Semiautomatic assessment of endothelial density and morphology in organ-cultured corneas - potential predictors for transplantation suitability and clinical outcome?

BACKGROUND: The quality of the endothelial cell layer is a major criterion for the approval of organ-cultured human donor-corneas for transplantation. We wanted to compare the predictive capacities of initial endothelial density and endothelium cell morphology for the approval of donor corneas for transplantation and for the clinical outcome after transplantation. METHODS: The endothelial density and endothelium morphology in organ culture were examined by semiautomatic assessment of 1031 donor corneas. We performed a statistical analysis for correlations of donor-data and cultivation parameters regarding their predictive capacities for the final approval of donor corneas for transplantation and the clinical outcome of 202 transplanted patients. RESULTS: Corneal endothelium cell density proved to be the only parameter with a certain predictive capacity with regard to the final decision, whether donor corneas are suitable for transplantation - however, the correlation was low (area under the curve [AUC] = 0.655). Endothelial cell morphology lacked any predictive power (AUC = 0.597). The clinical outcome regarding visual acuity seemed to be largely independent from both corneal endothelial cell density and morphology. Sub-analyses on transplanted patients stratified for their diagnoses vindicated these findings. CONCLUSIONS: Higher endothelial density (above a cut-off level of 2000 cells/mm2), as well as better endothelial morphology do not seem to be critical for transplant-corneal functionality in organ culture and up to 2 years after transplantation. Comparable long-term studies on graft survival are recommended to determine, whether the present endothelial density cut-off levels might be too stringent.

A new storage solution for the hypothermic preservation of corneal grafts: an experimental study.

In this experimental study we used for the first time Tiprotec® as a solution for corneal preservation and cold storage. We compared the resultant endothelial cell morphology and viability with this obtained after preservation of the ex-vivo corneas with both usual standard techniques: conventional cold storage (using Eusol-C) and organ culture. This prospective, in vitro, 3-armed parallel study was performed with the use of 90 porcine corneas (examined for their endothelial quality and transparency) randomly selected for preservation in three storage methods (each 30 corneas): organ culture, standard cold storage (Eusol-C) and experimental cold storage (Tiprotec®) Endothelium cell quantity and quality as well as corneal opacification were assessed. The degree of endothelial transparency was significantly reduced over time with all preservation media, without any significant difference among the three groups at any point of time. A reduction in endothelial cell density was also observed with all three preservation media after 30 days of storage without statistically significant differences between groups. The number of hexagonal and pentagonal endothelium cells was significantly reduced overtime in all media with significantly more hexagonal and pentagonal in the organ culture group compared to the cold storage groups. We could show that the cryopreservation medium Tiprotec®, used until now for the preservation of vascular grafts, was of similar quality compared to the medium Eusol-C for the hypothermic storage of corneal tissue for an extended period of time up to 30 days. In comparison to organic culture with culture medium KII, both Tiprotec® and Eusol-C were found less effective in preserving endothelial cell quality, as assessed by the morphometric analysis, and viability, as assessed by the degree of vacuolization at least up to the 30th day of storage. However, both, Tiprotec®- and Eusol-C-preserved corneas demonstrated a certain capacity to recover after their submission in organ culture.

[Photorefractive Keratectomy (PRK) as a Procedure for Correction of Residual Refractive Errors after Radial Keratotomy]. / Photorefraktive Keratektomie (PRK) zur Korrektur der Restfehlsichtigkeit nach radiärer Keratotomie.

BACKGROUND: A large number of myopic patients were treated by radial keratotomy (RK) in recent years. Despite being effective in many cases, the refractive results of this surgical intervention proved to be of limited predictability, and it frequently resulted in over- or under-correction in the long term. In this study, we discuss the intermediate and long-term results of a topography-guided photorefractive keratotomy (PRK) in a consecutive series of patients who were previously treated for myopia by radial keratotomy. MATERIALS AND METHODS: In this retrospective case series, we examined the refraction and visual acuity in a consecutive series of patients-16 eyes-who were treated by PRK for residual refractive errors after radial keratotomy in the past. Mean follow up was 41 months (min. 9, max. 96). RESULTS: All treated eyes showed an improvement in uncorrected visual acuity, and 56% had an improvement in corrected visual acuity. No serious or sight-threatening complications were recorded. Refraction was stable throughout the study period in all patients. CONCLUSIONS: In this case series, photorefractive keratotomy was shown to be an effective treatment method for secondary ametropia after radial keratotomy. Apart from the correct planning and execution of the PRK, it is of critical importance to inform the patients about the limitations and the anticipated refractive result of the procedure.

[Intravitreal Therapy Combining Dexamethasone and Bevacizumab in Treating Radiation Retinopathy and Opticopathy]. / Kombinierte intravitreale Therapie mit Dexamethason und Bevacizumab bei Strahlenoptikopathie und -retinopathie.

Radiation Retinopathy is a progressive chronic disease triggered by ionising radiation and is characterised by vascular endothelial damage that can lead to macular edema, optic disc edema and proliferative retinopathy. We discuss a case of a patient with radiation retinopathy and optic disc edema who we treated with a combination of intravitreal bevacizumab and dexamethasone. After 3 injections of bevacizumab, and one of dexamethasone, the patient experienced a resolution of optic disc edema and a marked increase of his visual acuity and remained stable throughout the follow-up period.

Donor Cornea Harvest Techniques: Comparison Between Globe Enucleation and In Situ Corneoscleral Disc Excision.

PURPOSE: To compare whole eye enucleation and corneoscleral disc (CD) excision as donor cornea harvesting techniques for possible effects on corneal cultivation and the clinical outcome of the corneal grafts after transplantation in 2929 cases. METHODS: A retrospective analysis was performed on the Hamburg Eye Bank database using comparative statistics. The standard method for donor cornea recovery at the Hamburg Eye Bank changed from enucleation of the whole eye to CD in situ excision in 2008. Corneas recovered between 2003 and 2013 were included in this study. We compared the contamination rate, the endothelial density after retrieval, endothelial cell loss during cultivation, and the clinical outcome (visual acuity, astigmatism, and refraction) of transplanted corneas. RESULTS: Once the retrieval method was changed from whole globe enucleation to in situ CD excision, the donation numbers increased (after several years of constant decrease). Furthermore, we observed slightly lower endothelial cell density after retrieval in corneas obtained by in situ CD excision compared with those from enucleated eyes, whereas endothelial cell loss during cultivation was similar. After changing the recovery procedure to in situ excision, initially a higher rate of contamination was observed, but but it eventually decreased. Finally, the corneas of both groups had a similar clinical outcome. CONCLUSIONS: After a transient technical learning period, in situ CD excision proved to be a method of donor cornea recovery with similar cultivation performance and clinical results compared with whole eye enucleation. It also may have led to higher willingness to donate, possibly because of better acceptance by the relatives of the deceased.

Endothelial Cell Count in Eye Bank Corneal Grafts: Impact of Death Cause and Donor Diseases.

PURPOSE: The purpose of this study is to analyze the impact of death causes and documented donor diseases on initial endothelial cell counts (after retrieval) and the development of corneal graft endothelia during organ culture. METHODS: The retrospective statistic analyses was conducted on a data set of 10,185 human corneas prepared at the Hamburg Eye Bank. RESULTS: Although we observed that death by gunshot trauma or alcoholism seems to be associated with marginally higher endothelium cell counts (independently from donor age), we could prove that only donor age is a relevant predictive parameter for the initial cell-density of the endothelium and its development in vitro. CONCLUSION: We conclude that an extension of prospective quality parameters for donor selection additional to donor age (such as individual causes of death) is not necessary.

Semi-quantitative assessments of dextran toxicity on corneal endothelium: conceptual design of a predictive algorithm.

Dextran is added to corneal culture medium for at least 8 h prior to transplantation to ensure that the cornea is osmotically dehydrated. It is presumed that dextran has a certain toxic effect on corneal endothelium but the degree and the kinetics of this effect have not been quantified so far. We consider that such data regarding the toxicity of dextran on the corneal endothelium could have an impact on scheduling and logistics of corneal preparation in eye banking. In retrospective statistic analyses, we compared the progress of corneal endothelium (endothelium cell loss per day) of 1334 organ-cultured corneal explants in media with and without dextran. Also, the influence of donor-age, sex and cause of death on the observed dextran-mediated effect on endothelial cell counts was studied. Corneas cultured in dextran-free medium showed a mean endothelium cell count decrease of 0.7% per day. Dextran supplementation led to a mean endothelium cell loss of 2.01% per day; this reflects an increase by the factor of 2.9. The toxic impact of dextran was found to be time dependent; while the prevailing part of the effect was observed within the first 24 h after dextran-addition. Donor age, sex and cause of death did not seem to have an influence on the dextran-mediated toxicity. Based on these findings, we could design an algorithm which approximately describes the kinetics of dextran-toxicity. We reproduced the previously reported toxic effect of dextran on the corneal endothelium in vitro. Additionally, this is the first work that provides an algorithmic instrument for the semi-quantitative calculation of the putative endothelium cell count decrease in dextran containing medium for a given incubation time and could thus influence the time management and planning of corneal transplantations.

Utilization of a genetically modified muscle flap for local BMP-2 production and its effects on bone healing: a histomorphometric and radiological study in a rat model.

AIM OF THE STUDY: We developed an experimental rat model to explore the possibility of enhancing the healing of critical-size bone defects. The aim of this study was to demonstrate the feasibility of this concept by achieving high local BMP-2 expression via a transduced muscle flap that would facilitate bony union while minimizing systemic sequelae. METHODS: The transduction potential of the adenoviral vector encoding for BMP-2 was tested in different cell lines in vitro. In vivo experiments consisted of harvesting a pedicled quadriceps femoris muscle flap with subsequent creation of a critical-size defect in the left femur in Sprague-Dawley rats. Next, the pedicled muscle flap was perfused with high titers of Ad.BMP-2 and Ad.GFP virus, respectively. Twelve animals were divided into three groups comparing the effects of Ad.BMP-2 transduction to Ad.GFP and placebo. Bone healing was monitored radiologically with subsequent histological analysis post-mortem. RESULTS: The feasibility of this concept was demonstrated by successful transduction in vitro and in vivo as evidenced by a marked increase of BMP-2 expression. The three examined groups only showed minor difference regarding bone regeneration; however, one complete bridging of the defect was observed in the Ad.BMP-2 group. No evidence of systemic viral contamination was noted. CONCLUSIONS: A marked increase of local BMP-2 expression (without untoward systemic sequelae) was detected. However, bone healing was not found to be significantly enhanced, possibly due to the small sample size of the study.

Anterior and posterior corneal changes after crosslinking for keratoconus.

PURPOSE: To evaluate anterior and posterior changes in corneal topography and tomography after corneal crosslinking (CXL) in eyes with progressive keratoconus. METHODS: Scheimpflug analyses (Pentacam, Oculus) of 20 eyes with keratoconus performed before and after corneal CXL were included into retrospective analysis. Mean follow-up was 2 years. Changes in topographic, tomographic, and pachymetric values were statistically analyzed applying analysis of variance. Further, the distance and direction between the anterior maximum keratometry (K(max)) and the apex as well as the distance and direction between the thinnest point in corneal thickness (TPCT) and the corneal apex before and after CXL were studied. RESULTS: Two years after CXL, a statistically significant reduction of the keratometry at the flat meridian (-0.8 D, p < 0.05), the steep meridian (-0.5 D, p < 0.05), the "index of surface variance" (-5.3, p < 0.05), and the "index of highest decentration" (-0.05, p < 0.05) could be demonstrated. While the elevation of the front surface at the apex decreased (-1.5 µm, p < 0.05), the back elevation at the apex (+2 µm, p < 0.05) increased. Although not reaching statistical significance, the maximum front and back elevation demonstrated the same trend; while maximum front elevation data remained stable (-0.3 µm, p = 0.961), maximum back elevation data increased (+6.7 µm, p = 0.122). The corneal thickness at the apex (-22.0 µm, p < 0.001) and the TPCT (-20.0 µm, p < 0.001) decreased, leading to an increase of the corneal thickness progression from the corneal apex to the periphery. The position of K(max) and TPCT remained stable. CONCLUSIONS: Corneal topography proved to be useful in the follow-up for CXL because of significant changes in the keratometry. Increasing posterior elevation values, despite a stabilized anterior corneal surface, might be a sign of ongoing ectatic changes in the posterior corneal surface.