Stable isotope study of rainfall, river drainage and hot springs of the kerguelen archipelago, SW Indian Ocean

International audienceThis study is the first synthesis of chemical composition and stable isotopes values for the Kerguelen archipelago waters. The stable isotope values for rainfall and river waters in the Kerguelen archipelago allow a calculation of the Local Meteoric Water Line (δD rainfall = 8.43 x δ18O rainfall + 11) and a summer runoff line (δD river drainage = 7.45 x δ18O river drainage + 6). Surface waters with low- ion concentrations, chlorine facies and stable isotope values infiltrate through fractures and lava flows recharging deeper groundwaters. Thermal groundwater with low- (7 to 50 °C) and high- (50 to 100 °C) temperatures emerges in different localities in the volcanic archipelago. The low-temperature thermal waters might represent a mixture of high-temperature water with rainfall, thermal gradient changes or shallower infiltration compared to that for high-temperature thermal waters. The Rallier du Baty and Val Travers areas contain geothermal fluids with high-temperature springs, fumaroles and a large water flow. In the Rallier du Baty, the major ion chemistry and O, H, C and S stable isotope ratio of low (7 to 50 °C) temperature spring waters in Rallier du Baty area demonstrate a geothermal-system recharged by meteoric water (δD H2O liquid = 7.0 x δ18O H2O liquid + 0.5) rather than sea water. The chemical and isotopic compositions of elevated temperature spring waters (50 to 100 °C) have a long and complex history of meteoric water interacting with cooling magmas (δD H2O liquid = 1.78 x δ18O H2O liquid –  23). Surficial precipitation of aragonite, kaolinite, pyrite, native sulfur attest to a long livied geothermal system. A temperature of the geothermal reservoir has been estimated between 193 and 259 °C by cation geothermometry. The combination of minerals observed, major ion composition of water with thermodynamic modeling and stable isotope data suggest a geothermal system with a series of water/rock interactions from 50 to 250 °C. The conductive cooling of rising of H2O−CO2-rich fluids have produced a H2O−CO2 phase separation with the precipitation of secondary minerals

Specular Microscopy of Human Corneas Stored in an Active Storage Machine

Purpose: Unlike corneas stored in cold storage (CS) which remain transparent and thin, corneas stored in organoculture (OC) cannot be assessed by specular microscopy (SM), because edema and posterior folds occur during storage and prevent from specular reflection. We previously developed an active storage machine (ASM) which restores the intraocular pressure while renewing the storage medium, thus preventing major stromal edema. Its transparent windows allow multimodal corneal imaging in a closed system. Aim: to present SM of corneas stored in this ASM. Methods: Ancillary study of two preclinical studies on corneas stored for one and three months in the ASM. A prototype non-contact SM was developed (CMOS camera, ×10 objective, collimated LED source, micrometric stage). Five non-overlapping fields (935 × 748 μm) were acquired in exactly the same areas at regular intervals. Image quality was graded according to defined categories (American Cornea Donor Study). The endothelial cell density (ECD) was measured with a center method. Finally, SMECD was also compared to Hoechst-stained cell nuclei count (HoechstECD). Results: The 62 corneas remained thin during storage, allowing SM at all time points without corneal deconditioning. Image quality varied depending on donors and days of control but, overall, in the 1100 images, we observed 55% of excellent and 30% of good quality images. SMECD did not differ from HoechstECD (p = 0.084). Conclusions: The ASM combines the advantages of CS (closed system) and OC (long-term storage). Specular microscopy is possible at any time in the ASM with a large field of view, making endothelial controls easy and safe

Specular Microscopy of Human Corneas Stored in an Active Storage Machine

Purpose: Unlike corneas stored in cold storage (CS) which remain transparent and thin, corneas stored in organoculture (OC) cannot be assessed by specular microscopy (SM), because edema and posterior folds occur during storage and prevent from specular reflection. We previously developed an active storage machine (ASM) which restores the intraocular pressure while renewing the storage medium, thus preventing major stromal edema. Its transparent windows allow multimodal corneal imaging in a closed system. Aim: to present SM of corneas stored in this ASM. Methods: Ancillary study of two preclinical studies on corneas stored for one and three months in the ASM. A prototype non-contact SM was developed (CMOS camera, ×10 objective, collimated LED source, micrometric stage). Five non-overlapping fields (935 × 748 μm) were acquired in exactly the same areas at regular intervals. Image quality was graded according to defined categories (American Cornea Donor Study). The endothelial cell density (ECD) was measured with a center method. Finally, SMECD was also compared to Hoechst-stained cell nuclei count (HoechstECD). Results: The 62 corneas remained thin during storage, allowing SM at all time points without corneal deconditioning. Image quality varied depending on donors and days of control but, overall, in the 1100 images, we observed 55% of excellent and 30% of good quality images. SMECD did not differ from HoechstECD (p = 0.084). Conclusions: The ASM combines the advantages of CS (closed system) and OC (long-term storage). Specular microscopy is possible at any time in the ASM with a large field of view, making endothelial controls easy and safe

Stable isotope study of rainfall, river drainage and hot springs of the kerguelen archipelago, SW Indian Ocean

International audienceThis study is the first synthesis of chemical composition and stable isotopes values for the Kerguelen archipelago waters. The stable isotope values for rainfall and river waters in the Kerguelen archipelago allow a calculation of the Local Meteoric Water Line (δD rainfall = 8.43 x δ18O rainfall + 11) and a summer runoff line (δD river drainage = 7.45 x δ18O river drainage + 6). Surface waters with low- ion concentrations, chlorine facies and stable isotope values infiltrate through fractures and lava flows recharging deeper groundwaters. Thermal groundwater with low- (7 to 50 °C) and high- (50 to 100 °C) temperatures emerges in different localities in the volcanic archipelago. The low-temperature thermal waters might represent a mixture of high-temperature water with rainfall, thermal gradient changes or shallower infiltration compared to that for high-temperature thermal waters. The Rallier du Baty and Val Travers areas contain geothermal fluids with high-temperature springs, fumaroles and a large water flow. In the Rallier du Baty, the major ion chemistry and O, H, C and S stable isotope ratio of low (7 to 50 °C) temperature spring waters in Rallier du Baty area demonstrate a geothermal-system recharged by meteoric water (δD H2O liquid = 7.0 x δ18O H2O liquid + 0.5) rather than sea water. The chemical and isotopic compositions of elevated temperature spring waters (50 to 100 °C) have a long and complex history of meteoric water interacting with cooling magmas (δD H2O liquid = 1.78 x δ18O H2O liquid –  23). Surficial precipitation of aragonite, kaolinite, pyrite, native sulfur attest to a long livied geothermal system. A temperature of the geothermal reservoir has been estimated between 193 and 259 °C by cation geothermometry. The combination of minerals observed, major ion composition of water with thermodynamic modeling and stable isotope data suggest a geothermal system with a series of water/rock interactions from 50 to 250 °C. The conductive cooling of rising of H2O−CO2-rich fluids have produced a H2O−CO2 phase separation with the precipitation of secondary minerals

Investigating the Role of TGF-β Signaling Pathways in Human Corneal Endothelial Cell Primary Culture

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial–mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-βs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-β signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-β1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-β-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-β1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-β receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-β1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD

Design and validation of a custom-made system to measure transepithelial electrical impedance in human corneas preserved in active storage machine

Corneal epithelial barrier represents one of the major limitations to ocular drug delivery and can be explored non-invasively through the evaluation of its electrical properties. Human corneas stored in active storage machine (ASM) could represent an interesting physiological model to explore transcorneal drug penetration. We designed a new system adapted to human corneas preserved in ASM to explore corneal epithelial barrier function ex-vivo. A bipolar set-up including Ag/AgCl electrodes adaptors to fit the corneal ASM and a dedicated software was designed and tested on freshly excised porcine corneas (n = 59) and human corneas stored 14 days in ASM (n = 6). Porcine corneas presented significant and proportional decrease in corneal impedance in response to increasing-size epithelial ulcerations and acute exposure to benzalkonium chloride (BAC) 0.01 and 0.05%. Human corneas stored 14 days in ASM presented a significant increase in corneal impedance associated with the restoration of a multi-layer epithelium and an enhanced expression of tight junctions markers zonula occludens 1, claudin 1 and occludin. These results support the relevance of the developed approach to pursue the exploration and development of human corneas stored in ASM as a physiological pharmacological model

Ex vivo model of herpes simplex virus type I dendritic and geographic keratitis using a corneal active storage machine

International audienceBackground: Herpetic keratitis (HK) models using whole human corneas are essential for studying virus-host relationships, because of high species specificity and the role of interactions between corneal cell populations that cell culture cannot reproduce. Nevertheless, the two current corneal storage methods (hypothermia and organ culture (OC)) do not preserve corneas in good physiological condition, as they are characterized by epithelial abrasion, stromal oedema, and excessive endothelial mortality.Methods: To rehabilitate human corneas intended for scientific use, we used an active storage machine (ASM) that restores two physiological parameters that are essential for corneal homeostasis: intraocular pressure and storage medium renewal (21mmHg and 2.6 μL/min, respectively). ASM storage regenerates a normal multilayer epithelium in 2 weeks. We infected six pairs of corneas unsuitable for graft by inoculating the epithelium with herpes simplex virus type 1 (HSV-1), and compared each ASM-stored cornea with the other cornea stored in the same medium using the conventional OC method.Results: Only corneas in the ASM developed a dendritic (n = 3) or geographic (n = 2) epithelial ulcer reproducing typical HSV-1-induced clinical lesions. Corneas in OC showed only extensive desquamations. None of the uninfected controls showed epithelial damage. Histology, immunohistochemistry, transmission electron microscopy and polymerase chain reaction on corneal tissue confirmed infection in all cases (excluding negative controls).Conclusions: The ASM provides an innovative ex vivo model of HK in whole human cornea that reproduces typical epithelial lesions

Femtosecond Laser Cutting of Human Crystalline Lens Capsule and Decellularization for Corneal Endothelial Bioengineering

The bioengineering of corneal endothelial grafts consists of seeding in vitro cultured corneal endothelial cells onto a thin, transparent, biocompatible, and sufficiently robust carrier which can withstand surgical manipulations. This is one of the most realistic alternatives to donor corneas, which are in chronic global shortage. The anterior capsule of the crystalline lens has already been identified as one of the best possible carriers, but its challenging manual preparation has limited its use. In this study, we describe a femtosecond laser cutting process of the anterior capsule of whole lenses in order to obtain capsule discs of 8 mm diameter, similar to conventional endothelial grafts. Circular marks made on the periphery of the disc indicate its orientation. Immersion in water for 3 days is sufficient to completely remove the lens epithelial cells and to enable the seeding of corneal endothelial cells, which remain viable after 27 days of culture. Therefore, this method provides a transparent, decellularized disc ready to form viable tissue engineered endothelial grafts