86 research outputs found
Acute corneal melt and perforation - A possible complication after riboflavin/UV-A crosslinking (CXL) in keratoconus.
Purpose
To report two cases of acute corneal melting and perforation requiring emergency penetrating keratoplasty after corneal crosslinking (CXL) in advanced keratoconus.
Observations
Case 1 was a 34 and case 2 was a 16-year old male, both with progressive keratoconus, who underwent CXL (Dresden protocol). After riboflavin imbibition, patients had a minimal pachymetry of 337 ÎŒm and 347 ÎŒm, and therefore required stromal swelling by hypoosmolar riboflavin resulting in pachymetries of 470 ÎŒm and 422 ÎŒm, prior to the 30 minute UV-irradiation with 3mW/cm2. In case 1, on the 7th postoperative day a 4mm linear perforation occurred. Extensive post-hoc examinations revealed no infectious cause. In case 2, a corneal melting developed within 24 hours, from which Staphylococcus aureus was cultured.
Conclusions and importance
Acute corneal melting and perforation may occur after CXL. Dysfunctional collagen metabolism, atopia, thin preoperative pachymetry and the use of hypoosmolar substances may have initiated this complication in our cases
Wound healing in rabbit corneas after flapless refractive lenticule extraction with a 345 nm ultraviolet femtosecond laser
Purpose
To characterize corneal wound healing in a rabbit model after flapless refractive lenticule extraction with a 345âŻnm ultraviolet femtosecond laser.
Setting
Departments of Ophthalmology and Anatomy II, University of Erlangen-NĂŒrnberg and Wavelight GmbH, Erlangen, Germany.
Design
Methods
Flapless refractive lenticule extraction was performed in 1 eye each of 20 New Zealand white rabbits (â5.0 diopters). Groups of 4 animals were euthanized after 48âŻhours, 1âŻweek, 2âŻweeks, 4âŻweeks, and 3âŻmonths, respectively. Corneal samples were prepared for histology and fluorescence microscopy. To assess corneal cell death, proliferation, and myofibroblastic transdifferentiation, terminal uridine deoxynucleotidyl nick end-labeling (TUNEL) assay as well as immunostaining for Ki67 and α-smooth muscle actin (αSMA) were performed on sagittal cryosections.
Results
Histology revealed a zone of keratocyte depletion with a thickness of approximately 50âŻÎŒm around the extraction site. At 48âŻhours, pronounced TUNEL staining of keratocytes was detected around the interface (159.9âŻcells/mmâŻÂ±âŻ18.4 [SD]), which steadily decreased to 74.9âŻÂ±âŻ19.8âŻcells/mm at 1âŻweek and 5.7âŻÂ±âŻ4.8âŻcells/mm at 2âŻweeks. Ki67 staining of keratocytes was evident at 48âŻhours (10.0âŻÂ±âŻ3.8âŻcells/mm), which then decreased at 1âŻweek (5.2âŻÂ±âŻ1.7âŻcells/mm) and 2âŻweeks (0.4âŻÂ±âŻ0.5âŻcells/mm). From 4âŻweeks onward, no TUNEL or Ki67 staining was detected. The corneal stroma was αSMA-negative at all timepoints.
Conclusion
Application of the 345âŻnm laser showed no signs of problematic repair processes in the cornea, which supports the initiation of the clinical phase
Global Health Education: a cross-sectional study among German medical students to identify needs, deficits and potential benefits (Part 1 of 2: Mobility patterns & educational needs and demands)
<p>Abstract</p> <p>Background</p> <p>In recent years, education and training in global health has been the subject of recurring debate in many countries. However, in Germany, there has been no analysis of the educational needs or demands of medical students, or the educational deficits or potential benefits involved in global health education. Our purpose is to analyse international health elective patterns of medical students enrolled at German universities and assess whether or how they prepare for their electives abroad. We examine the exposure of medical students enrolled at German universities to training courses in tropical medicine or global health and assess students' perceived needs and demands for education in global health.</p> <p>Methods</p> <p>Cross-sectional study among medical students in Germany including all 36 medical schools during the second half of the year 2007. All registered medical students were eligible to participate in the study. Recruitment occurred via electronic mailing-lists of students' unions. We developed a web-based, semi-structured questionnaire to capture students' international mobility patterns, preparation before electives, destination countries, exposure to and demand for global health learning opportunities.</p> <p>Results</p> <p>1126 online-replies were received and analysed from all registered medical students in Germany (N = 78.067). 33.0% of all respondents (370/1126) declared at least one international health elective and of these, 36.0% (133/370) completed their electives in developing countries. 36.0% (131/363) did not prepare specifically at all, 59.0% (214/363) prepared either by self-study or declared a participation in specific preparation programmes. 87.8% of 5<sup>th </sup>and 6<sup>th </sup>year students had never participated in a global health course and 72.6% (209/288) had not completed a course in tropical medicine. 94.0% (861/916) endorsed the idea of introducing global health into medical education.</p> <p>Conclusion</p> <p>Students in our sample are highly mobile during their studies. International health electives are common, also in developing countries. Formal preparation beyond self-study is virtually non-existent amongst our sample and the participation rate in courses of tropical medicine or global health is appallingly low. We have identified unmet perceived needs and the demand for more learning opportunities in global health in our sample, urging for reforms to adjust curricula to a globalising world.</p
Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells
yesThe gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a deïŹned substrate and is subject to biological variabil-ity and the potential to transmit disease. To better deïŹne HAM and mitigate the risk of disease transmis-sion, we sought to determine if decellularisation and/or c-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5 M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without c-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, DNp63, Ki67, CK12, and CK13). Æł-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and c-irradiation did not signiïŹcantly affect the LSC phe-notype, however LSCs expanded signiïŹcantly faster on Æł-irradiated SDS decellularised HAM (p < 0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efïŹciency assays showed a greater yield and proportion of holoclones in cells cultured on Æł-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efïŹcacious substrate for the expansion of LSCs and the use of a c-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer
Global Health Education: a cross-sectional study among German medical students to identify needs, deficits and potential benefits (Part 2 of 2: Knowledge gaps and potential benefits)
<p>Abstract</p> <p>Background</p> <p>In Germany, educational deficits or potential benefits involved in global health education have not been analysed till now.</p> <p>Objective</p> <p>We assess the importance medical students place on learning about social determinants of health (SDH) and assess their knowledge of global health topics in relation to (i) mobility patterns, their education in (ii) tropical medicine or (iii) global health.</p> <p>Methods</p> <p>Cross-sectional study among medical students from all 36 medical schools in Germany using a web-based, semi-structured questionnaire. Participants were recruited via mailing-lists of students' unions, all medical students registered in 2007 were eligible to participate in the study. We captured international mobility patterns, exposure to global health learning opportunities and attitudes to learning about SDH. Both an objective and subjective knowledge assessment were performed.</p> <p>Results</p> <p>1126 online-replies were received and analysed. International health electives in developing countries correlated significantly with a higher importance placed on all provided SDH (p †0.006). Participation in tropical medicine (p < 0.03) and global health courses (p < 0.02) were significantly associated with a higher rating of 'culture, language and religion' and the 'economic system'. Global health trainings correlated with significantly higher ratings of the 'educational system' (p = 0.007) and the 'health system structure' (p = 0.007), while the item 'politics' was marginally significant (p = 0.053).</p> <p>In the knowledge assessment students achieved an average score of 3.6 (SD 1.5; Mdn 4.0), 75% achieved a score of 4.0 or less (Q<sub>25 </sub>= 3.0; Q<sub>75 </sub>= 4.0) from a maximum achievable score of 8.0. A better performance was associated with international health electives (p = 0.032), participation in tropical medicine (p = 0.038) and global health (p = 0.258) courses.</p> <p>Conclusion</p> <p>The importance medical students in our sample placed on learning about SDH strongly interacts with students' mobility, and participation in tropical medicine and global health courses. The knowledge assessment revealed deficits and outlined needs to further analyse education gaps in global health. Developing concerted educational interventions aimed at fostering students' engagement with SDH could make full use of synergy effects inherent in student mobility, tropical medicine and global health education.</p
Transcription factor gene expression profiling and analysis of SOX gene family transcription factors in human limbal epithelial progenitor cells
To function as our âwindow to the worldâ, the cornea requires an intact epithelial surface. Epithelial stem/progenitor cells at the corneoscleral limbus are a reservoir for corneal epithelial homeostasis and repair. When these cells are lost, delayed wound healing, vascularisation and scarring may lead to painful loss of vision. Current treatment options include expansion of limbal epithelial progenitor cells (LEPCs) from a healthy donor eye through ex vivo culture and transplantation of these cells to the diseased ocular surface. However, the availability of autologous LEPCs for transplantation is limited in cases of systemic and/or bilateral disease. This has raised interest in the use of induced pluripotent cells or direct transdifferentiation of non-ocular cells towards a corneal epithelial phenotype. Transcription factors (TFs) are key players both in establishing pluripotency and in direct reprogramming. Understanding TF regulation of LEPCs and corneal epithelial homeostasis may aid in successfully using non-ocular cell sources to regenerate the corneal surface. Hence, this study aimed to identify differentially expressed TF genes in human limbal and corneal epithelial cells and to characterise their role for proliferation and differentiation of limbal epithelial cells.
LEPC clusters and central corneal epithelial cells were excised from cryosections of snap-frozen human post-mortem donor eyes using laser capture microdissection (LCM). RNA extracted from these specimens underwent linear amplification. Limbal and central corneal samples were screened for levels of expression of a panel of stem cell TF genes using real-time polymerase chain reaction (PCR) arrays. Four genes showed preferential limbal expression (at least two-fold elevated in limbal specimens compared to central cornea): DACH1, HOXA11, SOX9, and PPARG. Eleven genes were preferentially expressed in central corneal epithelial cells: FOXP2, RB1, MSX2, JUN, PCNA, SP1, SIX2, PAX6, FOXP3, SMAD2, and FOXP1. Validation experiments using real-time PCR hydrolysis probe assays confirmed statistically significant preferential limbal expression of SOX9 (with the highest fold change value of 428), DACH1 (272.5), HOXA11 (104.7) and PPARG (29.3).
Because SOX genes (Sry-related high mobility group box) encode TFs known to regulate cell fate and differentiation, a complete screen of SOX transcription factor gene expression was performed on LCM samples using real-time PCR. Preferential limbal expression was found for a number of SOX family genes, including all members of the SoxE, SoxF and SoxH groups.
Intracellular localization of their respective gene products was assessed using in-situ immunofluorescence. Here, SoxE proteins showed distinctive staining patterns, with predominantly cytoplasmic staining in basal limbal epithelial cells suggesting inhibition of its transcriptional program, and predominantly nuclear localisation in suprabasal and central corneal epithelial cells suggesting DNA binding and transcriptional activity. SOX9 was selected for further analyses given its strong expression and taking into consideration previous reports from other progenitor cell types. Using double-labeling, partial co-localisation was observed between SOX9 and putative limbal progenitor cell markers (Bmi1, OCT4, p63α, N-cadherin and Keratin 15).
SOX9 protein expression was also assessed in human corneas that had been subjected to a central epithelial wound in vitro. Increased expression and nuclear translocation of SOX9 was found in activated LEPCs and re-grown corneal epithelial cells compared to unwounded control eyes.
Next, mRNA expression of SOX9 was analyzed in primary cultures of limbal epithelial cells at different passages. Expression levels increased from P0 to P1 and P2. Immunofluorescent labeling of SOX9 in LEPC clones showed a nuclear staining pattern, with immunopositive cells being located predominantly towards the proliferating periphery of the clones.
Knockdown of SOX9-expression in cultured LEPCs was achieved using RNAi, and at 24 hours after transfection, effects on target gene regulation were monitored by real-time PCR. Expression of the progenitor cell marker gene KRT15 (Cytokeratin 15) was significantly reduced in cells after knockdown of SOX9. No significant changes were seen in expression of other progenitor cell marker genes such as CEBPD, ABCG2, p63α or CDH2. We did observe a trend towards upregulation of differentiation markers KRT3 and IVL but found no effect on expression of KRT12, PAX6 or MUC1. Also, we observed a trend towards upregulation of cyclin-dependent kinase inhibitors p21 and p57 and a trend towards downregulation of proliferation marker PCNA (Proliferating cell nuclear antigen). Using Western blot, reduced levels of Cytokeratin 15 and PCNA were detected in cultured cells following siRNA-mediated knockdown of SOX9. In line with downregulation of PCNA, proliferation rates (analyzed by BrdU incorporation) significantly decreased following knockdown of SOX9, in comparison to cells transfected with scramble siRNA.
In a nutshell, this study identified a number of TFs not previously known to be preferentially expressed in LEPCs. It also provided some evidence that SOX9, and potentially other SOX-family TFs, are expressed in LEPCs and may regulate corneal epithelial homeostasis. Our results suggest that SOX9 promotes proliferation and differentiation in transient amplifying cells following nuclear translocation, while supporting a progenitor cell phenotype and the continued expression of marker genes of putative LEPC by means of its cytoplasmic retention. Activation of SOX9 may assist clonal expansion, proliferation and differentiation of limbal epithelial cells in vitro for clinical applications. Therefore, SOX9 is a strong candidate gene, which, in combination with other factors, may form part of a strategy to achieve transdifferentiation and expansion of cells from non-ocular sources towards a corneal epithelial phenotype. The functional mechanisms underlying cytoplasmic retention and nuclear shuttling of SOX9 require further investigations
Implantation und Explantation von inaktiven, epiretinal fixierten Retina Implant Systemen am Minipig
Hereditary photoreceptor-degenerations of the retina, such as Retinitis pigmentosa, lead to progressive visual loss and often result in complete blindness of the patient. Until now it has not been possible to halt disease progression. For the purpose of visual rehabilitation of those affected, the development of an artificial visual prosthesis is being aimed at. Thanks to progress made in the fields of microtechnology and microsurgery different approaches emerge, particularly those where stimulation of the retina is performed via epiretinal, subretinal or transscleral electrodes. Preliminary studies suggest that electric stimulation at the level of the retina via a chronically implanted micro-contact-foil is able to induce stable visual phenomenons in patients over an extended period of time, which may be useful for spatial orientation. The subject-matter of the publication at hand is a system designed for complete intraocular implantation and wireless activation to receive and deliver electric signals. In the course of the development of this system the tolerability of the used materials inside the eye, feasibility of reversible epiretinal fixation via retinal tacks, the viability of implantation as well as cortical activation triggered by the system via wireless activation could be shown, among other features. The purpose of the trial described herein was to show that both implantation and explantation of the EPI-RETÂź III device could be conducted safely, and to investigate tissue compatibility via immunohistochemical examination. To attain this goal, the system was implanted in five Göttinger minipigs. The eyes of this species and those of humans are very much alike as far as size and anatomy are concerned. Following phacoemulsification of the lens and complete vitrectomy the implant could be inserted into the eye via a corneal incision. While the receiver was placed into the ciliary sulcus (or into the anterior chamber) the stimulator which is attached to it was placed onto the central retina. For explantation these steps were reversed, removing the implant from the eye as gently as possible. Finally, histopathologic examination of the study eyes was conducted. In the course of this examination, special attention was paid to the visualisation of potential inflammatory or proliferative changes. The use of known and established surgical materials and procedures has shown to be a special advantage of the completely intraocular concept of the EPI-RETÂź-prosthesis. Extensive bleeding, due to the very particular vascularisation of the ciliary body in pigs, has complicated especially the explantation, so that the prosthesis could not be recovered completely in all cases. However, this could be achieved through altering the implantation strategy and placing the receiver chip in loco alio (i.e. into the anterior chamber, instead of ciliary sulcus). In spite of these difficulties the chosen animal model has shown to be suitable for demonstrating the general viability of implantation and explantation of the EPI-RETÂź-system. The complications we came across using the minipigs are unlikely to be encountered to the same extend in humans. In the human eye there is some substantial experience with the implantation of technical devices (intraocular lenses). The exigencies of the German âMedizinproduktegesetzâ (national law to implement the European directive 90/385/EWG) for the safety of a human proband on the occasion of implantation and testing of the EPI-RETÂź III-system are fulfilled and a human trial can be deemed possible. This is supported by the absence of immunocompetent cells or a strong glial reaction in the histopathologic analysis of the study eyes
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