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Sheng, Huan. "Factors affecting corneal endothelial morphology." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1141395542.
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McGowan, Sara L. "Stem cell markers in the posterior limbus and cornea." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2007r/mcgowan.pdf.
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Arancibia, Carcamo Carolina Virgina. "Class II MHC on corneal endothelium : implications for corneal transplantation." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.395028.
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Parker, Douglas George Anthony, and park0290@flinders edu au. "Lentivirus-mediated gene expression in corneal endothelium." Flinders University. Medicine, 2008. http://catalogue.flinders.edu.au./local/adt/public/adt-SFU20081204.094431.
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Modulation of corneal transplant rejection using gene therapy shows promise in experimental models but the most appropriate vector for gene transfer is yet to be determined. The overarching aim of the thesis was to evaluate the potential of a lentiviral vector for use in human corneal transplantation. Specific aims were: (i) to assess the ability of an HIV-1-based lentiviral vector to mediate expression of the enhanced yellow fluorescent protein (eYFP), and a model secreted protein interleukin-10 (IL10), in ovine and human corneal endothelium; and (ii) to examine the influence of lentivirus-mediated IL10 expression on the survival of ovine corneal allografts.
Four lentiviral vectors expressing eYFP under the control of different promoters, were tested: the simian virus type-40 (SV40) early promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor-1alpha (EF) promoter, and the cytomegalovirus (CMV) promoter. Two lentiviral vectors expressing IL10 were tested: one containing the SV40 promoter and another containing a steroid-inducible promoter (GRE5). Lentivirus-mediated expression in transduced ovine and human corneal endothelium was assessed by fluorescence microscopy, real-time quantitative RT-PCR and ELISA, following alterations of transduction period duration (224 hr) and vector dose, as well as in the presence or absence of polybrene or dexamethasone (GRE5 vector). It was also compared to expression mediated by adenoviral vectors. Orthotopic transplantation of ex vivo transduced donor corneas was performed in outbred sheep. Allografts were reviewed daily for vascularisation and signs of immunological rejection.
Lentivirus-mediated eYFP expression was delayed in ovine corneal endothelium compared to human. However, in both species the final transduction rate was greater than 80% and expression was stable for at least 14 d in vitro. Lentivirus-mediated expression in ovine and human corneal endothelium was higher with the viral promoters in comparison to the mammalian promoters. A 24 h transduction of ovine corneal endothelium with the lentiviral vector encoding IL10 resulted in expression levels which were increasing after 15 d of organ culture but logarithmically lower than those achieved by adenovirus. Shortening the lentiviral transduction period to 2 h led to a reduction in expression, but the addition of polybrene (40 micrograms / ml) to the transduction mixture restored expression to levels comparable to those attained after a 24 h transduction period. Lentivirus-mediated IL10 expression was higher and more rapid in human corneal endothelium compared to ovine corneas. Dexamethasone-responsive transgene expression was observed in both ovine and human corneal endothelium using the lentiviral vector containing the GRE5 promoter. Lentivirus-mediated expression in ovine corneal endothelium was stable for 28 d in vivo. A modest prolongation of ovine corneal allograft survival (median of 7 d) was achieved by transduction of donor corneas for 23 h with the lentivirus expressing IL10. Attempts to increase the expression of IL10 by the addition of polybrene (40 micrograms / ml) to the transduction mixture, resulted in a toxic effect on corneal allografts which abrogated the beneficial effect of IL10.
The lentiviral vector shows potential for the stable expression of therapeutic transgenes in human corneal transplantation. However, the mechanisms underlying the species-specific differences in HIV-1-mediated transgene expression will need to be elucidated and overcome if the ovine preclinical model is to provide justification for a clinical trial.
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Martins, Luís Carlos [UNESP]. "Avaliação ultra-estrutural do endotélio corneal de ratos normais e de diabéticos aloxânicos." Universidade Estadual Paulista (UNESP), 2002. http://hdl.handle.net/11449/88910.
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martins_lc_me_botfm.pdf: 1919468 bytes, checksum: d4a42d395d3517a07ebafa18c26a4e02 (MD5)O objetivo do estudo foi avaliar a influência do diabetes experimental sobre a ultra-estrutura do endotélio corneal de ratos. O estudo foi prospectivo, utilizando 20 ratos da raça Wistar, com 3 meses de idade, divididos em: grupo controle (GC), contendo 10 ratos, e grupo diabético (GD), contendo 10 ratos. A indução do diabetes foi feita com injeção de Aloxana endovenosa 42 mg/kg de peso (M0), após o que os animais foram observados por 15 dias para confirmar a presença de diabetes grave (M1). Um mês após M1 (M2) e 12 meses após M1 (M3) os animais foram sacrificados, sendo removidos e preparados os olhos para avaliação à microscopia eletrônica de transmissão. Os animais do GD mostraram importante redução de peso, aumento da injestão hídrica e aumento da diurese em relação aos ratos do GC. Na avaliação morfológica observou-se nos animais do GC corpos densos e figuras de Mielina no M3. Os ratos do GD apresentaram as mesmas alterações encontradas no GC em M3, em maior intensidade, com alterações nucleares e citoplasmáticas, como mitocôndrias bastante alteradas na forma e tamanho, rarefação do citoplasma e aumento de vesículas. Os ratos do GD em M3 apresentaram mais alterações que os do GDM2. Conclui-se que o diabetes experimental causa dano ultra-estrutural no endotélio corneal de ratos e que as alterações são evolutivas.
The objective of study was to make na assessment of experimental diabetes influence on of rats corneal endothelium ultra-structure. The study was prospective, using 20 Wistar 3-month-old rats, divided (by draw) into groups: control group (GC), with 10 rats, and diabetic group (GD), with 10 rats. The diabetes induction was made by means of intravenous injection of Aloxan 42 mg/Kg weigth. After the diabetes induction (M1), the animals had been observed for 15 days, and then, 1 month after M1 (M2) and 12 months after M1 (M3) to confirm the diagnosis of severe diabetes. At experimental moments M2 and M3, the animals eyes enucleation and preparation were carried out for assessment trough transmission eletronic microscopy. GD animals had shown significant reduction of weigth, increasing of hydric and nourishing injection and increasing of diuresis in relation to GC rats. In the morphological assessment, dense bodies and Myelin figures were observed in M3 GC animals. GC rats had presented the same alterations found in GC animals in M3, in major intensity, beyond mitochondrias rather modified in their form and size, cytoplasm rarefaction, vesicles increasing and nuclear alterations. It is concluded that experimental diabets causes ultra-structural of rats corneal endothelium.
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Martins, Luís Carlos. "Avaliação ultra-estrutural do endotélio corneal de ratos normais e de diabéticos aloxânicos /." Botucatu : [s.n.], 2002. http://hdl.handle.net/11449/88910.
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Orientador: Silvana Artioli SchelliniResumo: O objetivo do estudo foi avaliar a influência do diabetes experimental sobre a ultra-estrutura do endotélio corneal de ratos. O estudo foi prospectivo, utilizando 20 ratos da raça Wistar, com 3 meses de idade, divididos em: grupo controle (GC), contendo 10 ratos, e grupo diabético (GD), contendo 10 ratos. A indução do diabetes foi feita com injeção de Aloxana endovenosa 42 mg/kg de peso (M0), após o que os animais foram observados por 15 dias para confirmar a presença de diabetes grave (M1). Um mês após M1 (M2) e 12 meses após M1 (M3) os animais foram sacrificados, sendo removidos e preparados os olhos para avaliação à microscopia eletrônica de transmissão. Os animais do GD mostraram importante redução de peso, aumento da injestão hídrica e aumento da diurese em relação aos ratos do GC. Na avaliação morfológica observou-se nos animais do GC corpos densos e figuras de Mielina no M3. Os ratos do GD apresentaram as mesmas alterações encontradas no GC em M3, em maior intensidade, com alterações nucleares e citoplasmáticas, como mitocôndrias bastante alteradas na forma e tamanho, rarefação do citoplasma e aumento de vesículas. Os ratos do GD em M3 apresentaram mais alterações que os do GDM2. Conclui-se que o diabetes experimental causa dano ultra-estrutural no endotélio corneal de ratos e que as alterações são evolutivas.
Abstract: The objective of study was to make na assessment of experimental diabetes influence on of rats corneal endothelium ultra-structure. The study was prospective, using 20 Wistar 3-month-old rats, divided (by draw) into groups: control group (GC), with 10 rats, and diabetic group (GD), with 10 rats. The diabetes induction was made by means of intravenous injection of Aloxan 42 mg/Kg weigth. After the diabetes induction (M1), the animals had been observed for 15 days, and then, 1 month after M1 (M2) and 12 months after M1 (M3) to confirm the diagnosis of severe diabetes. At experimental moments M2 and M3, the animals eyes enucleation and preparation were carried out for assessment trough transmission eletronic microscopy. GD animals had shown significant reduction of weigth, increasing of hydric and nourishing injection and increasing of diuresis in relation to GC rats. In the morphological assessment, dense bodies and Myelin figures were observed in M3 GC animals. GC rats had presented the same alterations found in GC animals in M3, in major intensity, beyond mitochondrias rather modified in their form and size, cytoplasm rarefaction, vesicles increasing and nuclear alterations. It is concluded that experimental diabets causes ultra-structural of rats corneal endothelium.
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Jones, Frances E. "The corneal endothelium in development, disease and surgery." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/49911/.
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Aims: The cornea is a tough, transparent tissue providing the primary refractive element of the eye. The stroma consists of specially arranged collagen required for corneal transparency. Correct stromal hydration is important in the maintenance of transparency, a feature controlled by the endothelial cells on the posterior surface of the cornea. The aims of this research were firstly to investigate the morphology of corneal endothelial cells and their expression of the sodium bicarbonate cotransporter during avian embryonic development and secondly, to clarify the effect of disease, surgery and drugs on the posterior cornea including in particular the corneal endothelium. Methods: The corneal endothelial cell morphology and posterior stroma were examined using transmission electron microscopy to determine the ultrastructure of the cells and collagen fibril arrangement in the stroma in all results chapters. Immunohistochemistry and A-scan ultrasonography were employed to identify the presence of the Na+HCO3- cotransporter and to determine the thickness changes in embryonic chick cornea, respectively. Electron tomography was also used to determine the collagen arrangement in Descemet’s membrane. Results: The expression of the Na+HCO3- cotransporter was identified in the endothelial layer of the embryonic chicks at all stages imaged. Central corneal thickness increased in the initial stages of development before a plateau between the E12-E15 developmental period followed by a steady thickness decrease. The ultrastructure of Descemet’s membrane was determined using electron tomography of transverse and en face resin embedded sections from which a model was produced. Polygonal and elongated structures were observed with proteoglycans present at the intermodal regions of the collagenous structures. The polygonal lattice visualised in en face sections appeared to be composed of stacked globular domains which were integrated into the collagen type VIII model. Predominant changes in the Col8a2 knock-in mouse models were observed in the posterior cornea. Differences included increased proteoglycans at the Descemet’s endothelial interface, dilated rough endoplasmic reticulum and focal posterior oedema. This animal model exhibits features similar to those seen in the human form of early-onset Fuchs’ endothelial corneal dystrophy, unlike previous models reported. The final chapter is concerned with regeneration of the corneal endothelial cells. Tissue from posterior corneal surgery examined using electron microscopy revealed the presence of the host endothelial cells and fibrous tissue at the interface in non-Descemet’s membrane stripping automated endothelial keratoplasty and interface haze in Descemet’s membrane stripping automated endothelial keratoplasty. However, these features did not appear to interfere with the adhesion of the graft nor the clarity. Finally, ultrastructural analysis of Rho-kinase inhibited cells showed cells with typical morphology when compared with the untreated group Conclusions: 1) The Na+HCO3- cotransporter is present in the embryonic cornea. It is possible that the cotransporter is involved in the developmental stages and probably the thickness changes we observe during this period. 2) The ultrastructure of Descemet’s membrane appears to be composed of stacked globular domains arranged in a polygonal lattice alongside more elongated structures interspersed with proteoglycans within the internodal regions. 3) Our studies have helped validate Col4a2 mice as a promising Fuchs’ endothelial corneal dystrophy model. 4) Our investigation into posterior corneal surgery revealed ultrastructural changes that occur post-surgery at the graft interface.
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Painter, Geoffrey Thomas. "Corneal Protection in Cataract Surgery." Thesis, The University of Sydney, 2019. https://hdl.handle.net/2123/21214.
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Protection of the corneal endothelium is one of the most important aspects of cataract surgery. This thesis describes two studies that examine new products and techniques to protect the cornea in cataract surgery. The first study examines the corneal protective effect of a new viscoelastic, DisCoVisc, compared to two established products in a randomised control trial of 180 patients. While no objective difference in corneal protection could be found between DisCoVisc and the other two viscoelastics, DisCoVisc compared more favourably in subjective surgical behaviour when compared to Healon although no difference could be found when compared to Amvisc Plus. Complications during surgery have the potential to adversely affect the corneal endothelium. The second study is a retrospective analysis of 1589 cases of Femtosecond Laser Assisted Cataract Surgery (FLACS) using the LenSx femtosecond laser with the SoftFit patient interface. It found the rate of capsular complications using the LenSx femtosecond laser with the SoftFit patient interface was notably lower than published rates of capsular complications with manual phacoemulsification. It compares favourably with earlier studies using the LenSx platform as well as earlier studies using other femtosecond laser platforms. This study’s result is in disagreement with a meta-analysis published in 2016 which found a higher capsular complication rate with FLACS and is more in keeping with recent published studies of the safety of FLACS using the LenSx platform.
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Painter, Geoffrey Thomas. "Corneal Protection in Cataract Surgery." Thesis, The University of Sydney, 2019. https://hdl.handle.net/2123/21389.
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Protection of the corneal endothelium is one of the most important aspects of cataract surgery. This thesis describes two studies that examine new products and techniques to protect the cornea in cataract surgery. The first study examines the corneal protective effect of a new viscoelastic, DisCoVisc, compared to two established products in a randomised control trial of 180 patients. While no objective difference in corneal protection could be found between DisCoVisc and the other two viscoelastics, DisCoVisc compared more favourably in subjective surgical behaviour when compared to Healon although no difference could be found when compared to Amvisc Plus. Complications during surgery have the potential to adversely affect the corneal endothelium. The second study is a retrospective analysis of 1589 cases of Femtosecond Laser Assisted Cataract Surgery (FLACS) using the LenSx femtosecond laser with the SoftFit patient interface. It found the rate of capsular complications using the LenSx femtosecond laser with the SoftFit patient interface was notably lower than published rates of capsular complications with manual phacoemulsification. It compares favourably with earlier studies using the LenSx platform as well as earlier studies using other femtosecond laser platforms. This study’s result is in disagreement with a meta-analysis published in 2016 which found a higher capsular complication rate with FLACS and is more in keeping with recent published studies of the safety of FLACS using the LenSx platform.
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Ojo, Victor Temidayo. "Postnatal Cell Shape development of the Corneal Endothelium in Mice." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etd/3301.
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Corneal endothelial cells have been shown to possess a uniform polygonal and complex multipolar shape at their apical and basolateral surface respectively. We established a morphological timetable to study how this complex shape arises postnatally. Corneas were collected from mice between postnatal day 8 to postnatal day 35 and labelled with antibodies specific for ZO-1 and NCAM at apical and basolateral region, respectively. Images were collected using wide-field fluorescence microscopy and morphometrically analyzed. Results showed that apical cell sizes increase linearly over the first 3 weeks, plateauing at 4-5 weeks postnatally with increased regularity. Basolateral membrane surfaces remained relatively smooth prior to eyelid opening and thereafter begins developing showing differences in development from periphery to the center until about 4 weeks postnatally when the wavy processes get vivid. Results indicate concurrent and independent development at both poles of the corneal endothelium, with more complexity seen at the basolateral surface.
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Bosch, Canals Begoña María. "A bioengineering approach for corneal endothelial regeneration." Doctoral thesis, Universitat Internacional de Catalunya, 2019. http://hdl.handle.net/10803/667398.
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Nowadays, there are approximately 10 million people worldwide with visual impairment due to corneal diseases. Currently, the main therapeutic solution is the transplant of a donor's cornea. The great majority of transplants is due to some failure in the inner layer of the cornea, which is called the corneal endothelium and this is mainly related with the inability of this layer to regenerate in vivo. However, transplants present several limitations such as the low number of healthy donors or immunological rejection by the patient.
In order to overcome these problems, several researchers have focused in culturing corneal endothelial cells (CEC) to subsequently replace non-functional CEC. However, cell therapy is still very recent and still presents a series of drawbacks. For instance, using animal CEC or cells from other patients has shown to lead into immunological rejection. In order to avoid this, it is possible to use stem cells from the same patient, which have the ability to differentiate into many cell types, including the corneal endothelium. Currently, the stem cells used to regenerate CEC are mainly pluripotent stem cells, either embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC), which are derived from adult cells. Despite their great potential for treating diseases, these types of stem cells present major limitations such as the risk of teratoma formation. In addition, they present other disadvantages such as ethical problems associated with the use of ESCs, safety problems related to iPSC since they requires the use of virus for their production hence limiting its clinical application.
For this reason, and in order to solve the current problems in the regeneration of corneal endothelium, this thesis project uses dental pulp stem cells (DPSC) for the formation of CEC. DPSC are an accessible source derived from the same patient, avoiding possible future problems of rejection. In addition, the use of DPSC avoids the ethical and security problems associated with ESC and iPSC. Furthermore, DPSC and CEC have the same embryological origin, as they both arise from neural crest stem cells. In fact, DPSC express neural crest stem cells markers, which facilitates their differentiation into neural crest stem cells (NCSC), which is an intermediate step for the formation of CEC. Therefore, this thesis project uses a two-step protocol, where DPSC are differentiated into NCSC and, subsequently, NCSC are derived into CEC.
Because the use of cell therapies alone may present limited cell viability once it is injected, the field of tissue engineering is a new discipline that has appeared to overcome this limitation. Tissue engineering combines the use of cells, biomaterials and biological molecules. It has been demonstrated that the use of different topographies in cell culture modulates cell behavior, and may have an effect on their functionality, cell distribution or cell size. Therefore, this thesis project applies tissue engineering as another strategy for the generation of functional CEC with its characteristic phenotype and morphology. For doing this, we have mimicked the natural CEC environment by cultivating the cells on substrates with different curvatures, composition or topographies that are able to mimic those of the human eye.
In conclusion, this thesis project proposes the use of bioengineering, by differentiating CEC from stem cells derived from the patient and the use of biomaterials with different topographies and curvatures, for the regeneration of corneal endothelium.
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Shivanna, Mahesh. "Breakdown of the barrier integrity in corneal endothelium by inflammatory stress." [Bloomington, Ind.] : Indiana University, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3386723.
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Thesis (Ph.D.)--Indiana University, School of Optometry, 2009.Title from PDF t.p. (viewed on Jul 22, 2010). Source: Dissertation Abstracts International, Volume: 70-12, Section: B, page: 7504. Adviser: Sangly P. Srinivas.
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Hünning, Paula Stieven. "Avaliação da morfologia pós-operatória das células do endotélio corneano de coelhos na região periférica perincisional comparativamente à região central." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2011. http://hdl.handle.net/10183/30846.
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A manutenção da morfologia normal do endotélio da córnea é um importante indicador da integridade funcional. A reparação endotelial frente a um trauma, em coelhos, ocorre por migração, hipertrofia e mitose celular. Objetivou-se comparar a morfologia das células do endotélio, da região periférica perincisional à região central, da córnea de coelhos (Oryctolagus cuniculus) em diferentes períodos pós-operatórios. Foram designados três grupos, com 5 animais cada, para avaliação pós-operatória, sendo G1 (7 dias); G2 (15 dias) e G3 (45 dias). Trinta bulbos dos olhos de coelhos, da raça Nova Zelândia, foram submetidos à incisão de córnea clara uniplanar com 3,2 mm. Ao fim dos períodos determinados, procedeu-se a avaliação da morfologia endotelial valendose da microscopia eletrônica de varredura. Realizaram-se seis eletromicrografias de varredura, de cada região da córnea, com aumento de 1000 vezes. Para análise do percentual do número de lados celular, foram analisadas 100 células endoteliais. Na região periférica perincisional, avaliada ao 7° dia de pós-operatório, foram encontradas células com 6 lados (47,8%), 5 lados (31,3%), 7 lados (13,9%), 3 lados (0,1%), 4 lados (4,9%), 8 lados (1,8%) e 9 lados (0,2%). Na avaliação ao 15° dia de pós-operatório, observaram-se células com 6 lados (45,6%), 5 lados (32,6%), 7 lados (17,4%), 4 lados (1,7%) e 8 lados (2,7%). No 45° dia de pós-operatório, verificou-se a presença de células com 6 lados (57%), 5 lados (24%), 7 lados (17,2%), 4 lados (0,1%), 8 lados (1,6%) e 9 lados (0,1%). Na área central, ao 7° dia de pós-operatório, detectaram-se células com 6 lados (75,6%), 5 lados (13,3%), 7 lados (10,8%) e 8 lados (0,3%). Na avaliação, ao 15° dia de pós-operatório, foi possível observar células com 6 lados (78,9%), 5 lados (11,5%) e 7 lados (9,6%). No 45° dia de pós-operatório identificaram-se células com 6 lados (74,8%), 5 lados (13,6%) e 7 lados (11,6%). Os resultados demonstraram que na região periférica perincisional ocorreu diminuição das células com seis lados e aumento do número de células com cinco e sete lados. Na região central manteve-se o padrão regular de hexagonalidade das células endoteliais nos diferentes períodos pós-operatórios. Conclui-se que houve alteração na morfologia das células endoteliais, da região periférica perincisional comparada à região central, da córnea de coelhos nos diferentes períodos pós-operatórios.The maintenance of the normal corneal endothelium morphology is an important indicator of its functional integrity. In rabbits, endothelial repair in the event of traumas is made through cell migration, hypertrophy and mitosis. The purpose of this study was to compare the morphology of endothelial cells of the perincisional area with the central area of the cornea of rabbits (Oryctolagus cuniculus), in different post-operative periods. Three groups containing 5 animals each were designed for post-operative evaluation: G1 (7 days); G2 (15 days) and G3 (45 days). The clear cornea of thirty New Zealand rabbits was subjected to a single-planed incision of 3.2 mm. At the end of the established periods, a morphological evaluation of the endothelium was carried out using scanning electron microscopy. Six scanning electron micrographs of each corneal area were performed using a magnification of 1000 x. One hundred endothelial cells were analyzed to obtain the cell side count percentage. In the perincisional peripheral area, which was evaluated at the 7 post-operative day, 6-sided (47.8%), 5-sided (31.3%) and 7-sided (13.9%) cells were found, in addition to, 3-sided cells (0.1%), 4-sided cells (4.9%), 8-sided cells (1.8%) and 9-sided cells (0.2%). In the evaluation made on the 15th post-operative day, 6-sided (45.6%), 5-sided (32.6%) and 7-sided (17.4%) cells were observed, as well as 4-sided (1.7%) and 8-sided cells (2.7%). On the 45th postoperative day, the presence of 6-sided (57%), 5-sided (24%), 7-sided (17.2%), 4-sided (0,1%), 8-sided (1.6%) and 9-sided cells (0.1%) was verified. On the 7th post-operative day, 6-sided (75.6%), 5-sided (13.3%), 7-sided (10.8%) and 8-sided cells (0.3%) were observed in the central area .Upon evaluation made on the 15th post-operative day, it was possible to observe 6-sided (78.9%), 5-sided (11.5%) and 7-sided (9.6%) cells. Results have shown that there was a reduction of six-sided cells and an increase in the number of five and seven-sided cells in the perincisional peripheral area. The regular hexagonal standard of the endothelial cells was maintained in the central area in different post-operative periods. In comparison to the central area, there was a morphological alteration of the endothelial cells of the peripheral perincisional area in different post-operative periods of the cornea of rabbits.
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Mgwebi, Thandiswa. "Morphological investigations into the development of the mammalian corneal endothelium using the mouse model." Doctoral thesis, University of Cape Town, 2004. http://hdl.handle.net/11427/3268.
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Includes bibliographical references (leaves 83-89).The corneal endothelium (CE), a mesenchyme-derived tissue, is a monolayer of squamous cells on the inner corneal surface. In Foxc1-1• mice, the CE fails to form. The understanding of the cause of this defect has implications for the study of human eye disorders that are related to FOXC1 mutations. To understand the basis of CE defects in Foxc1-1- mice, an analysis of normal CE development was performed using scanning electron microscopy. Results showed that in normal mice the transformation from mesenchyme to endothelium was initiated at embryonic day (E) 12.5 and was characterised by a change from stellate to cobblestone shape and the formation of junctions. In FoxcN- mice, the process was initiated but a cobblestone shape not attained. The expression of adherens (N-cadherin) and tight junction (Z0-1) proteins was investigated by immunoflouresence microscopy. In the normal embryo, the expression of N-cadherin was initially in cytoplasmic vesicles and later at the cell membranes. ZO-l was first detected at the cell peripheries at E13.5. In Foxct-I- mice, N-cadherin peripheral bands failed to form. ZO-l was not expressed. These results suggest that the failure to form a monolayered CE in Foxc1 mice is due to incomplete mesenchyme-endothelial conversion. Junction formation was further investigated in vitro. N-cadherin was cytoplasmic in pre-confluent cells and at cell edges in confluent cells. ZO-l was not detected. These results suggest that in vitro, these cells are either unable to form tight junctions or the culture medium does not contain the appropriate signalling molecules.
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Rudnick, David Jr. "Hyperadaptation - Another Missing Term in the Science of Form." Thesis, Virginia Tech, 1997. http://hdl.handle.net/10919/36886.
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In a 1982 paper, Gould and Vrba argue that a conflation of the two components of adaptation of a feature, historical development of the feature and present utility, has caused evolutionists to overlook a missing term in the science of form, which they call 'exaptation'. In the present project, I show that evolutionary biology still contains a confusion in the use of 'adaptation' due to an inappropriate perception of the interaction between the two components of adaptation. Because of the confusion, evolutionists have missed another term in the science of form. Evolutionary theory, specifically the treatment of adaptation, would profit from the introduction of a term referring to features that have a selective history which causes them to appear overly well adapted to their present function. I suggest we refer to these features as hyperadaptations, since they appear to be hyperbolized adaptations. By introducing hyperadaptation into the conceptual framework of adaptation, we can sharpen our understanding of related concepts (adaptation to function, exaptation, maladaptation, etc.) and remove or reduce some confusion regarding the interplay between analysis of historical pathways and ascriptions of (current) function in the diagnosis of adaptation. Furthermore, the improved framework should allow evolutionists to more adequately explain biological phenomena.Master of Arts
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Pedemonte, Sarrias Eduard. "Tècnica de Muraine per a DMEK: anàlisi comparativa amb la tècnica estàndard." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/405259.
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La queratoplàstia endotelial de la membrana de Descemet (DMEK) és la tècnica d'elecció actual per al tractament de l’edema corneal irreversible. Després que Melles la desenvolupés el 2006, Muraine va proposar el 2013 una tècnica alternativa per a la dissecció i implantació de l’empelt. Aquesta tècnica aportava dues novetats: la hidrodissecció amb trepanació parcial i curvatura corneal invertida del teixit donant i el plegament de l’empelt amb l'endoteli a la seva cara interna, que afavoria la protecció de l’endoteli i la tendència natural de l’empelt a desplegar-se a la cambra anterior del receptor.
L’objectiu d’aquesta tesi doctoral és comparar la tècnica de Muraine amb la tècnica estàndard analitzant-ne la densitat de cèl·lules endotelials (DCE) i agudesa visual (AV) postoperatòries, els temps quirúrgics i les complicacions intraoperatòries i postoperatòries. Es va dur a terme un estudi de cohorts prospectiu observacional multicèntric en la pràctica clínica habitual a l’Hospital Universitari MútuaTerrassa i l’Institut de Microcirurgia Ocular. El seguiment postoperatori va ser de sis mesos, amb controls com a mínim l’endemà, al cap d’una setmana i al cap d’un, tres i sis mesos de la cirurgia.
Es van incloure 27 ulls de 20 pacients al grup de la tècnica Estàndard i 42 ulls de 40 pacients al grup intervingut amb la tècnica Muraine. La DCE als sis mesos va ser de 1488 (1337-1679) cèl·lules/mm2 al grup Estàndard i de 1170 (734-1614) cèl·lules/mm2 al grup Muraine (P=0.10). L’AV mitja als sis mesos va ser de 0.89 al grup Estàndard i 0.79 al grup Muraine, en l’escala decimal (P=0.19). Al voltant del 80% van assolir una AV de 0.5 o més i el 50-70%, 0.8 o més.
Es va observar que la DCE i el percentatge de pèrdua de DCE al mes de la cirurgia eren equivalents als de la tècnica estàndard. El percentatge de pèrdua de DCE als sis mesos va ser superior amb la tècnica de Muraine, si bé la DCE va ser clínicament comparable. L’AV assolida als sis mesos va ser equivalent. La tècnica de Muraine va ser tan segura com la tècnica estàndard per a l’obtenció de l’empelt. La incidència de complicacions intraoperatòries amb l’empelt preparat amb la tècnica de Muraine en ulls amb facoemulsificació no complicada no va ser estadísticament superior. La dissecció de l’empelt amb la tècnica de Muraine va ser més lenta. El desplegament, per contra, va ser lleugerament més ràpid. Totes dues tècniques van tenir una taxa elevada de supervivència de l’empelt. La complicació postoperatòria més freqüent en ambdós grups va ser l’edema macular quístic. Els empelts dissecats amb la tècnica de Muraine van tenir una major incidència de necessitat de reinsuflació.Descemet’s membrane endothelial keratoplasty (DMEK) is the current gold standard treatment for irreversible corneal oedema. After Melles developed this technique in 2006, Muraine proposed in 2013 an alternative technique for the dissection and implantation of the graft. Its main contributions were: hidrodissecting the graft from a partially trephined, inverted donor tissue, and folding the graft over the endothelial side, which favoured the protection of endothelial cells and the graft’s natural tendency to unfold in the receptor’s anterior chamber. The purpose of this doctoral thesis is to compare Muraine’s technique to the Standard through analysis of the postoperative endothelial cell density (ECD) and visual acuity (VA), surgical time, and intraoperative and postoperative complications. An observational, multicentric, prospective, cohorts trial was carried out in Hospital Universitari MútuaTerrassa and Institut de Microcirurgia Ocular in a daily praxis basis. There were follow-up controls over the six months following the surgery, at least at day one, first week and first, third and sixth months. Twenty-seven eyes from 20 patients were included in the Standard technique group. Forty-two eyes from 40 patients were included in the Muraine’s technique group. The ECD at six months was 1488 (1337-1679) cells/mm2 for the Standard group and 1170 (734-1614) cells/mm2 for Muraine’s group. The mean VA at six months was 0.89 for the Standard group and 0.79 for Muraine’s group, in the decimal scale (P=0.19). Around 80% of the eyes reached a VA of 0.5 or higher and 50-70%, 0.8 or higher. The ECD and the percentage of ECD loss with Muraine’s technique at the first month after surgery were equivalent to the Standard technique’s. The percentage of ECD loss at six months was higher with Muraine’s technique, although the ECD was clinically comparable. The VA achieved at six months was equivalent. Muraine’s technique was as safe as the Standard technique for the graft dissection. The incidence of intraoperative complications among the eyes with uncomplicated phacoemulsification was not statistically higher with Muraine’s technique. The graft dissection with Muraine’s technique was slower. Conversely, the unfolding was slightly faster. Both techniques had a high graft survival rate. The most frequent postoperative complication in both groups was cystoid macular oedema. The grafts dissected with Muraine’s technique had a higher incidence of need for rebubbling.
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Mueller, Andreas. "Assessment of eye growth-related changes in the corneal endothelium of children and young teenagers." Thesis, Glasgow Caledonian University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341552.
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Kocaba, Viridiana. "Tissue engineering pour la reconstruction cornéenne." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1078.
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En France, les dysfonctions endothéliales représentent environ la moitié des indications de greffes de cornée réalisées chaque année. Cependant, les problématiques liées à la pénurie de greffon, aux difficultés des techniques chirurgicales de greffes endothéliales ainsi qu’aux risques d’échec ou de rejet de greffe poussent les chercheurs à développer de nouvelles thérapies moins invasives et plus efficaces. La thérapie cellulaire cornéenne endothéliale est une des voies de recherche actuellement explorées dont le but est de s’affranchir des aléas de la greffe de cornée. La cornée humaine est un tissu idéal pour la thérapie cellulaire. Grâce à ses caractéristiques d’organe à la fois avasculaire et immunitairement privilégié, les cellules transplantées sont ainsi bien mieux tolérées par rapport aux autres tissus et organes vascularisés. Les avancées dans le domaine des cellules souches, de l'ingénierie, particulièrement avec l’arrivée des greffes de cellules souches épithéliales pour le traitement des pathologies sévères de la surface oculaire, ont suscité un intérêt massif afin d’adapter ces techniques aux cellules endothélialesIn France, around half of all corneal keratoplasties are performed to treat corneal endothelial dysfunction each year. However, the use of endothelial keratoplasty is limited by the technical difficulty of the procedure, a shortage of available grafts, and the potential for graft failure or rejection. These limitations are driving researchers to develop new, less invasive, and more effective therapies. Corneal endothelial cell therapy is being explored as a potential therapeutic measure, to avoid the uncertainty associated with grafting. The human cornea is an ideal tissue for cell therapy as owing to its avascular characteristics, transplanted cells are better tolerated compared with other vascularized tissues and organs. Advances in the field of stem-cell engineering, particularly the development of corneal epithelial stem cell therapy for the treatment of severe diseases of the ocular surface, have aroused a massive interest in adapting cell-therapy techniques to corneal endothelial cells
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PINA, Fábio Luiz Silva. "Preservação de córneas de felinos domésticos (Felis catus - Linnaeus, 1758) em conservante utilizando a água de coco como meio nutritivo." Universidade Federal Rural de Pernambuco, 2007. http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/5619.
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Corneal endotelium cells present the important function of osmotic pump and barrier, thus keeping it transparent. The loss of these cells, either by any reason, becomes the cornea cloudy, influencing negativety in the visual capacity of the individual. For this fact, the conservation of corneas for transplant ends has evolved sufficiently to the long one of the years. Had to the characteristics desirable physicist-chemistries and the low cost of the coconut water, the aim of this work was to investigate the possibility of the use of the coconut water as nutritious medium in corneal preservation medium. A total of 9 cats, without no corneal pathology in course, had been sacrified. One cornea of each pair was evaluated immediately, being the other evaluated after preservation on the medium with coconut on days 3, 7 and 14. The corneas had been processed for optic microscopy. The corneas had become impracticable since day 3 of conservation, having presented morphologic alterations, signals of cellular death and estromal thickness increased, indicating important edema. We conclude that the coconut water did not serve as nutritious medium for corneal storage medium of domestic felines, however the corneas, due to absence of contamination of the medium, can be used in tectonic transplants and lamellar keratoplasty.
As células do endotélio corneal apresentam a importante função de bomba osmótica e barreira, mantendo assim a córnea transparente. A perda destas células, seja por qualquer motivo, tornam a córnea opaca, influenciando negativamente na capacidade visual do indivíduo. Por este fato, a conservação de córneas para fins de transplante tem evoluído bastante ao longo dos anos. Devido às características físico-químicas desejáveis e ao baixo custo da água de coco, o objetivo deste trabalho foi investigar a possibilidade do uso da água de coco como meio nutritivo em conservantes de córnea. Um total de 9 gatos, sem nenhuma patologia corneal em curso, foram eutanasiados. Uma córnea de cada par foi avaliada imediatamente, sendo a outra avaliada após a preservação no meio com água de coco nos dias 3, 7 e 14. As córneas foram processadas para microscopia óptica. As córneas tornaram-se inviáveis a partir do dia 3 de conservação, apresentando alterações morfológicas, sinais de morte celular e espessura estromal aumentada, indicando edema importante. Concluiu-se que a água de coco não serviu como meio nutritivo para conservantes de córneas de felinos domésticos, porém as córneas, devido à ausência de contaminação do meio, podem ser utilizadas em transplantes tectônicos e ceratoplastias lamelares.
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Oblak, Emil. "Assessment of welding ultraviolet radiation on the corneal endothelium using a newly developed computerized morphometric system." Thesis, Glasgow Caledonian University, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289519.
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Zhang, Yan. "Studies on the calcium-regulated bicarbonate ion permeability in the apical membrane of bovine corneal endothelium." [Bloomington, Ind.] : Indiana University, 2004. http://wwwlib.umi.com/dissertations/fullcit/3162273.
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Thesis (Ph.D.)--Indiana University, School of Optometry, 2004.Title from PDF t.p. (viewed Dec. 2, 2008). Source: Dissertation Abstracts International, Volume: 66-01, Section: B, page: 0050. Chair: Joseph A. Bonanno.
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Al, Abdulsalam Najla Khaled S. "Evaluation of silk fibroin as a scaffold for cultured corneal endothelial cell implants." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/122231/1/Najla%20Khaled%20S_Al%20Abdulsalam_Thesis.pdf.
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Corneal transplants are a safe and effective treatment for corneal disease, but are hampered by the limited supply and quality of donor tissue. The goal of this project was therefore to evaluate the use of membranes prepared from silk protein as a scaffold on which to grow corneal tissue substitutes in the laboratory from corneal endothelial cells.
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Huang, Lan. "Endothelial Colony Forming Cells (ECFCs): Identification, Specification and Modulation in Cardiovascular Diseases." Thesis, Connect to resource online, 2009. http://hdl.handle.net/1805/2063.
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Thesis (Ph.D.)--Indiana University, 2009.Title from screen (viewed on February 2, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Mervin C. Yoder, Jr., David A. Ingram, Jr., Lawrence A. Quilliam, Mark D. Pescovitz. Includes vitae. Includes bibliographical references (leaves 171-194).
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Corrêa, Luis Felipe Dutra. "Morfologia e morfometria das células do endotélio corneal de ovinos (Ovis aires) em diferentes faixas etárias obtidas pela microscopia especular." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2011. http://hdl.handle.net/10183/132917.
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Baseado na carência de documentação com respeito ao endotélio da córnea de ovinos, a pesquisa teve como objetivo documentar o endotélio corneal de ovinos em diferentes faixas etárias – jovem, adulto e velho – utilizando a microscopia especular. Logo, buscou-se investigar o efeito da idade sobre a morfologia e morfometria do endotélio da córnea de ovinos. O endotélio corneano foi avaliado com auxilio do microscópio especular de contato. No total, foram avaliados 18 ovinos (Ovis aires) neste estudo. Os animais foram distribuídos em três grupos de 6 animais cada um em função da idade. O grupo I continha animais com idade de 6 meses; o grupo II, animais com idade de 2 a 4 anos; e o grupo III continha animais de 5 a 8 anos. Para a avaliação do endotélio corneal foram levados em consideração a densidade de células endoteliais (DCE), a área celular média, o polimegatismo e o pleomorfismo. Os resultados encontrados revelaram a diminuição na DCE em córneas de ovinos normais com o avanço da idade bem como o aumento da área das células endoteliais e do pleomorfismo. O presente trabalho mostrou que os parâmtros endoteliais valiados sofrem alterações decorrentes da idade.Based on the lack of information on the corneal endothelium of ovine, the objective of this research was to evaluate the corneal endothelium of sheep in different age groups – young, adult and old - using the specular microscope. So the aim of this study was to investigate the effect of age on morphology and morphometry corneal endothelium of sheep. The corneal endothelium was evaluated with the help of a contact specular microscope. A total of 18 sheep (Ovis aires) were evaluated in this study. The sheep mob was divided into three groups of six animals each according to age. Group I contained animals 6 months old, group II contained animals from 2 to 4 years old and group III contained animals from 5 to 8 years old. To evaluate corneal endothelium we estimated the endothelial cell density (ECD), the average cell area, polymegathism and pleomorphism. The results revealed on ECD decrease in corneas of normal sheep with advancing age, as well as a corresponding increase in endothelial cell area and pleomorphism. The present work suggests that the endothelial parameters evaluated change with advancing age.
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Hammadi, Shumoos T. H. "Novel medical imaging technologies for processing epithelium and endothelium layers in corneal confocal images. Developing automated segmentation and quantification algorithms for processing sub-basal epithelium nerves and endothelial cells for early diagnosis of diabetic neuropathy in corneal confocal microscope images." Thesis, University of Bradford, 2018. http://hdl.handle.net/10454/16924.
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Diabetic Peripheral Neuropathy (DPN) is one of the most common types of diabetes that can affect the cornea. An accurate analysis of the corneal epithelium nerve structures and the corneal endothelial cell can assist early diagnosis of this disease and other corneal diseases, which can lead to visual impairment and then to blindness. In this thesis, fully-automated segmentation and quantification algorithms for processing and analysing sub-basal epithelium nerves and endothelial cells are proposed for early diagnosis of diabetic neuropathy in Corneal Confocal Microscopy (CCM) images. Firstly, a fully automatic nerve segmentation system for corneal confocal microscope images is proposed. The performance of the proposed system is evaluated against manually traced images with an execution time of the prototype is 13 seconds. Secondly, an automatic corneal nerve registration system is proposed. The main aim of this system is to produce a new informative corneal image that contains structural and functional information. Thirdly, an automated real-time system, termed the Corneal Endothelium Analysis System (CEAS) is developed and applied for the segmentation of endothelial cells in images of human cornea obtained by In Vivo CCM. The performance of the proposed CEAS system was tested against manually traced images with an execution time of only 6 seconds per image. Finally, the results obtained from all the proposed approaches have been evaluated and validated by an expert advisory board from two institutes, they are the Division of Medicine, Weill Cornell Medicine-Qatar, Doha, Qatar and the Manchester Royal Eye Hospital, Centre for Endocrinology and Diabetes, UK.
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Franzen, Angela Aguiar [UNESP]. "Morfologia e morfometria do endotélio corneal de gatos (Felis catus - Linnaeus, 1758) de diferentes idades à microscopia especular." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/89021.
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franzen_aa_me_jabo.pdf: 432317 bytes, checksum: 6e76f84b35fefc0d8afc5bb52873a43f (MD5)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
O endotélio corneal é essencial para a manutenção da transparência da córnea, é necessário um número mínimo de células endoteliais para a manutenção desta transparência. As alterações morfológicas e morfométricas do endotélio da córnea de gatos decorrentes do avanço da idade não estão ainda compreendidas e elucidadas. Objetivou-se estudar os parâmetros morfológicos e morfométricos do endotélio da córnea de gatos de diferentes idades, utilizando-se a microscopia especular. Avaliaram-se a densidade celular, a área celular média, o pleomorfismo e o polimegatismo. Empregaram-se 18 animais da espécie felina (Felis catus - LINNAEUS, 1758), machos ou fêmeas, subdivididos em três grupos com 6 animais cada, designados por: G1 (animais com idade entre 1 a 3 meses); G2 (animais com idade entre 5 a 12 meses) e G3 (animais com idades entre 24 a 40 meses). A microscopia especular de contato revelou que com o avanço da idade a densidade celular endotelial diminuiu, a área celular média e o pleomorfismo aumentaram e o polimegatismo apresentou valores constantes nos 3 grupos estudados.
The corneal endothelium is essential to maintain the corneal transparency. Morphological and morphometric changes in the feline corneal endothelium as a function of age are little known. The purpose of this study was investigating the effect of age on endothelial morphology and morphometry in cats. The corneal endothelium was studied throught contact specular microscopy. The exam presented data as cell density, average cell area, polymegathism and pleomorphism. A total of 16 cats were evaluated in this study. The subjects were divided in three groups with six cats (Felis catus - LINNAEUS, 1758): G1 (cats with 1 to 3 months old); G2 (cats with 5 to 12 months old); G3 (cats with 24 to 40 months old). The specular microscopy revealed decrease of cell density with age. The cell area increase and pleomorphism increase as function of age. The polymegathism showed constants values on the three groups.
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Franzen, Angela Aguiar. "Morfologia e morfometria do endotélio corneal de gatos (Felis catus - Linnaeus, 1758) de diferentes idades à microscopia especular /." Jaboticabal : [s.n.], 2008. http://hdl.handle.net/11449/89021.
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Resumo: O endotélio corneal é essencial para a manutenção da transparência da córnea, é necessário um número mínimo de células endoteliais para a manutenção desta transparência. As alterações morfológicas e morfométricas do endotélio da córnea de gatos decorrentes do avanço da idade não estão ainda compreendidas e elucidadas. Objetivou-se estudar os parâmetros morfológicos e morfométricos do endotélio da córnea de gatos de diferentes idades, utilizando-se a microscopia especular. Avaliaram-se a densidade celular, a área celular média, o pleomorfismo e o polimegatismo. Empregaram-se 18 animais da espécie felina (Felis catus - LINNAEUS, 1758), machos ou fêmeas, subdivididos em três grupos com 6 animais cada, designados por: G1 (animais com idade entre 1 a 3 meses); G2 (animais com idade entre 5 a 12 meses) e G3 (animais com idades entre 24 a 40 meses). A microscopia especular de contato revelou que com o avanço da idade a densidade celular endotelial diminuiu, a área celular média e o pleomorfismo aumentaram e o polimegatismo apresentou valores constantes nos 3 grupos estudados.Abstract: The corneal endothelium is essential to maintain the corneal transparency. Morphological and morphometric changes in the feline corneal endothelium as a function of age are little known. The purpose of this study was investigating the effect of age on endothelial morphology and morphometry in cats. The corneal endothelium was studied throught contact specular microscopy. The exam presented data as cell density, average cell area, polymegathism and pleomorphism. A total of 16 cats were evaluated in this study. The subjects were divided in three groups with six cats (Felis catus - LINNAEUS, 1758): G1 (cats with 1 to 3 months old); G2 (cats with 5 to 12 months old); G3 (cats with 24 to 40 months old). The specular microscopy revealed decrease of cell density with age. The cell area increase and pleomorphism increase as function of age. The polymegathism showed constants values on the three groups.
Orientador: José Luiz Laus
Coorientador: João Antonio Tadeu Pigatto
Banca: Marcia Rita Fernandes Machado
Banca: Aline Adriana Bolzan
Mestre
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Albuquerque, Luciane de. "Avaliação das repercussões do transplante lamelar posterior sobre o endotélio corneano de suínos utilizando a microscopia eletrônica de varredura. Estudo in vitro." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2014. http://hdl.handle.net/10183/103436.
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O transplante endotelial tem sido cada vez mais utilizado como uma alternativa ao transplante penetrante no tratamento das desordens do endotélio da córnea. No entanto, ao consultar a literatura não foram encontradas referências identificando em qual momento da cirurgia de transplante lamelar posterior ocorre maior dano às células endoteliais do botão doador. Objetivou-se avaliar e comparar as repercussões de duas etapas do transplante lamelar posterior sobre o endotélio da córnea de suínos utilizando a microscopia eletrônica de varredura. Utilizaram-se 30 bulbos oculares de 20 suínos, mestiços (1/2 Large White ½ Landrace), machos, com seis meses de idades e com peso médio de 100 kg. Foram designados dois grupos experimentais. No G1 constituído por 10 bulbos oculares, foi avaliado o dano ao endotélio da córnea após a confecção do botão doador. No G2, formado por 20 bulbos oculares, foi avaliado o dano ao endotélio da córnea após a inserção do botão doador no leito receptor utilizando introdutor de Busin. Perdas celulares ocorreram e variaram de acordo com o grupo estudado. A perda endotelial média do G1 foi 8,41% e no G2 17,31%. As diferenças foram estatisticamente significativas entre os grupos estudados. No presente estudo e nas condições experimentais realizadas foi possível concluir que a inserção do botão doador induziu maiores danos ao endotélio corneano de suínos, comparativamente à sua criação.Endothelial keratoplasty has been adopted as an alternative to penetrating keratoplasty in the treatment of corneal endothelial disorders. However, references identifying at what time of posterior lamellar transplant surgery occurs greater damage to the endothelial cells of the donor button were not found, when consulting the literature. The aim of this study was to assess and compare the effects of two stages of posterior lamellar keratoplasty on the corneal endothelium of swine using scanning electron microscopy. A total of 30 eyes were evaluated in this study. The eyes were divided in two groups of ten each eye: G1 (evaluated after delamination and preparation of the donor button) and G2 (evaluated after delamination and preparation and insertion of the donor button using Busin glide). Cell loss occurred and varied according to the study group. The average endothelial cell loss was 8.41% in G1 and 17.31% in G2. The differences were statistically significant between the groups studied. Scanning electron microscopy demonstrated damage in either group. The average endothelial cell loss was 8.41% in G1 and 17.31% in G2. The differences were statistically significant between the groups studied. It was concluded in the present study and also in the experimental conditions that the insertion of the donor button has induced greater damage to the corneal endothelium of swine compared to its creation.
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Faganello, Cláudia Skilhan. "Morfologia celular endotelial de diferentes regiões da córnea de equinos (Equus caballus) com a coloração vermelho de alizarina." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2014. http://hdl.handle.net/10183/108179.
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Objetivo: O endotélio é uma monocamada de células achatadas, poligonais e interligadas que recobrem a superfície posterior da córnea, sendo fundamental na manutenção da transparência desta estrutura. Objetivou-se avaliar a morfologia de diferentes regiões da córnea de equinos após coloração com vermelho de alizarina utilizando a microscopia óptica. Procedimentos: Foram estudados 16 bulbos oculares de oito equinos, machos ou fêmeas, de diferentes faixas etárias. O endotélio da córnea foi corado com o corante vital vermelho de alizarina (Alizarin Red S, Sigma Aldrich), dissolvido previamente em solução isotônica (0,2g/100mL), com pH ajustado para 4,2 com ácido clorídrico, e após examinado com microscópio óptico e fotografado. Foi avaliada a morfologia endotelial das regiões central, superior, inferior, temporal e nasal da córnea. De cada região da córnea, foram analisadas 100 células endoteliais. Foi realizada a análise de variância (ANOVA). Resultados: A porcentagem média de células hexagonais na região superior da córnea foi de 57,78 ± 3,14 %, na região inferior foi de 58,62 ± 6,413 %, na região temporal foi de 56,14 ± 6,749 %, na região nasal foi de 56,88 ± 6,296 %, na região central dos equinos foi de 55,43 ± 4,464 %. O percentual de células com menos de seis lados foi 22,72 ± 3,04 % para a região central, 20,81 ± 3,534 % para a região superior, 20,14 ± 3,82 % para a região inferior, 21,66 ± 4,04 % para a região temporal, 21,60 ± 3,04 % para a região nasal. O percentual de células com mais de seis lados foi de 21,85 ± 3,99 % para a região central, 21,31 ± 3,81 % para a região superior, 21,24 ± 4,08 % para a região inferior, 22,2 ± 4,88 % para a região temporal, 21,52 ± 4,71 % para a região nasal. Com relação à morfologia não houve diferença estatisticamente significante entre as regiões da córnea avaliadas. Conclusão: A microscopia óptica e a coloração com vermelho de alizarina possibilitaram a análise e a documentação do endotélio da córnea de equinos. No que diz respeito à morfologia, não existem diferenças entre as regiões da córnea de equinos.Objective: The endothelium is a single layer of flattened, interlocking polygonal cells lining the posterior surface of the cornea; its main function is to maintain the transparency of this structure. The objective was to evaluate the morphology of different regions of the equine cornea by optical microscopy after staining with alizarin red. Procedures: 16 eye bulbs of eight horses, male or female, of different ages were studied. The corneal endothelium was stained with alizarin red vital dye (Alizarin Red S, Sigma Aldrich), previously dissolved in isotonic solution (0.2g / 100 mL) with pH adjusted to 4.2 with hydrochloric acid. The corneal endothelium was examined by optical microscope and photographed. Endothelial morphology of central, superior, inferior, temporal and nasal cornea was evaluated. One hundred endothelial cells of each cornea region were analyzed. Analysis of variance (ANOVA) was performed. Results: The percentage of hexagonal cells in the upper region was 57,78 ± 3,14 %, in the lower region was 58,62 ± 6,413 %, in temporal region was 56,14 ± 6,749 %, in the nasal region was 56,88 ± 6,296 %, in the central region was 55,43 ± 4,464 %. The percentage of cells with less than six sides was 22,72 ± 3,04 % for the region central, 20,81 ± 3,534 % for the upper region, 20,14 ± 3,82 % for the lower region, 21,66 ± 4,04 % for the temporal region, 21,60 ± 3,04 % for the nasal region. The percentage of cells with more than six sides was 21,85 ± 3,99 % for the region central, 21,31 ± 3,81 % for the upper region, 21,24 ± 4,08 % for the lower region, 22,2 ± 4,88 % for the temporal region, 21,52 ± 4,71 % for the nasal region. Regarding to morphology there was no statistically significant difference between the regions of the evaluated corneas. Conclusion: Optical microscopy and staining with Alizarin red enabled the analysis and documentation of the corneal endothelium of horses. There are no differences in endothelial cell morphology in different regions of the cornea of horses.
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Valtink, Monika, Rita Gruschwitz, Richard H. W. Funk, and Katrin Engelmann. "Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-136199.
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Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCECDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Valtink, Monika, Rita Gruschwitz, Richard H. W. Funk, and Katrin Engelmann. "Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics." Karger, 2008. https://tud.qucosa.de/id/qucosa%3A27701.
Full textAbstract:
Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Almeida, Ana Carolina da Veiga Rodarte de. "Avaliação clínica e pela microscopia eletrônica de varredura do adesivo de fibrina, comparativamente ao fio de sutura na oclusão da incisão de córnea: estudo experimental em coelhos." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2009. http://hdl.handle.net/10183/16293.
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As técnicas de remoção de catarata evoluíram nas últimas décadas. Na tentativa de oclusão da córnea após incisão para remoção da catarata, diversas técnicas têm sido propostas. Objetivou-se avaliar experimentalmente a viabilidade do emprego do adesivo de fibrina na oclusão da incisão de córnea em coelhos. Além disso, comparar os efeitos do adesivo de fibrina e do fio de sutura na oclusão da incisão de córnea em coelhos, utilizando-se os aspectos clínicos, a microscopia eletrônica de varredura e a morfometria. Dezesseis coelhos (Oryctolagus cuniculus) da raça Nova Zelândia foram submetidos à incisão de córnea bilateral. Para a oclusão da incisão utilizou-se aleatoriamente em um bulbo do olho, adesivo de fibrina e no seu contralateral, fio de sutura. Os períodos de avaliação foram de 7 e 15 dias. As repercussões dos procedimentos foram avaliadas utilizando-se exame oftálmico. Ao final dos períodos determinados, procedeu-se à avaliação da área perincisional desprovida de células endoteliais por meio da microscopia eletrônica de varredura da morfometria e a análise estatística inferencial foi obtida pelo teste t de Student para amostrar pareadas. Clinicamente, observaram-se melhores resultados nas amostras ocluídas com fio de sutura. No que se refere à área perincisional desprovida de células endoteliais, comparando-se os dois tipos de oclusão, a área das amostras seladas com fibrina apresentou-se maior que a área ocluída com fio de sutura. Neste estudo, ambas as técnicas foram eficazes na oclusão da córnea de coelhos. Porém, a avaliação valendo-se da microscopia eletrônica de varredura e da morfometria das eletromicrografias das áreas perincisionais do endotélio da córnea desprovidas de células ocluídas com fio de sutura demonstrou maior nível de significância quando comparada ao adesivo de fibrina.The techniques for cataract removal had developed in the last decades. As an attempt to repair the cornea after incision, different techniques are proposed for corneal sealing. The objective of this study was to evaluate experimentally the viability of the use of fibrin adhesive in occlusion of the incision of the cornea in rabbits. Also compare clinically and by scanning electron microscopy and morphometry the fibrin adhesive and the suture on the sealing the cornea incision in rabbits. In this study, 16 rabbits (Oryctolagus cuniculus) New Zealand breed were used. It was performed bilateral corneal incision. In one eye the incision was sealed with suture, in the other eye, with fibrin adhesive randomly. The periods of evaluation varied from 7 to 15 days. The repercussions at the eye were studied using the ophthalmic exam. At the end of the determinate period, it was performed the evaluation of the perincisional area without endothelial cell by means of scanning electron microscopy, morphometry and the inferential statistical analysis was made by Student t test for paired samples. Comparing the two types of sealing, the perincisional average area without endothelial cells was higher in the fibrin tissue than wired in both groups. In this study, both techniques had corneal sealing, however, the evaluation by scanning electron microscopy,and electromicrographs morphometry of the perinsional areas of the corneal endothelium without devoid cells occluded with suture wire has higher level significance when compared with fibrin adhesive.
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Marcon, Alexandre Seminoti. "Influência da espessura corneana na acuidade visual corrigida após transplante de córnea endotelial lamelar profundo (TCELP)." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/5/5149/tde-16102014-085907/.
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Objetivo: Analisar a influência da espessura corneana central na acuidade visual (AV) corrigida após transplante de córnea endotelial lamelar profundo (TCELP). Métodos: Foram estudados de forma prospectiva 155 olhos de 127 pacientes portadores de ceratopatia bolhosa ou distrofia endotelial de Fuchs no sexto mês de pós-operatório do TCELP, entre março de 2000 e março de 2005. Foram excluídos pacientes com outras alterações oculares que justificassem baixa AV. Todos os pacientes foram submetidos à avaliação oftálmica, quando foram determinadas AV corrigida, por meio de exame refratométrico, e espessura corneana central, através da paquimetria ultra-sônica. As técnicas usada foram previamente descritas. Os olhos foram agrupados de acordo com as medidas de AV: grupo I (20/20 - 20/30), grupo II (20/40 - 20/50), grupo III (20/60 - 20/80), grupo IV (20/100 - 20/400). Para correlação com paquimetria e análise estatística, as medidas de AV foram convertidas da tabela de Snellen para a tabela logarítmica (logMAR). Foram criadas variáveis categóricas para expressar status de faixa de normalidade de espessura corneana (entre 495 e 651 ?m), usando como pontos de corte valores encontrados na literatura. Resultados: A média, o desvio padrão e a variação da paquimetria foi: grupo I (n=38) 571 ±80 um, 408 a 784 um; grupo II (n=79) 598 ±80 um, 437 a 816 um; grupo III (n=30) 605 ±99 um, 454 a 945 um e grupo IV (n=8) 607 ±120 ?m, 410 a 781 ?m. Analisando o resultado da AV e a porcentagem de casos com espessura corneana acima de 651 um, foi observada associação linear significativa (P=0,037; ?2 de tendência linear) entre o aumento da paquimetria e a piora da AV. Analisando a associação entre os grupos de AV e a porcentagem de casos com espessura corneana abaixo da faixa de normalidade (<495 um), não foi encontrada significância estatística (P=0,92; x2 de Pearson). Quando analisado o resultado visual do grupo I em relação ao resultado dos grupos II+III+IV em conjunto, observou-se que somente 13% dos casos do grupo I e 30% dos casos dos demais grupos apresentaram espessura corneana maior do que 651 ?m. Essa correlação demonstrou significância estatística limítrofe (P=0,066; x2 de Pearson com correção de Yates). Conclusão: Observou-se associação linear significativa entre piora da AV corrigida e aumento da espessura corneana central. Quando analisados somente casos com paquimetria abaixo da faixa de normalidade, não foi observada associação significativa entre piora da AV corrigida e espessura corneana centralPurpose: To analyze the influence of central corneal thickness in the corrected visual acuity (VA) after deep lamellar endothelial corneal keratoplasty (DLEK). Methods: Retrospective study of 155 eyes of 127 patients 6 months post-op DLEK between March 2000 and March 2005. These patients had been previously diagnosed with either bullous keratopathy or Fuch\'s endothelial dystrophy. Patients with other ophthalmic conditions that could cause loss of vision were excluded. All patients underwent ophthalmic evaluation to determine corrected VA by means of refraction and central corneal thickness by means of ultrasonic pachymetry. Eyes were grouped according to visual acuity into 4 groups: I (20/20 - 20/30), II (20/40 - 20/50), III (20/60 - 20/80), IV (20/100 - 20/400). For statistical analysis and corelation with pachymetry, VA measurements were converted to logMAR. Categorical variables were created to express normal range corneal thickness status (from 495 to 651 um) using values published on the literature. Results: Mean and standart deviation pachymetry values were: group I (n=38) 571 ±80 ?m, ranging from 408 to 784 um; group II (n=79) 598 ±80 um, ranging from 437 to 816 ?m; group III (n=30) 605 ±99 um, ranging from 454 to 945 um and group IV (n=8) 607 ±120 um, ranging from 410 to 781 ?m. Analyzing the VA results and the percentage of cases with corneal thickness above 651 um, a significant linear correlation between higher pachymetry and worse VA was observed (P=0.037; linear trend). Analyzing the association between the different groups and the percentage of cases with corneal thickness bellow 495 um, there was no statistical significance (P=0.92; Pearson\'s x2). When analyzing the visual results of group I compared to groups II+III+IV together, it was observed that only 13% of group I cases and 30% of cases from the other groups presented corneal thickness greater then 651 um. This correlation showed borderline statistical significance (P=0.066; Pearson\'s x2 with Yates\' correction). Conclusions: A significant linear correlation between increased corneal thickness and worse VA was observed. When analyzing only cases bellow normal pachymetry, there was no correlation between corneal thickness and worse VA
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Andrade, Maria Cristina Caldart de. "Avaliação do endotélio corneano de equinos após exposição ao corante azul brilhante 0,05% - estudo in vitro." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2016. http://hdl.handle.net/10183/143947.
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Os corantes vitais têm sido utilizados em diversas áreas da oftalmologia para melhorar a visibilização de diferentes tecidos. Com o objetivo de avaliar os efeitos imediatos do corante Azul brilhante a 0,05% no endotélio da córnea de equinos, foram utilizadas 38 córneas de 19 cavalos, machos ou fêmeas, de diferentes idades e provenientes de abatedouro comercial licenciado. As córneas foram aleatoriamente divididas em dois grupos. Grupo1: o endotélio da córnea foi exposto a solução salina balanceada por 60 segundos. Grupo 2: o endotélio da córnea foi exposto ao corante Azul brilhante 0,05% (Opht-Blue, Ophthalmos, São Paulo, Brazil) por 60 segundos seguido por lavagem com solução salina balanceada (BSS, Ophthalmos, São Paulo, Brazil). As córneas então foram excisadas com trépano de 8mm e preparadas para avaliação com microscopia óptica (24 córneas) e microscopia eletrônica de varredura (14 córneas). As áreas de dano endotelial foram mensuradas. Devido ao aparecimento de resíduos não normais na utilização de comparação de médias via ANOVA, optou-se pelo modelo linear generalizado e, com 5% de significância, o teste Qui-quadrado demonstrou que o fator tratamento não difere do controle. Conclui-se que o corante Azul brilhante em concentração de 0,05% não provocou danos ao endotélio da córnea de equinos.Vital dyes have been used in many areas of ophthalmology for improving visualization of different tissues. With the objective of evaluating the immediate effects of 0,05% brilliant blue on corneal endothelium of horses, 38 corneas of 19 horses, both male and female of different ages obtained from a licensed Brazilian commercial slaughterhouse were studied. Corneas were randomly divided into two groups. In group 1, corneal endothelium was perfused with BSS for 60 seconds. In group 2, corneal endothelium was perfused with 0.3 mL of brilliant blue 0.05% (Opht-Blue, Ophthalmos, São Paulo, Brazil) for 60 seconds followed by rinsing with a balanced salt solution (BSS, Ophthalmos, São Paulo, Brazil). The corneas were, subsequently, excised with an 8mm trephine and prepared to analyze posterior endothelial surface using a light microscope (24 corneas) and a scanning electron microscope (14 corneas). Due to non-normal residuals at ANOVA mean comparison, a generalized linear model was utilized at 5% level of significance. Qui-square test stated that treatment and control group were not different statistically. The 0.05 % brilliant blue did not damage equine corneal endothelium.
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Silva, Vanessa Ruiz Moura da. "Avaliação do endotélio corneano de equinos após exposição à indocianina verde 0,5% : estudo in vitro." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2014. http://hdl.handle.net/10183/108171.
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A capsulotomia curvilínea contínua (CCC) é uma das etapas mais importantes da técnica de facoemulsificação. Em cataratas brancas e com reflexo de fundo de olho deficiente ou ausente, a identificação da cápsula anterior do cristalino é dificultada, e é necessária a utilização de substâncias, como os corantes vitais, para permitir a sua diferenciação. Contudo, antes da utilização de substâncias intraoculares, é necessário determinar se elas são seguras para as estruturas do globo ocular, principalmente para as células do endotélio corneano, que podem sofrer lesões irreversíveis. A indocianina verde é uma substância capaz de corar a cápsula do cristalino, tendo seu uso relatado em humanos, mas não existem dados sobre sua utilização em equinos. Objetivou-se determinar o efeito agudo da exposição do endotélio corneano de equinos à indocianina verde 0,5%. Foram estudadas 24 córneas provenientes de 12 equinos divididos em 2 grupos: 12 córneas dos bulbos oculares direitos (grupo controle) e 12 córneas dos bulbos oculares esquerdos (grupo tratamento). As córneas do grupo tratamento foram expostas à indocianina verde durante 1 minuto, e após lavadas com solução salina balanceada. Posteriormente, as córneas foram coradas pela técnica de coloração vital com alizarina vermelha e azul de tripano, visualizadas ao microscópio óptico e fotografadas. As córneas do grupo controle também foram coradas com alizarina vermelha e azul de tripano, visualizadas e fotografadas. Não foram encontradas áreas de perda celular com a coloração pela alizarina vermelha, e nem células com núcleo corado pelo azul de tripano, não havendo diferenças entre grupo controle e grupo tratamento. Baseado nos resultados do presente estudo foi possível concluir que a indocianina verde não induziu dano às células do endotélio da córnea de equinos.The continuous curvilinear capsulotomy (CCC) is one of the most important steps on the phacoemulsification technique. In white cataract combined with poor or absent red reflex the identification of the anterior capsule is hampered, thus requiring the use of substances such as vital dyes to allow their differentiation. However, before the use of intraocular substances it is necessary to determine whether these substances are safe to the structure of the ocular globe, mainly to the corneal endothelium cells, which may suffer irreversible damage. The indocyanine green is a substance capable of staining the lens capsule with documented use in humans, but there are no data on its use in horses. This study aimed to determine the acute effect of exposure of equine corneal endothelium to indocyanine green 0.5%. The sample consisted of 24 corneas from 12 horses divided into 2 groups: 12 corneas of right eye bulbs (control group) and 12 corneas of left eye bulbs (treatment group). The corneas belonging to the treatment group were exposed to the indocyanine green for 1 minute, and then washed with balanced saline solution. Subsequently, the corneas were stained by the technique of vital staining with alizarin red and trypan blue, and so visualized and photographed under an optical microscope. The corneas of the control group were also stained with alizarin red and trypan blue, visualized and photographed. No areas of cell loss were found with alizarin red staining, and no cell nuclei stained with trypan blue was visualized, with no difference between control group and treatment group. Based on the results of this study, we concluded that the indocyanine green did not induce damage to equine corneal endothelium.
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Freitas, Luciana Vicente Rosa Pacicco de. "Avaliação do endotélio da córnea de galinhas (Gallus gallus domesticus) em diferentes faixas etárias utilizando a microscopia especular." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2012. http://hdl.handle.net/10183/55971.
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O endotélio corneano é uma monocamada de células poligonais localizadas na face posterior da córnea e é essencial para a manutenção da transparência corneana. Não foram encontradas referências na literatura a respeito dos parâmetros morfológicos e morfométricos do endotélio da córnea de galinhas, apesar destes animais serem amplamente utilizados como modelo experimental em estudos oftálmicos, devido à similitude com a córnea de humanos. O objetivo deste estudo foi o de avaliar os parâmetros morfométricos e o pleomorfismo da região central do endotélio da córnea de galinhas (Gallus gallus domesticus) de diferentes faixas etárias, utilizando a microscopia especular de contato. Avaliaram-se a densidade, a área celular média e o pleomorfismo das células do endotélio da córnea de 60 olhos de 30 galinhas da raça Leghorn branca. Os animais foram divididos em três grupos composto por 10 animais cada: G1 (animais com 30 dias de idade), G2 (animais com 45 dias de idade) e G3 (animais com 60 dias de idade). O presente estudo revelou que o endotélio da córnea de galinhas é composto por células poligonais de padrão regular, com predomínio de formato hexagonal. O endotélio corneano de galinhas sofreu alterações decorrentes da idade no que tange a morfometria, mas no que diz respeito ao pleomorfismo, não ocorreram alterações em resposta ao envelhecimento.The corneal endothelium is a monolayer of polygonal cells located on the posterior face of the cornea and it is essential for the maintenance of corneal transparency. We found no references in the literature concerning the morphological and morphometric parameters of the corneal endothelium of chickens, although these animals are widely used as experimental model in ophthalmic studies due to the similarity with the human cornea. The objective of this study was to evaluate the morphometric parameters and the pleomorphism of central corneal endothelium of chickens (Gallus gallus domesticus) of different ages using the contact specular microscopy. The density, the average cell area and the pleomorphism of the corneal endothelial cells were evaluated on 60 eyes of 30 white Leghorn chickens. The animals were divided into three groups of 10 animals each: G1 (animals with 30 days of age), G2 (animals with 45 days of age) and G3 (animals with 60 days of age). The present study revealed that the corneal endothelium of chickens is composed of regular polygonal cells, with predominance of hexagonal shape. The corneal endothelium of chickens has changed due to age in respect to morphometry, but in regard to pleomorphism, no changes occurred in response to aging.
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Tessarioli, Mariana. "Avaliação do endotélio corneano suíno por microscopia eletrônica de varredura após aplicação de azul brilhante a 0,05% na câmara anterior – Estudo in vitro." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2013. http://hdl.handle.net/10183/76541.
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Diversos corantes vitais vêm sendo estudados e utilizados para a facilitação da capsulotomia curvilínea contínua (CCC) nas cirurgias de catarata no homem e nos animais. Além de corar adequadamente a cápsula anterior da lente e favorecer um melhor desempenho do cirurgião durante a realização da CCC, os corantes vitais devem ser seguros quanto aos seus efeitos sobre as estruturas oculares, em especial ao endotélio corneano, quando empregados com esta finalidade. O azul brilhante é um corante vital já empregado em cirurgias oculares do segmento posterior para coloração da retina e atualmente estudado sobre seu potencial de utilização em cirurgias de catarata para coloração da cápsula anterior da lente. Com o objetivo de avaliar os efeitos do uso intracameral do azul brilhante 0,05% na ultra-estrutura do endotélio corneano de suínos, vinte córneas de suínos foram avaliadas divididas em dois grupos: córneas dos bulbos oculares direitos (grupo controle) e esquerdos (grupo experimental). Todos os bulbos oculares foram previamente avaliados por microscopia especular. No grupo experimental foi realizada injeção intracameral de 0,2ml do corante azul brilhante 0,05% (OPTH-blue®) que permaneceu por um minuto antes de ser removido pela aplicação de solução salina balanceada. As córneas de ambos os grupos foram excisadas e avaliadas por microscopia eletrônica de varredura. Não houve diferença entre as imagens endoteliais obtidas em ambos os grupos. O uso intracameral do azul brilhante 0,05% não causou efeitos deletérios ao endotélio corneano dos suínos e pode, portanto, ser considerado uma escolha segura para a coloração da cápsula anterior da lente para cirurgias de catarata.Several vital dyes have been studied and used to help the continuous curvilinear capsulotomy (CCC) in cataract surgery in men and animals. Besides staining the anterior capsule of the lens properly and providing the surgeons a better performance in the CCC, the vital dyes must be safe for their effects on ocular structures, particularly the corneal endothelium when used for this purpose. The brilliant blue is a vital dye already employed in the posterior segment eye surgeries for retinal staining and currently studied about its potential use in cataract surgeries to stain the anterior capsule of the lens. In order to evaluate the effects of the use of 0.05% intra-cameral brilliant blue in the ultra-structure of the corneal endothelium of pigs, twenty swine corneas were evaluated in two groups: right eye bulb corneas (control group) and left eye bulb corneas (experimental group). All eye bulbs were previously evaluated by specular microscopy. In the experimental group, a 0.2 ml intra-cameral injection of 0.05% brilliant blue dye (blue-OPTH ®) was given, which remained for a minute before being removed by the application of balanced salt solution. The corneas of both groups were excised and evaluated by scanning electron microscopy. There was no difference between the endothelial images obtained in both groups. The use of 0.05% intra-cameral brilliant blue caused no detrimental effects to the corneal endothelium of pigs and can therefore be considered a safe choice for staining the anterior capsule of the lens for cataract surgery.
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Bollu, Lakshmi Reddy. "The Effect of Endothelin-1 on the expression of CDK Inhibitors p21 & p27 in Bovine Corneal Endothelial Cells." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/112.
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Mammalian corneal endothelial cells are considered to be non-proliferative due to the arrest of cells at the G1 phase of the cell cycle. The purpose of this study was to determine whether the down regulation of cyclin dependant kinase inhibitors (p21cip1 and p27kip1) levels by Endothelin-1 (ET-1), would overcome the G1 phase arrest and promote cell cycle progression and proliferation in cultured BCECs (Bovine corneal endothelial cells). BCECs were isolated from bovine corneas and cultured in DMEM supplemented with 10% serum. 5-Bromo 2-deoxy Uridine (BrdU) incorporation was determined in serum starved cultures in 24-well plates as a measure of cell proliferation. Confluent serum starved cells grown in T-25 flasks were treated with 100nM Endothelin-1 in DMEM. The control cells were left untreated in serum free medium. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of p21cip1 and p27kip1 proteins relative to β-actin was determined by western blotting technique. Immuno fluorescent localization of p27kip1 was performed using polyclonal anti-p27kip1 and anti-p21cip1 antibodies in confluent and growing cells. An increase in cell proliferation was observed in sub-confluent cultures with Endothelin-1 treatment. This evidence was supported by an increase (~18%) in BrdU incorporation in response to Endothelin-1. Densitometry analysis of immunoblots revealed an increase in the expression of p27kip1 in confluent cell cultures when compared to sub-confluent, dividing cells. p21cip1 was almost undetectable in sub-confluent, actively dividing cultures. Immuno fluorescent analysis revealed that the nuclear staining of p27kip1 was apparently decreased with ET-1 treatment. In conclusion, Endothelin-1 treatment resulted in decrease in p27kip1 and p21cip1 expression in confluent cultures that was greatest at 30 hr of post incubation with Endothelin-1. Endothelin-1 appears to promote cell proliferation. Expression of p27kip1 and p21cip1 was greatly reduced in actively dividing BCECs. Endothelin-1 treatment down-regulated these cyclin dependent kinase inhibitors and may promote cell cycle progression via this mechanism.
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Bethi, Akhila. "Endothelin-1 Induced Phosphorylation of ERK1/2 in Bovine Corneal Endothelial Cells." TopSCHOLAR®, 2012. http://digitalcommons.wku.edu/theses/1191.
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The purpose of this study was to determine whether Endothelin-1 (ET-1) induced cellular responses in bovine corneal endothelial cells (BCECs) involves MAPK pathway by phosphorylating ERK1/2 protein kinase and to find out the phosphorylation patterns of ERK1/2 in confluent and sub-confluent cells. BCECs were isolated from bovine corneas and cultured in medium supplemented with 10% serum. Confluent (contact inhibited) and sub-confluent (actively growing cells) serum starved cells grown in T-75 flasks were treated with 10nM Endothelin-1. The control cells were left untreated. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of phosphorylated ERK1/2 (pp44, pp42) proteins relative to overall ERK1/2 (p44, p42) was determined by western blotting technique. Densitometry analysis of immunoblots revealed differential phosphorylation patterns in confluent and sub-confluent cultures. The pERK1/2 levels were significantly increased at 15 min and 24 hrs after post incubation with ET-1, whereas following the initial rise levels declined to 6hrs of incubation with ET-1 in confluent cultures. In sub-confluent cultures pERK1/2 levels increased gradually to 6hrs of incubation with ET-1, returning to pre-incubation levels at 24hrs. In conclusion, ET-1 treatment was shown to induce phosphorylation of ERK1/2 in BCEC. ET-1 treatment in confluent and sub confluent BCEC exhibited time dependent phosphorylation of ERK1/2. ET-1 treatment affected the phosphorylation pattern distinctively in confluent and sub-confluent BCEC. These observations led to the conclusion that ET-1 induced cellular events in BCEC may involve the MAPK cascade and that these ET-1 induced MAPK cascades may exhibit a negative feedback mechanism, suggested by a distinctive oscillations in pERK 1/2 levels. The contrasting effects of ET-1 in confluent and subconfluent cells may suggest a density dependent phosphatase activity.
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Silva, Géssica Maria Ribeiro da. "Morfologia e morfometria das células do endotélio da córnea de equinos utilizando a microscopia eletrônica de varredura." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2018. http://hdl.handle.net/10183/181327.
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O conhecimento da morfologia endotelial nas diferentes regiões da córnea é de suma importância para avaliação de endotélios corneanos saudáveis e doentes e de suas respostas ao uso de medicações. O objetivo do presente estudo foi descrever a morfologia do endotélio nas regiões central e periférica superior da córnea saudável de equinos, medir a área média das células pentagonais, hexagonais e heptagonais presentes nas regiões avaliadas, calcular o polimegatismo de cada tipo celular e correlacionar estes parâmetros entre os diferentes formatos celulares. Foram estudados dez equinos, machos ou fêmeas, de diferentes idades, provenientes de um abatedouro licenciado. Imagens da superfície posterior do endotélio da córnea foram obtidas com uso de microscopia eletrônica de varredura. A morfologia do endotélio de diferentes regiões da córnea foi avaliada. Além disso, foi correlacionada a variabilidade do tamanho celular médio com a morfologia endotelial. A análise estatística foi conduzida usando o teste de análise de variância (ANOVA) seguido do teste de Tukey (Post-Hoc), com nível de significância de 5%. As amostras avaliadas foram compostas em sua maioria por células hexagonais (60,5%), pentagonais (21,4%) e heptagonais (16,9%), células com quatro, oito ou nove lados compuseram 1,3%. A área celular média das células pentagonais foi 203,58 μm², das hexagonais foi 223,84 μm² e nas heptagonais foi 270,54 μm². O polimegatismo encontrado foi de 16% nas células pentagonais e hexagonais e de 20% nas heptagonais. A morfologia das células endoteliais de equinos saudáveis não diferiu entre as regiões central e periférica superior da córnea, sugerindo que a região central é representativa da região periférica. As células com sete lados apresentaram polimegatismo maior em relação às células de seis e de cinco lados.The knowledge of the endothelial morphology in the different regions of the cornea is important for the evaluation of healthy and sick corneal endothelium and its response to drugs. In order to describe the endothelial morphology in the central and superior peripheral regions of the equines’ cornea, evaluate area from pentagonal, hexagonal and heptagonal cells present in the evaluated regions, calculate the polimegathism and correlate these parameters, two healthy corneas were collected of ten equine, male or female, of different ages. Images of the posterior surface of the corneal endothelium were taken with scanning electron microscope. The endothelial morphology was studied in the different regions of the equines’ cornea. In addition, the polimegathism and morphology was also correlated. A statistical analysis was conducted using the Analysis of Variance (ANOVA) followed by Tukey's Test (Post-Hoc), with a 5% level of significance. In the central region, the endothelium consisted of 58.8% hexagonal cells, 22.6% pentagon, 17.1% heptagonal and 1.4% cells with either four, eight or nine sides. In the superior peripheral region, 62.1% of the cells were hexagonal, 20.2% pentagon, 16.6% heptagonal and 1.1% cells with four or eight sides. The average cell area of the pentagonal cells was 210.77 ± 31.53 μm² in the central region and 196.39 ± 34.35 μm² in the superior peripheral region; in hexagonal cells, the average cell area found in the central region was 216.12 ± 37.09 μm² and 231.56 ± 34.95 μm² in the superior peripheral region; and in cells with seven sides, the average cell area was 261.76 ± 55.29 μm² in the central region and in the superior peripheral region was found 279.32 ± 52.37μm². When not considering the corneal regions, the polymegathism found was 16% in the pentagonal and hexagonal cells and 20% in the heptagonal cells. The morphology results obtained did not differ between the central and peripheral regions of the cornea, suggesting that the central region is representative of the peripheral region. The highest coefficient of variation was seen in cells with seven sides.
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Holzchuh, Ricardo. "Estudo da reprodutibilidade do exame de microscopia especular de córnea em amostras com diferentes números de células." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/5/5149/tde-01122011-111704/.
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INTRODUÇÃO: O endotélio corneal exerce papel primordial para a fisiologia da córnea. Seus dados morfológicos gerados pelo microscópio especular (MEC) como densidade endotelial (DE), área celular média (ACM), coeficiente de variação (CV) e porcentagem de células hexagonais (HEX) são importantes para avaliar sua vitalidade. Para interpretar estes dados de forma padronizada e reprodutível, foi utilizado um programa estatístico de análise amostral, Cells Analyzer PAT. REQ.(CA). OBJETIVO: Demonstrar valores de referência para DE, ACM, CV e HEX. Demonstrar o percentual de células endoteliais marcadas e desconsideradas no exame ao marcar-se 40, 100 e 150 células em uma única imagem do mosaico endotelial e o perfil do intervalo de confiança (IC) das variáveis estudadas ao se considerar 40, 100, 150 e tantas células quantas indicadas pelo CA. Demonstrar o erro amostral de cada grupo estudado. MÉTODOS: Estudo transversal. Os exames de MEC foram realizados com o aparelho Konan NONCON ROBO® SP-8000, nos 122 olhos de 61 portadores de catarata (63,97 ± 8,15 anos de idade). As imagens endoteliais caracterizaram se pelo número de células marcadas e consideradas para cálculo dos seguintes dados: DE, ACM, CV e HEX. Os grupos foram formados de 40, 100, 150 células marcadas numa única imagem endotelial e Grupo CA em que foram marcadas tantas células quanto necessárias em diferentes imagens, para obter o erro relativo calculado inferior ao planejado (0,05), conforme orientação do programa CA. Estudou-se o efeito do número de células sobre IC para as variáveis endoteliais utilizadas. RESULTADOS: A média dos valores de referência encontrados para DE foi 2395,37 ± 294,34 cel/mm2; ACM 423,64 ± 51,09 m2; CV 0,40 ± 0,04 e HEX 54,77 ± 4,19%. O percentual de células endoteliais desconsideradas no Grupo 40 foi 51,20%; no Grupo 100, 35,07% e no Grupo 150, 29,83%. O número médio de células calculado inicialmente pelo CA foi 247,48 ± 51,61 e o número médio de células efetivamente incluídas no final do processo amostral foi 425,25 ± 102,24. O erro amostral dos exames no Grupo 40 foi 0,157 ± 0,031; Grupo 100, 0,093 ± 0,024; Grupo 150, 0,075 ± 0,010 e Grupo CA, 0,037 ± 0,005. O aumento do número de células diminuiu a amplitude do IC nos olhos direito e esquerdo para a DE em 75,79% e 77,39%; ACM em 75,95% e 77,37%; CV em 72,72% e 76,92%; HEX em 75,93% e 76,71%. CONCLUSÃO: Os valores de referência da DE foi 2395,37 ± 294,34 cel/mm2; ACM foi 423,64 ± 51,09 m2; CV foi 0,40 ± 0,04 e HEX foi 54,77 ± 4,19%. O percentual de células endoteliais desconsideradas no Grupo 40 foi 51,20%; no Grupo 100 foi 35,07% e no Grupo 150 foi 29,83%. O programa CA considerou correto os exames nos quais 425,25 ± 102,24 células foram marcadas entre duas e cinco imagens (erro relativo calculado de 0,037 ± 0,005). O aumento do número de células diminuiu a amplitude do IC para todas as variáveis endoteliais avaliadas pela MECINTRODUCTION: Corneal endothelium plays an important role in physiology of the cornea. Morphological data generated from specular microscope such as endothelial cell density (CD), average cell area (ACA), coefficient of variance (CV) and percentage of hexagonal cells (HEX) are important to analyze corneal status. For a standard and reproducible analysis of the morphological data, a sampling statistical software called Cells Analyzer PAT. REC (CA) was used. PURPOSE: To determine normal reference values of CD, ACA, CV and HEX. To analyze the percentage of marked and excluded cells when the examiner counted 40, 100, 150 cells in one endothelial image. To analyze the percentage of marked and excluded cells according to the statistical software. To determine the confidence interval of these morphological data. METHODS: Transversal study of 122 endothelial specular microscope image (Konan, non-contact NONCON ROBO® SP- 8000 Specular Microscope) of 61 human individuals with cataract (63.97 ± 8.15 years old) was analyzed statistically using CA. Each image was submitted to standard cell counting. 40 cells were counted in study Group 40; 100 cells were counted in study Group 100; and 150 cells were counted in study Group 150. In study group CA, the number of counted cells was determined by the statistical analysis software in order to achieve the most reliable clinical information (relative error < 0,05). Relative error of the morphological data generated by the specular microscope were then analyzed by statistical analysis using CA software. For Group CA, relative planned error was set as 0.05. RESULTS: The average normal reference value of CD was 2395.37 ± 294.34 cells/mm2, ACA was 423.64 ± 51.09 m2, CV was 0.40 ± 0.04 and HEX was 54.77 ± 4.19%. The percentage of cells excluded for analysis was 51.20% in Group 40; 35.07% in Group 100; and 29.83% in Group 150. The average number of cells calculated initially by the statistical software was 247.48 ± 51.61 cells and the average number of cells included in the final sampling process was 425.25 ± 102.24 cells. The average relative error was 0.157 ± 0.031 for Group 40; 0.093 ± 0.024 for Group 100; 0.075 ± 0.010 for Group 150 and 0.037 ± 0.005 for Group CA. The increase of the marked cells decreases the amplitude of confidence interval (right and left eyes respectively) in 75.79% and 77.39% for CD; 75.95% and 77.37% for ACA; 72.72% and 76.92% for CV; 75.93% and 76.71% for HEX. CONCLUSION: The average normal reference value of CD was 2395.37 ± 294.34 cells/mm2, ACA was 423.64 ± 51.09 m2, CV was 0.40 ± 0.04 and HEX was 54.77 ± 4.19%. The percentage of excluded cells for analysis was 51.20% in Group 40; 35.07% in Group 100 and 29.83% in Group 150. CA software has considered reliable data when 425.25 ± 102.24 cells were marked by the examiner in two to five specular images (calculated relative error of 0.037 ± 0.005). The increase of the marked cells decreases the amplitude of confidence interval for all morphological data generated by the specular microscope
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Chan, Giulia. "Regulation of viability in corneal endothelial cells." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1444592/.
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The major cause of corneal opacity and resultant visual loss is a critical decrease in corneal endothelial cell density. Due to the fact that corneal endothelial cells do not generally proliferate, cell densities gradually decrease in the corneal endothelium with age. Before we can begin to aid patients with decreasing endothelial cell densities, by inhibiting cell death or stimulating cell proliferation, it is necessary to understand the basic cell signalling that underlies these processes. As human tissue is difficult to obtain, a representative animal model of corneal endothelium was devised. Analysis of cell morphology and expression of ct1 type VIII collagen were used to verify that primary cells derived from an explant model of mouse corneal cells were endothelial. A range of phenotypic and functional cellular features were also analysed to assess the usefulness of primary mouse corneal endothelial cultures as a model for human corneal endothelium. These included tight junction protein localization, proliferative responses to growth factor stimulation and ERK1/2 activation following growth factor stimulation. From these analyses, it was shown that primary mouse corneal endothelial cultures provide a representative model of the human corneal endothelium. A similar comparison was also made between primary mouse corneal endothelial cultures and an SV40 transformed mouse corneal endothelial cell line. Studies revealed significant differences in propensity to proliferate, junctional integrity, ERK1/2 activation, expression of apoptotic proteins and sensitivity to staurosporine-induced apoptosis between primary cells and the SV40 transformed cell line, suggesting that the SV40 transformed cell line is a less appropriate model for primary mouse corneal endothelial cells. In conclusion, the derivation and characterisation of mouse primary corneal endothelial cells provides a better model of the corneal endothelium, that offers greater understanding of cellular responses and which may eventually lead to the development of alternative therapies for primary corneal endotheliopathies.
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Rolev, Kostadin Dimov. "Evaluation of corneal endothelial cell therapy using an in vitro human corneal model." Thesis, Anglia Ruskin University, 2017. http://arro.anglia.ac.uk/702583/.
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Aim: To establish an in vitro human corneal decompensation model and to use it for the evaluation of a cell-therapy approach for treating corneal endothelial (CE) disorders and to test the expression profile of positive regulatory domain proteins (PRDMs) as potential markers for corneal endothelial cells (CECs). Materials and Methods: Human cadaveric corneas were obtained from Bristol and Manchester Eye Banks, UK. A CE decompensation model was established by removal of the Descemet’s membrane (DM)/Endothelium complex from donor corneas and placing them in air-interface organ culture. The corneal thickness was used as a surrogate measure of CE function and was measured using Optical Coherence Tomography (OCT). Decompensated corneas were subjected to cultured endothelial cell therapy using immortalized HCEC -12 cells (group 1), primary human corneal endothelial cells (hCECs) at 0 passage (group 2) and hCECs at passage 2 (group 3) with defined seeding cell density. The effect on stromal de-swelling in cell therapy treated corneas was assessed 3, 7 and 10 days post-transplantation followed by histological evaluation. In addition, expression of PRDM genes in the corneal endothelium was undertaken using reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and immunohistochemistry. Results: Organ culture of human cadaveric corneas in air-interface following the selective removal of the DM/Endothelium complex resulted in stromal thickness of 903.6 ± 86.51 μm, whereas normal corneas maintained a physiological thickness of 557.51 ± 72.64 μm. When transplanted directly onto the posterior corneal stroma the human CECs were able to attach and achieved physiological corneal thickness of 458.91 ± 90.07 μm, 489.65 ± 94.62 μm and 613.7 ± 94.62 μm for cell therapy groups -1, -2 and -3 respectively. The study identified PRDMs 1, 2, 4, 5 and 10 in the human CE and revealed a differential expression between normal CE and cultured hCECs. Conclusion: Removal of the DM/Endothelium complex from cadaveric human corneas held in air interface organ culture resulted in corneal endothelial decompensation. Direct transplantation of cultured primary hCECs to bare posterior corneal stroma devoid of DM resulted in the formation of an endothelial monolayer and restoration of stromal hydration to physiological thickness, substantiating the role of cell therapy to treat corneal endothelial disorders. The identification of PRDM proteins in the human corneal endothelium paves the way for future studies to understand their role in hCEC proliferation control.
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Wenzel, Daniel Alexander [Verfasser]. "Auswirkungen von Perfluorbutylpentan (F4H5) auf corneale Endothelzellen im porcinen Hornhautmodell : Effects of perfluorobutylpentan (F4H5) on corneal endothelial cells / Daniel Alexander Wenzel." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2019. http://d-nb.info/1223621057/34.
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Elzawia, Omar Rajab. "Corneal graft outcome, endothelial monolayer survival following corneal grafting : a prospective and retrospective study." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322567.
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Dannowski, Haike. "Gentransfer in korneale Endothelzellen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2004. http://dx.doi.org/10.18452/15137.
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Bei der Hornhauttransplantation (Keratoplastik) handelt es sich um die häufigste Transplantation humanen Gewebes. Das Hornhautendothel, eine empfindliche Zellschicht auf der Innenseite der Kornea, verfügt nicht über die Fähigkeit, Zellverluste durch Proliferation auszugleichen. Zwei grundsätzliche Probleme ergeben sich aus dieser Eigenschaft des kornealen Endothels für die Keratoplastik: ein Endothelzellverlust tritt zum einen während der Hornhautkonservierung vor Keratoplastik auf und führt zu einem Ausschluss vieler Transplantate, zum anderen ist er oftmals im Zuge von immunvermittelten Abstoßungsreaktionen oder in Folge chronischer Vorgänge nach Keratoplastik zu beobachten. Aus diesem Grund ist eine möglichst hohe Endothelzelldichte auf kornealen Transplantaten eine Voraussetzung für den Erfolg einer Keratoplastik. Das erste Ziel dieser Arbeit war deshalb die Übertragung des Gens für den aziden FGF (aFGF) in korneale Endothelzellen mit Hilfe des nicht-viralen Gentransfers. Dazu wurden verschiedene Lipid-Formulationen für den Gentransfer in humane korneale Endothelzellen in vitro optimiert. Der Einsatz von DAC-30 und Lipofectin für den aFGF-Gentransfer führte zu einer deutlichen Proliferationssteigerung (um ca. 50 %) der Zellen, womit der Einsatz dieses Wachstumsfaktors eine gute Möglichkeit darzustellen scheint, die prä-operative Ausgangssituation kornealer Transplantate zu verbessern. Der zweite Teil dieser Arbeit befasste sich mit Untersuchungen zur Immunmodulation nach Keratoplastik. Eine Hauptrolle bei der Abstoßung kornealer Transplantate spielen CD4+ T- Lymphozyten. In anderen Transplantationsmodellen konnte durch die lokale Überexpression immunmodulatorischer Zytokine die Entstehung und Aktivierung dieser Zellen gehemmt und eine verlängerte Transplantatüberlebenszeit erzielt werden. Mit Hilfe gentherapeutischer Vektoren (Adenoviren, Liposomen) wurden die immunmodulatorischen Zytokine vIL-10 und rIL-4 ex vivo in korneale Transplantate eingebracht. Nach erfolgreichen in vitro- Untersuchungen zur Genexpression wurden die transduzierten/transfizierten Hornhäute in einem starken Abstoßungsmodell der Ratte transplantiert, was jedoch zu keiner signifikanten Verlängerung der Transplantatüberlebenszeit führte. Diese Arbeit liefert Hinweise darauf, dass der lokale Gentransfer von vIL-10 in der Keratoplastik nicht für eine Immunmodulation geeignet ist, und dass sowohl die Zytokindosis, als auch der Zeitpunkt und der Ort der Vektorapplikation eine entscheidende Rolle für den Transplantationserfolg spielen.Keratoplasty is the most common transplantation of human tissue. The corneal endothelium constitutes a damagable cell layer on the inner surface of the cornea, unable to proliferate. From this, two general problems arise for the outcome of keratoplasty: loss of corneal endothelial cells occurs on the one hand during corneal long time storage before keratoplasty and enforces the lack of donor tissue, on the other hand it is often correlated with immune mediated rejections as well as with chronic processes after keratoplasty. Therefore an endothelial cell number as high as possible on corneal grafts displays a requirement for successful keratoplasties. The first aim of this study was non-viral gene transfer of the aFGF (acidic FGF) gene in corneal endothelial cells. Different lipid formulations were optimized for gene transfer in human corneal endothelial cells in vitro. Application of DAC-30 and Lipofectin for aFGF gene transfer clearly showed a stimulating effect on cell proliferation (approximately 50 %). Thus, the use of aFGF seems to be a good possibility for improving the pre-operative situation of corneal allografts. The second part of this study deals with the immune modulation after keratoplasty. CD4+ T- lymphocytes play a key role in rejection processes after keratoplasty. Local over expression of immunomodulatory cytokines in different transplantation models could inhibit the development and activation of these cells and was able to prolong the allograft survival time. Using different gene therapeutic vectors (adenoviruses, liposomes) the immunomodulatory cytokines vIL-10 and rIL-4 were transferred ex vivo in corneal allografts. After successfully determined gene expression in vitro, the transduced/transfected corneal allografts were transplanted in a strong rejection model of the rat. However, this was not sufficient in prolonging the graft survival time significantly. This study provides indications, that local gene transfer of vIL-10 in keratoplasty is not suitable for an immune modulation, and that both cytokine dose as well as time point and site of vector application play an important role for successful transplantation.
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Yu, Wing-yan, and 余泳欣. "Study of stem/progenitor cells located in the posterior limbus of the eye." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/197119.
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Some stem-like cells that have received limited attention but may be of considerable clinical value reside in the transition zone between the corneal endothelium (CE) and trabecular meshwork (TM) at the posterior limbus of the eyes. A growing body of evidence has revealed that these cells may be able to rejuvenate the CE, TM or both. However, these stem-like cells have not been clearly defined and we have thus named them PET cells (Progenitor cells of the Endothelium and Trabeculum).
Both the CE and TM cells are unique cell types in the eye that do not normally replace themselves once they are lost in ageing or diseases, such as Fuch’s endothelial dystrophy and primary open angle glaucoma. To date, no therapy exists that targets the rejuvenation of the compromised CE or TM in diseases. Therefore, the potential to repair or replace the diseased CE or TM through a cell repopulation approach is an important area that needs to be explored. The use of PET cells for tissue rejuvenation may represent an attractive therapeutic direction for the regeneration of the CE and/or TM. As a good animal model for PET cells is lacking, we sought to identify whether bovine eyes could serve as a good large tissue model for the studies of PET cells.
The comparative anatomy of the human and bovine posterior limbus was studied using light, confocal and scanning electron microscopy. Immunohistochemical studies were performed to localize the stem cell niche for the PET cells. Sphere culture was used to isolate and amplify progenitor cells from the CD and TM respectively. A detailed characterization of the spheres and their progenies was performed with immunocytochemistry, quantitative reverse-transcription polymerase chain reaction and differentiation and functional assays.
We showed the presence of stem or progenitor cells in the bovine CE, transition zone and TM in situ. The bovine TM insert region may house a stem cell niche that is comparable to that observed in humans. The progenitor cells isolated from the bovine CE and TM that grew as floating spheres demonstrated similar phenotypes in terms of stem cell marker expression. In addition, both the CE and TM spheres were bipotent, highly proliferative and had limited self-renewal capacity. However, they showed a high propensity to differentiate back into the cell type of their tissue of origin. We speculated that the PET cells become more tissues-specific as they migrate away from their niche towards the CE or TM. This may be a reasonable explanation why the CE and TM spheres respectively adopted their original lineage upon non-directed differentiation.
Taken together, our results support the hypotheses that PET cells are present in the posterior limbus of bovine eyes and that bovine eyes may serve as a good large tissue model for the studies of these cells. The PET cells represent an attractive target for developing new treatments to regenerate both the CE and TM, thereby reducing the requirement for donor corneas and invasive treatments in glaucomatous patuents.published_or_final_version
Ophthalmology
Doctoral
Doctor of Philosophy
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Valtink, Monika, Mirko Nitschke, Thomas Götze, Katrin Engelmann, and Carsten Werner. "Kultivierung transplantierbarer Zellverbände aus cornealem Endothel." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1223722766870-16827.
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Die In-vitro-Kultivierung von cornealem Endothel, einer funktionalen, beim Menschen nicht regenerierbaren Schicht in der Hornhaut des Auges, eröffnet weitreichende Möglichkeiten zum Zell- und Gewebeersatz. Dieser Artikel beschreibt aktuelle und künftige Optionen für zellbasierte Therapieansätze sowie die Bedeutung unbegrenzt proliferationsfähiger (immortalisierter) Zellpopulationen als Modellsystem für die Entwicklung neuartiger Methoden. In diesem Zusammenhang werden schaltbare Zellkulturträger als Möglichkeit zur schonenden Gewinnung transplantierbarer „cell sheets“ vorgestellt. Darüber hinaus wird die serumfreie Kultivierung als wichtige Voraussetzung für eine Anwendung am Menschen diskutiertThe in vitro cultivation of corneal endothelium – a functional, non-regenerable layer of the human cornea – is a promising approach for cell and tissue replacement. This paper introduces options for cell-based therapies and points out the importance of immortalised cell populations as a model system to develop tissue engineering strategies. In particular, the use of stimuli-responsive cell culture carriers for the gentle harvesting of “cell sheets” is described. Furthermore, serum-free cultivation is discussed as a prerequisite for future applications
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Laverne-Acquart, Sophie. "Banque de cornée : 10 ans d’innovations en contrôle qualité du greffon cornéen et projets d’avenir." Thesis, Saint-Etienne, 2013. http://www.theses.fr/2013STET012T/document.
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Le contrôle qualité réalisé sur les cornées en organoculture dans les banques de cornées est primordial pour l'évaluation de la qualité des greffons. Dans une première partie, nous avons montré, sur 505 cornées en routine, que les paramètres mesurés avec notre analyseur d'images Sambacornea (CV de taille et hexagonalité cellulaire endothéliale) étaient tout à fait cohérents avec ceux mesurés en microscopie spéculaire in vivo chez les patients. Notre dispositif fournit une méthode fiable, validée pour la mesure de la densité cellulaire endothéliale DCE et de la morphométrie cellulaire. Dans une deuxième partie, nous avons analysé les erreurs inhérentes à la méthode de comptage à cadre fixe (grille calibrée ou réticule) lors de la mesure de la DCE par les banques de cornées. Nous avons mesuré sur 3000 zones, la DCE de 20 mosaïques cornéennes gravées, en utilisant des grilles dont la maille variait de 50 à 300 µm. La méthode était répétable avec des grilles de 200 µm à condition de considérer la moyenne sur 10 comptages et présentait une variabilité résiduelle de 5 % vs la DCE réelle. Les grilles classiques de 100 µm utilisées en comptage manuel devraient être abandonnées et remplacées par des grilles de 200 µm. L'analyse d'images numériques demeure cependant la meilleure solution. Dans une troisième partie, le développement d'un dispositif de mesure de la transparence et des diamètres utiles en greffe de cornée (cornée claire et diamètre total) est décrit. La transparence est appréciée via la mesure de la fonction de modulation de transfert à travers la cornée sur une mire originale dessinée dans notre laboratoire. Les mesures étaient reproductibles. D’après l’analyse de 358 cornées en routine, la transparence n'était pas corrélée à l’âge du donneur, ni à la DCE, et baissait au fil de la conservation. Ce dispositif simple et objectif permet de standardiser la «transparence» ; il devra être redéployé sur notre bioréacteur, et pourrait mener à la définition d'un seuil de transparenceThe corneal quality control (QC) in eye banks is essential for the assessment of the quality of grafts. In the first part, we showed on 505 consecutive corneas, that the parameters measured by our image analyzer Sambacornea (size CV and hexagonality of endothelial cells) were entirely consistent with those measured by specular microscopy in vivo in patients. Our device provides a reliable validated method for the measurement of the Endothelial Cell Density ECD and cell morphometry. In a second part, we analyzed the inherent errors of the fixed-frame counting method (calibrated grid or graticule) for corneal ECD in eye banks. We measured in 3000 areas, the 20 corneal mosaic ECD using grids ranged from 50 to 300 µm. The method was repeatable with 200 µm long grids if the average of 10 counts were used: it showed a residual variability of 5 % vs the real ECD. Conventional 100 µm long grids used in manual counting should be abandoned and replaced by 200 µm grids. The digital image analysis, however, is the best solution. In the third part, the development of a device for measuring the transparency and effective diameters in corneal transplantation (clear cornea and total diameter) is described. Transparency is assessed by measuring the modulation transfer function through the cornea on an original pattern designed in our laboratory. The measurements were reproducible. Based on the analysis of 358 corneas routine, transparency was not correlated with donor age, or the ECD, and decreased with duration of stockage. This simple and objective device standardizes the QC "transparency" and it should be used with our bioreactor, and could lead to the definition of a level for transparency
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JOANNOT, BERNARD. "Phakoemulsification et endothelium corneen : etude prospective a propos de 69 cas." Nantes, 1994. http://www.theses.fr/1994NANT253M.
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