Features of the action of ICA on the endothelial pump function of the cornea made it possible to formulate the rules for the use of topical ICA in patients with an insufficient function of the corneal endothelium.

Spontaneous lens luxation into anterior chamber: why is it important to intervene immediately?

A 70-year-old woman, with a history of cataract surgery 2 years ago, presented to the ophthalmology clinic with 3 months of decreased vision, pain and redness in his right eye. Visual acuity was limited to hand motion and intraocular pressure was normal. Slit lamp examination showed a lens luxation into the lower part of the anterior chamber, diffuse corneal edema and corneal neovascularization. Fundus examination was unremarkable. Specular microscopy showed severe endothelial cell loss in the right eye and moderate in the left eye, testifying preexistent corneal endothelial dystrophy. Anterior segment optical coherence tomography showed increased thickness in the affected eye. Postural measures have been recommended, and the lens implant was extracted rapidly. Endothelial keratoplasty was performed later. Corneal edema may occur due to endothelial pump failure because of the direct contact between the corneal endothelium and the lens. Others causes have been incriminated such as chemical injury, infection, inflammation, corneal dystrophy. The management of corneal edema depends upon the underlying cause and visual potential, starting from the medical management to penetrating keratoplasty. This case highlighted the importance of early management in case of lens luxation into the anterior chamber (AC) in order to minimize the risk of endothelial cell loss.

RNA sequencing uncovers alterations in corneal endothelial metabolism, pump and barrier functions of Slc4a11 KO mice

Slc4a11 KO mice show significant corneal edema, altered endothelial morphology, and mitochondrial ROS at an early age without a decrease in endothelial cell density. We examined the differential gene expression profile between wild type (WT) and KO with the goal of finding pathways related to corneal endothelial metabolic, pump and barrier function that can explain the corneal edema. Freshly dissected Corneal Endothelium-Descemet's Membrane (CEDM) and cultured Mouse Corneal Endothelial Cells (MCEC) were obtained from WT and Slc4a11 KO mice. RNA sequencing Ingenuity Pathway Analysis (IPA) predicted activation, inhibition or differential regulation of several pathways. QPCR and Western analysis validated downregulation of Glycolytic enzymes, Mitochondrial complex components and Ion transporters. Functional testing revealed decreases in endothelial lactate production, Extracellular Acidification Rate (ECAR), glutaminolysis, and Oxygen Consumption Rate (OCR) of KO CEDM in the presence of Glutamine (Gln) that was not compensated by fatty acid oxidation. Stromal lactate was significantly elevated in KO along with decreased expression of MCT1 and MCT4 lactate transporters in endothelial cells. ATP levels were 2x higher in KO CEDM, concomitant with a 3-fold decrease in Na-K-ATPase activity and reduced basolateral membrane localization. Genes for cholesterol biosynthesis, glutathione metabolism and tight and adherens junctions were elevated. Alteration of tight junction structure and cortical cytoskeleton is evident in KO corneal endothelium with a significant increase in trans-endothelial fluorescein permeability. We conclude that Slc4a11 KO induces a coordinated decrease in glycolysis, glutaminolysis, lactate transport and Na-K-ATPase activity. These changes together with an altered barrier function cause an accumulation of stromal lactate in Slc4a11 KO mice leading to chronic corneal edema.

Aqueous humour cytokine profiles after Descemet\u2019s membrane endothelial keratoplasty

The aim of this study was to compare aqueous humour (AqH) cytokine profiles before and after Descemet’s membrane endothelial keratoplasty (DMEK) in eyes with bullous keratopathy (BK) and age-matched normal eyes. A total of 49 participants (mean age 75.0 ± 13.5 years) were divided into three groups: (1) BK patients scheduled for DMEK (BK group); (2) patients after DMEK (DMEK group; average postoperative time 1,018 ± 460 days); and (3) control participants without corneal endothelial disease scheduled for cataract surgery (control group). AqH (0.2 mL) was collected from all participants, and the levels of various AqH cytokines were simultaneously measured using a multiplex bead immunoassay. The DMEK group exhibited significantly lower concentrations of several pro-inflammatory cytokines, such as IL-1β, IL-5, IL-6, IL-10, and IL-8, and granulocyte colony stimulating factor than the BK group. In addition, the levels of IL-1β and IL-5 were significantly lower in the DMEK group than in the control group. The AqH levels of several pro-inflammatory cytokines were significantly reduced after DMEK, indicating that regeneration of the endothelial pump owing to the transplantation of a healthy endothelium might have a positive effect on anterior chamber inflammation.

Non invasive backscattering light to detection of endothelial cells activity for graft sorting: Proof of consent

In tissue bank, one of the key steps in the corneal grafts sorting is the analysis of the deepest cell layer of the cornea, the endothelium. This analysis consists of counting the density of living cells using trypan blue staining. However, this dye is toxic and the measurement is localized. Then no information is given on the cell’s viability of the entire cell layer. This preliminary study proposes to track the kinetics of variation of scattered light during the deturgescence process. The backscattered light of two different sets of cornea, with and without endothelial cells, are measured and the decrease of this parameter as a function of the deturgescence time are different. This noninvasive technique could be used as a criterion for attesting the presence or absence of endothelial cells. These specificities make it possible to preserve the endothelial layer compared to the techniques. The ultimate objective would be to use this measurement as a sorting criterion characterizing endothelial pump activity.

Potential Functional Restoration of Corneal Endothelial Cells in Fuchs Endothelial Corneal Dystrophy by ROCK Inhibitor (Ripasudil)

Rho-associated kinase (ROCK) inhibitors have been successfully used as a rescue strategy in eyes that failed to clear after descemetorhexis without endothelial graft for treatment of Fuchs endothelial corneal dystrophy (FECD). The functional mechanisms by which ROCK inhibitors modulate corneal endothelial cell regeneration in FECD patients have, however, not been clarified. Here, we analyzed the effect of the ROCK inhibitor ripasudil on corneal endothelial cells of FECD patients and normal donors using exvivo tissue and invitro cellular models. Experimental study: laboratory investigation. This institutional study used endothelial cell-Descemet membrane lamellae from FECD patients (n= 450) undergoing Descemet membrane endothelial keratoplasty (FECD exvivo model), normal research-grade donor corneas (n= 30) after scraping off central endothelial cells (exvivo wound healing model), normal donor corneas (n= 20) without endothelial injury, and immortalized cell lines (n= 3) generated from FECD patients (FECD invitro model). Descemet membrane lamellae were dissected into halves and incubated for 24-72 hours in storage medium with or without a single dose of 30μM ripasudil. The effects of ripasudil on expression of genes and proteins related to endothelial cell proliferation, migration, functionality, and endothelial-to-mesenchymal transition were analyzed and complemented by functional assays on FECD cell lines. A single dose of ripasudil induced significant upregulation of genes and proteins related to cell cycle progression, cell-matrix adhesion and migration, as well as endothelial barrier and pump function up to 72 hours, whereas classical markers of endothelial-to-mesenchymal transition were downregulated in both FECD and normal specimens compared to unstimulated controls exvivo. In addition to stimulation of proliferation and migration, ripasudil-induced changes in expression of functional signature genes could be also verified in FECD cell lines invitro. These data support the concept that inhibition of ROCK signaling represents a potent tool in regenerative therapies in FECD patients through reactivation of cell proliferation and migration as well as restoration of endothelial pump and barrier function without inducing adverse phenotypic changes.

Bicarbonate activates glycolysis and lactate production in corneal endothelial cells by increased pHi

Recent studies have shown that lactate coupled water flux is the underlying mechanism of the corneal endothelial pump, which is highly dependent on the presence of bicarbonate. In this study we test the hypothesis that the increased intracellular pH (pHi) caused by bicarbonate stimulates glycolytic activity and the production of lactate by endothelial cells. Primary cultures of bovine corneal endothelial cells (BCEC) were incubated in bicarbonate-free (BF) ringer, a high [HEPES] ringer, and bicarbonate-rich (BR) ringer all at pH 7.5. Lactate production and glucose consumption were greatest in BR>HEPES >BF. Similarly, pHi was greatest in BR>HEPES>BF. Increasing pHi with NH4Cl also increased lactate production in BF or BR, indicating that the increased lactate production in BR is not due to HCO3− itself. Glucose transport capacity, as measured by 2-N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino-2-Deoxyglucose (2-NBDG) uptake was unaffected by the three incubation conditions. Using Laconic, a FRET sensor for lactate, we found that intracellular [lactate] increased immediately and transiently when cells were switched from BF to BR perfusion indicating increased lactate production with subsequent matching of efflux. Moreover, induction of acute lactate influx by perfusion pulses of 10 mM lactate increased intracellular [lactate] significantly faster in BF than in BR, consistent with higher lactate production and efflux in BR. In summary, our results indicate that glycolytic flux and lactate production increase in BR due to increased pHi, consistent with the well-known pH sensitivity of phosphofructokinase, the rate limiting enzyme in glycolysis.

Energy Shortage in Human and Mouse Models of SLC4A11-Associated Corneal Endothelial Dystrophies.

PurposeTo elucidate the molecular events in solute carrier family 4 member 11 (SLC4A11)-deficient corneal endothelium that lead to the endothelial dysfunction that characterizes the dystrophies associated with SLC4A11 mutations, congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy 4.MethodsComparative transcriptomic analysis (CTA) was performed in primary human corneal endothelial cells (pHCEnC) and murine corneal endothelial cells (MCEnC) with normal and reduced levels of SLC4A11 (SLC4A11 KD pHCEnC) and Slc4a11 (Slc4a11−/− MCEnC), respectively. Validation of differentially expressed genes was performed using immunofluorescence staining of CHED corneal endothelium, as well as western blot and quantitative PCR analysis of SLC4A11 KD pHCEnC and Slc4a11−/− MCEnC. Functional analyses were performed to investigate potential functional changes associated with the observed transcriptomic alterations.ResultsCTA revealed inhibition of cell metabolism and ion transport function as well as mitochondrial dysfunction, leading to reduced adenosine triphosphate (ATP) production, in SLC4A11 KD pHCEnC and Slc4a11−/− MCEnC. Co-localization of SNARE protein STX17 with mitochondria marker COX4 was observed in CHED corneal endothelium, as was activation of AMPK–p53/ULK1 in both SLC4A11 KD pHCEnC and Slc4a11−/− MCEnC, providing additional evidence of mitochondrial dysfunction and mitophagy. Reduced Na+-dependent HCO3− transport activity and altered NH4Cl-induced membrane potential changes were observed in Slc4a11−/− MCEnC.ConclusionsReduced steady-state ATP levels and subsequent activation of the AMPK–p53 pathway provide a link between the metabolic functional deficit and transcriptome alterations, as well as evidence of insufficient ATP to maintain the Na+/K+-ATPase corneal endothelial pump as the cause of the edema that characterizes SLC4A11-associated corneal endothelial dystrophies.

Plasma Homocysteine and Cardiovascular Organ Damage in a Population with a High Prevalence of Risk Factors.

It is unclear whether plasma homocysteine (Hcy) has a direct noxious impact on the cardiovascular (CV) system or whether its association with cardiovascular events (CVEs) is mediated by established risk factors. To explore the role of Hcy in CV impairment, the study evaluated cross-sectional relationships between plasma Hcy and indices of CV organ damage together with the associations of these indices with the history of CVEs. In 269 patients with a high prevalence of diabetes, dyslipidemia, and hypertension, the carotid intima-media thickness, ankle-brachial index (ABI), reactive hyperemic index, carotid-femoral pulse wave velocity (cfPWV), left ventricular (LV) mass, and cardiac index were measured. 132 patients had carotid plaque, 31 ABI < 0.90, 126 endothelial dysfunction, 66 increased cfPWV, 125 LV hypertrophy (LVH), 153 decreased cardiac index, and 115 a history of CVEs. Plasma Hcy levels were related to LV mass and ABI, after adjustment for covariates and creatinine. Significantly higher Hcy levels were found in patients with LVH (8.5 [4.4] vs 7.6 [2.8] μmol/L; adjusted P = .001) and ABI < 0.9 (10.4 [3.8] vs 7.9 [3.4] μmol/L; adjusted P = .001) than in those with LV mass and ABI within limits. Hcy levels were comparable between patients with and without carotid plaques, increased arterial stiffness, impaired endothelial, and LV pump function. Within markers of CV organ damage, only LVH was associated with a history of CVEs. This study demonstrated an independent association between Hcy and LV mass as well as between LVH and a history of CVEs and suggests that LVH may represent 1 of the pathophysiologic links between Hcy and CV risk.

Protective Effects Oncorneal Endothelium During Intracameral Irrigation Using N-(2)-l-alanyl-l-Glutamine.

Corneal endothelial disease is a global sight-threatening disease, and corneal transplantation using donor corneas remains the sole therapeutic option. A previous work demonstrated that N (2)-alanyl-glutamine (Ala-Gln) protected against apoptosis and cellular stress, and maintained intestinal tissue integrity. In this pursuit, the present study aimed to examine the effect of Ala-Gln in the protection of the corneal endothelium and expand its range of potential clinical applications. Mice in the control group were intracamerally irrigated with Ringers lactate injection, whereas those in the experimental group were irrigated with Ringers lactate injection containing Ala-Gln. The mean intraocular pressure increased to 44 ± 3.5 mm Hg during intracameral irrigation (normal range 10.2 ± 0.4 mmHg). In vivo confocal microscopy results showed that the addition of Ala-Gln protected the morphology, structure, and density of the corneal endothelial cells. Optical Coherence Tomography (OCT) measurements showed that corneal thickness was not significantly different between the two groups, because of the immediate corneal edema after irrigation, but the addition of Ala-Gln obviously promoted the recovery of the corneal edema. Scanning electron microscopy indicated that the corneal endothelial cells were severely ruptured and exfoliated in the Ringer’s group accompanied with cellular edema, when compared with the Ala-Gln group. The intracameral irrigation using Ala-Gln protected the structure and expression of cytoskeleton and Na-K-ATPase, which exhibited a regular distribution and significantly increased expression in comparison to Ringer’s group. Furthermore, Ala-Gln maintained the mitochondrial morphology and increased the activity of mitochondria. Moreover, transmission electron microscopy showed that intracameral irrigation of Ala-Gln reversed the ultrastructural changes induced by the acute ocular hypertension in mice. Our study demonstrates that the intracameral irrigation of Ala-Gln effectively maintained the corneal endothelial pump function and barrier function by protecting the mitochondrial function and preventing the rearrangement of cytoskeleton in acute ocular hypertension in mice.

Corneal Endothelial Pump Coupling to Lactic Acid Efflux in the Rabbit and Mouse.

PurposeConfirm that the corneal endothelial pump uses a lactate-coupled water efflux mechanism.MethodsCorneal thickness, lactate efflux, and stromal [lactate] were measured in de-epithelialized swollen and nonswollen ex vivo-mounted rabbit corneas perfused with bicarbonate-rich and bicarbonate-free Ringers, ouabain, or acetazolamide to determine if the relationships among these parameters were similar to previous data using intact corneas. The role of barrier function was tested by perfusion with calcium-free EGTA. Predictions of [lactate] in endothelial dystrophy were examined in the Slc4a11 knock out mouse.ResultsDe-epithelialized corneal swelling, lactate efflux, and stromal [lactate] in response to bicarbonate-free Ringers, ouabain, and acetazolamide perfusion had the same relationship as in intact corneas. The absolute amount of lactate efflux and stromal [lactate] in the de-epithelialized corneas was about half of intact corneas. De-epithelialized, swollen corneas deswelled fully with bicarbonate-rich, partially in the presence of acetazolamide, but continued to swell with bicarbonate-free or ouabain. The relationship among corneal thickness, lactate efflux, and [lactate] was the same as with nonswollen de-epithelialized corneas. In intact corneas swollen by perfusion with calcium-free EGTA, the relationship between swelling and lactate flux was the inverse of control corneas. The relationship between corneal swelling and [lactate] of intact corneas exposed to ouabain, but perfused with 7 mM lactate to simulate aqueous humor, was the same as without lactate. Corneal [lactate] in Slc4a11 knock out was twice that of wild type.ConclusionsThe corneal endothelial pump works via a lactate efflux mechanism that requires an intact osmotic barrier.

Severe Corneal Edema in a Dog Naturally Infected by Leishmania spp.

Background: Visceral leishmaniasis (VL) is an infectious disease caused by the protozoan Leishmania infantum that is transmitted to dogs and humans by sandflies. The incidence of eye injuries in VL is high. They occur in 20 to 81% of infected dogs and include blepharitis, granulomatous conjunctivitis, scleritis, keratitis, anterior uveitis, keratoconjunctivitis sicca, and secondary glaucoma. However, some dogs present only the clinical signs of eye damage. The main objective of this manuscript is to report a case of anterior uveitis with severe corneal edema in a dog with VL that underwent clinical and surgical ophthalmic treatments after miltefosine therapy.Case: An 8-month-old, intact male Labrador Retriever with brown fur presented with pruritus, diffuse desquamation, and conjunctival hyperemia on physical evaluation. On the basis of an ophthalmic examination, nodular conjunctivitis and uveitis were diagnosed in both eyes. Moreover, laboratory examination results showed hyperproteinemia, increased serum alkaline phosphatase activity, and positive reactions to immunochromatographic tests for VL. Clinical treatment was instituted from the moment of diagnosis, when miltefosine and allopurinol were prescribed. At the end of treatment, based on laboratory examination results, only allopurinol was prescribed at a lower dose than initially prescribed for treatment continuation. Topical medications (prednisolone eye drops and sodium hyaluronate) were recommended for the ophthalmic changes. One week after the start of topical treatment, the dog showed an improvement in eye inflammation but still had bilateral corneal edema. A hyperosmotic agent was prescribed to improve edema, and a surgical procedure was recommended if there was no improvement. The physician opted for the surgical procedure in one of the eyes that had not shown significant improvement after the clinical treatment.Discussion: VL is a zoonosis, and the domestic dog is the main reservoir. These animals often have dermatological conditions, and the ophthalmic changes observed can be unilateral or bilateral, with more than one change in the same eye. Lymphoplasmacytic or granulomatous anterior uveitis is the most prevalent change, as the uvea and conjunctiva are important lymphoid areas of the eye; this also explains the high incidence of uveitis and conjunctivitis in dogs with VL. In uveitis, corneal edema is driven by endothelial cell damage induced by prostaglandins, which interfere with the function of the endothelial pump and interrupt the normal dehydrated state of the cornea. Severe corneal edema can result in the formation of fluid-filled multifocal bubbles in the corneal stroma—a condition called bullous keratopathy. These bubbles accumulate under or inside the corneal epithelium, and they can burst spontaneously, leading to corneal erosions or ulcerations. Drug therapy with hyperosmotic agents could, in principle, reduce the formation of bubbles. Surgical options to decrease edema and blistering include a permanent conjunctival graft or thermokeratoplasty. Thermokeratoplasty induces the formation of superficial scars in the corneal stroma, applying multiple cauterization foci to the stroma exposed in the areas of bullous keratopathy and epithelial ulceration. In conclusion, the surgical therapeutic choice results in better visual quality in patients who do not respond well to clinical treatment.

Innovative corneal active storage machine for long-term eye banking.

Optimal ex vivo corneal storage in eye banks is crucial to increase both the number of corneas suitable for graft and their intrinsic quality, mainly the number of viable endothelial cells, which dictates graft survival in recipients. With both passive storage methods used worldwide (short-term cold storage in the United States, long-term organ culture in Europe), significant endothelial cell loss is inevitable. Here we show that, with an active storage machine, also called a bioreactor, which restores 2 fundamental physiological parameters, intraocular pressure and medium renewal, endothelial cell survival is improved by 23% compared with organ culture after 4weeks' storage. Also observed in the bioreactor is a 4-fold higher expression of Na+ /K+ ATPase, which supports one of the major endothelial cell pumping functions. In addition, corneas remain thin and transparent, so they are suitable for surgery at any time. This new active eye banking method may help to reduce the severe global scarcity of donor corneas.

Ophthalmic Nonsteroidal Anti-Inflammatory Drugs as a Therapy for Corneal Dystrophies Caused by SLC4A11 Mutation.

SLC4A11 is a plasma membrane protein of corneal endothelial cells. Some mutations of the SLC4A11 gene result in SLC4A11 protein misfolding and failure to mature to the plasma membrane. This gives rise to some cases of Fuchs' endothelial corneal dystrophy (FECD) and congenital hereditary endothelial dystrophy (CHED). We screened ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs) for their ability to correct SLC4A11 folding defects. Five ophthalmic NSAIDs were tested for their therapeutic potential in some genetic corneal dystrophy patients. HEK293 cells expressing CHED and FECD-causing SLC4A11 mutants were grown on 96-well dishes in the absence or presence of NSAIDs. Ability of NSAIDs to correct mutant SLC4A11 cell-surface trafficking was assessed with a bioluminescence resonance energy transfer (BRET) assay and by confocal microscopy. The ability of mutant SLC4A11-expressing cells to mediate water flux (SLC4A11 mediates water flux across the corneal endothelial cell basolateral membrane as part of the endothelial water pump) was measured upon treatment with ophthalmic NSAIDs. BRET-assays revealed significant rescue of SLC4A11 mutants to the cell surface by 4 of 5 NSAIDs tested. The NSAIDs, diclofenac and nepafenac, were effective in moving endoplasmic reticulum-retained missense mutant SLC4A11 to the cell surface, as measured by confocal immunofluorescence. Among intracellular-retained SLC4A11 mutants, 20 of 30 had significant restoration of cell surface abundance upon treatment with diclofenac. Diclofenac restored mutant SLC4A11 water flux activity to the level of wild-type SLC4A11 in some cases. These results encourage testing diclofenac eye drops as a treatment for corneal dystrophy in patients whose disease is caused by some SLC4A11 missense mutations.