Pan-Corneal Endothelial Viability Assessment: Application to Endothelial Grafts Predissected by Eye Banks

Pipparelli, A.; Thuret, Gilles; Toubeau, D.; Hé, Zhiguo; Piselli, S; Lefèvre, Sabine; Gain, Philippe; Muraine, M.

doi:10.1167/iovs.10-6641

Cited by 53 publications

(46 citation statements)

References 63 publications

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“…We therefore combined in vitro tests on human cells and ex-vivo assessment on porcine corneas. Furthermore, complementary methods were used to assess deswelling efficacy and toxicity: in vitro MTT assay on the three corneal cell types, CCT measurement, automated quantitative assessment of transparency with an innovative device, and live/dead assay using both trypan blue staining for endothelial dead areas and calcein-AM/ethidium homodimer assay [30]. The combination of these techniques allowed an objective and reliable comparison of poloxamines with the control dextran T500.…”

Section: Discussionmentioning

confidence: 99%

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Poloxamines for Deswelling of Organ-Cultured Corneas

et al. 2012

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Background: Poloxamines are amphiphilic tetrofunctional block copolymers composed of four polyoxyethylene-polyoxypropylene arms joined to a central ethylene diamine bridge. Their safe profile allows diverse pharmaceutical and biomedical applications. Aim: To assess their use for corneal deswelling using a porcine model of organ culture (OC). Methods: Five poloxamines (T90R4, T904, T908, T1107 and T1307) were dissolved in a standard commercial OC medium (control) to reach 350 mosm kg^–1. In vitro cytotoxicity was tested using MTT assay on human corneal epithelial and endothelial cell (EC) lines and on primary human corneal fibroblasts. Paired porcine corneas stored in OC for 3 days were assigned for 48 h to a poloxamine medium or to a standard deswelling medium containing 5% dextran T500. Corneal EC density, morphometry, mortality, stromal thickness and transparency were evaluated before and after deswelling. Post-deswelling, EC viability/mortality was determined using a fluorescent live/dead assay. Results: Besides similar corneal thickness reduction and transparency improvement, T908, T1107 and T1307 decreased EC loss (5.4 ± 1.7% vs. 9.9 ± 2.6% in controls (p < 0.001)) and mortality, improved EC morphometry and reduced endothelial lesions compared to dextran. Conclusion: On this porcine model, poloxamines T908, T1107 and T1307 appear as good candidates to replace dextran for the deswelling. Experiments on human corneas are now necessary to confirm their efficiency and safety profile in OC.

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Section: Discussionmentioning

confidence: 99%

“…In each photograph, the green-stained surface of viable area and that of the dead area, stained red, were analyzed using the Cell P imaging software (Olympus) [30]. The mean total endothelial area of each cornea analyzed was 2.9 mm 2 .…”

Section: Methodsmentioning

confidence: 99%

Poloxamines for Deswelling of Organ-Cultured Corneas

et al. 2012

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“…The remaining sections were left in PBS at room temperature during the incubation. TX-100-treated (EMD Millipore) and untreated wedges were incubated with 0.3 lM calcein AM [33][34][35] and 1 lg/mL PI [37][38][39] and/or 1 lg/mL Hoechst 33342 for 30 minutes at 378C and 8% CO 2 . TPEF was performed immediately after incubation.…”

Section: Methodsmentioning

confidence: 99%

Analyzing Live Cellularity in the Human Trabecular Meshwork

González

Tan

2013

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. To directly visualize the live cellularity of the intact human trabecular meshwork (TM) and quantitatively analyze tissue viability in situ.METHODS. Human donor corneoscleral rims were sectioned immediately before intravital dye incubation to label nuclei (Hoechst 33342 & propidium iodide [PI]); cytosol (CellTracker Red CMTPX, calcein AM); and membranes (octadecyl rhodamine B chloride [R18]), followed by 2-photon microscopy. Viability was assessed by counting cells in tissue colabeled with PI and Calcein AM. Some tissues were exposed to Triton X-100 to establish dead tissue controls. Fresh postmortem eyes (within 48 hours of death) represented viable tissue controls. Tissues with live cellularity exceeding 50% were considered viable.RESULTS. Hoechst nuclear labeling was seen throughout the TM, among the autofluorescent beams, plate-like structures and fibers of the meshwork, and within tissue gaps and pores. CellTracker-labeled live cells were attached to autofluorescent TM structures and filled corneoscleral meshwork pores. R18-labeling revealed the membrane distributions of interconnected cells. Calcein-positive cells were visible in all TM layers, but not in tissues killed by Triton X-100 exposure. Dead control tissues showed PI staining in the absence of Calcein-positive cells. Two-thirds of the standard donor tissues we received possessed viable TM, having a mean live cellularity of 71% (n ¼ 14), comparable with freshly postmortem eyes (76%; n ¼ 2). Mean live cellularity of nonviable tissue was 11% (n ¼ 7). CONCLUSIONS.We have visualized and quantified the live cellularity of the TM in situ. This provided unique perspectives of live cell-matrix organization and a means of assaying tissue viability. (Invest Ophthalmol Vis Sci.

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“…We recently optimized triple labeling with Hoechst-ethidium-calcein (HEC) for experimental assessment of endothelial viability over the whole endothelial area [26]. The endothelial side was exposed for 1 minute to 0.4% trypan blue solution (Sigma) or submitted to HEC triple labeling as previously described [26]. After two rinses in BSS to remove excess dye, the cornea was fixed for 2 minutes in 4% PFA.…”

Section: Observation Of Flat-mounted Corneas (En Face Observation)mentioning

confidence: 99%

Revisited Microanatomy of the Corneal Endothelial Periphery: New Evidence for Continuous Centripetal Migration of Endothelial Cells in Humans

et al. 2012

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The control of corneal transparency depends on the integrity of its endothelial monolayer, which is considered nonregenerative in adult humans. In pathological situations, endothelial cell (EC) loss, not offset by mitosis, can lead to irreversible corneal edema and blindness. However, the hypothesis of a slow, clinically insufficient regeneration starting from the corneal periphery remains debatable. The authors have re-evaluated the microanatomy of the endothelium in order to identify structures likely to support this homeostasis model. Whole endothelia of 88 human corneas (not stored, and stored in organ culture) with mean donor age of 80 6 12 years were analyzed using an original flatmounting technique. In 61% of corneas, cells located at the extreme periphery (last 200 lm of the endothelium) were organized in small clusters with two to three cell layers around Hassall-Henle bodies. In 68% of corneas, peripheral ECs formed centripetal rows 830 6 295 lm long, with Descemet membrane furrows visible by scanning electron microscopy. EC density was significantly higher in zones with cell rows. When immunostained, ECs in the extreme periphery exhibited lesser differentiation (ZO-1, Actin, Na/ K ATPase, CoxIV) than ECs in the center of the cornea but preferentially expressed stem cell markers (Nestin, Telomerase, and occasionally breast cancer resistance protein) and, in rare cases, the proliferation marker Ki67. Stored corneas had fewer cell clusters but more Ki67-positive ECs. We identified a novel anatomic organization in the periphery of the human corneal endothelium, suggesting a continuous slow centripetal migration, throughout life, of ECs from specific niches.

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Pan-Corneal Endothelial Viability Assessment: Application to Endothelial Grafts Predissected by Eye Banks

Cited by 53 publications

References 63 publications

Poloxamines for Deswelling of Organ-Cultured Corneas

Poloxamines for Deswelling of Organ-Cultured Corneas

Analyzing Live Cellularity in the Human Trabecular Meshwork

Revisited Microanatomy of the Corneal Endothelial Periphery: New Evidence for Continuous Centripetal Migration of Endothelial Cells in Humans

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