Purpose: We evaluate solutions for an applanating surface modification to the Goldmann tonometer prism, which substantially negates the errors due to patient variability in biomechanics. Methods: A modified Goldmann or correcting applanation tonometry surface (CATS) prism is presented which was optimized to minimize the intraocular pressure (lOP) error due to corneal thickness, stiffness, curvature, and tear film. Mathematical modeling with finite element analysis (FEA) and manometric lOP referenced cadaver eyes were used to optimize and validate the design. Results: Mathematical modeling of the optimized CATS prism indicates an approximate 50% reduction in each of the corneal biomechanical and tear film errors. Manometric lOP referenced pressure in cadaveric eyes demonstrates substantial equivalence to GAT in nominal eyes with the CATS prism as predicted by modeling theory. Conclusion: A CATS modified Goldmann prism is theoretically able to significantly improve the accuracy of lOP measurement without changing Goldmann measurement technique or interpretation. Clinical validation is needed but the analysis indicates a reduction in CCT error alone to less than +/- 2 mm Hg using the CATS prism in 100% of a standard population compared to only 54% less than +/- 2 mm Hg error with the present Goldmann prism. Translational Relevance: This article presents an easily adopted novel approach and critical design parameters to improve the accuracy of a Goldmann applanating tonometer.

Syfte: Att jämföra mätvärden från tre instrument som används för topografiska mätningar på två olika patientgrupper: en med friska ögon och en med corneala tillstånd. Instrumenten som användes var Javal Schiøtz keratometer, Topcon CA-200 topograf och Bon Sirius Scheimpflugkamera. Material och metod: Tre mätningar med respektive topograf och en mätning med keratometern utfördes på samtliga 27 patienter som deltog i studien och sammanlagt 54 ögon mättes av. Medelåldern på deltagarna med friska ögon (Grupp 1) var 25 år (21-44 år) och på de med corneala tillstånd (Grupp 2) var medelåldern 34,4 år (22-58 år). Krökningsradie, styrka och gradtal på de två huvudmeridianerna mättes med keratometern. Vid mätning med topograferna noterade värden från Sim-k och för 3, 5 och 7mm; dessa värden jämfördes sedan med varandra. Keratometern jämfördes med topografernas Sim-k värden. Resultat: Resultatet av studien visar ingen signifikant skillnad mellan de tre instrumenten (p=0,48 1-vägs ANOVA för upprepade mätningar) varken för Grupp 1 eller Grupp 2. Sim-k värden som visar astigmatism var lägst enligt Topcon för samtliga mätområden (Sim-k, 3, 5 och 7mm). Bon Sirius visade högre grader av uppmätt astigmatism än Topcon, men lägre än de uppmätta med keratometern. Korrelationen mellan instrumenten var bra för 5mm-zonen och visade signifikant skillnad för Grupp 2 (p=0,03). Jämförelse mellan Javal keratometer och Sirius visade också signifikant skillnad för Grupp 2 (p=0,02). Javal keratometer visade högst standardavvikelse av samtliga instrument. Slutsats: Det fanns ingen signifikant skillnad mellan de olika instrumenten, med undantag för jämförelse mellan Sim-k-värden för Javal keratometer och Bon Sirius topograf, samt för 5mm-zonen mellan Topcon CA-200 och Bon Sirius i gruppen med corneala dystrofier. Detta visar att instrumenten är jämförbara när ögonen inte lider av extrema, onormala formförändringar.

Corneal transplantation is performed to restore vision or to relieve pain in patients with damaged or diseased corneas. However, approximately 40% of corneal allografts fail after 10 years. The most common cause of graft failure is irreversible immunological rejection, primarily mediated by CD4+ T cells, despite the topical application of glucocorticosteroids. The aim of this project was to investigate the anatomic site of antigen presentation during corneal transplantation in the rat, by using a lentiviral vector to express an anti-CD4 antibody fragment at potential sites of antigen presentation, including the donor corneal endothelium, the anterior segment of the eye and the cervical lymph nodes. Dual-gene lentiviral vectors were constructed by inserting the 2A self-processing sequence between two transgenes. This allowed expression of two transgenes within a single open reading frame. In vitro characterisation of the dual-gene vectors was performed in cell culture experiments, which showed that transgenic proteins were expressed at lower levels from dual-gene vectors compared to the expression from single-gene vectors and expression was lowest when the transgene was situated downstream of the 2A self-processing sequence. To locate the anatomic site of antigen presentation during corneal transplantation in rats, a lentiviral vector carrying an anti-CD4 antibody fragment was delivered to the corneal endothelium either immediately prior to corneal transplantation by ex vivo transduction of the donor corneas, or 5 days prior to corneal transplantation by anterior chamber injection into both the recipient and the donor rats. A separate group of recipient rats received intranodal injections of the lentiviral vector carrying an anti-CD4 antibody fragment into the cervical lymph nodes 2 days prior to corneal transplantation. Another group of rats underwent bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Corneal allografts were scored daily for opacity, inflammation and neovascularisation. Expression of the anti-CD4 antibody fragment from transduced tissues was detected using flow cytometry and polymerase chain reaction. Modest, but significant prolongation of corneal allograft survival was experienced by rats that received ex vivo transduction of the donor corneas with a lentiviral vector carrying an anti-CD4 antibody fragment immediately prior to corneal transplantation, but all grafts did eventually reject. Anterior chamber injection of the lentiviral vector carrying the anti-CD4 antibody fragment 5 days prior to corneal transplantation into both recipient and donor eyes did not prolong allograft survival. Intranodal injection of a lentiviral vector carrying an anti-CD4 antibody fragment did not prolong the survival of the corneal allografts, nor did bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Neither expression of the anti-CD4 antibody fragment in the cervical lymph nodes nor the removal of these nodes was able to prolong corneal allograft survival in rats, suggesting that T cell sensitisation could potentially occur elsewhere in the body. However, expression of the anti-CD4 antibody fragment from the donor corneal endothelium was able to prolong corneal allograft survival, suggesting that some antigen presentation might occur within the anterior segment of the eye. Based on the findings described in this thesis and those of others, I propose that antigen presentation in the rat occurs within anterior segment of the eye and within the secondary lymphoid tissues such as the cervical lymph nodes, and that inhibiting antigen presentation at one of these sites will delay graft rejection. However, to completely abolish antigen presentation during corneal transplantation in the rat, I hypothesise that antigen presentation within both the anterior segment of the eye and within the secondary lymphoid tissues must be inhibited.

Thesis (M.S.)--New England College of Optometry, 2009. / Includes bibliographical references (p. 94-96).

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 156-180). Also available online.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Pathology. Includes bibliographical references.

Εισαγωγή: η οφθαλμική νεοαγγείωση αποτελεί την πρωταρχική αιτία τύφλωσης για μεγάλο εύρος ασθενειών του οφθαλμού. Ο ακριβής υποκείμενος παθογενετικός μηχανισμός της οφθαλμικής νεοαγγείωσης δεν είναι ακόμη απόλυτα κατανοητός αλλά θεωρείται, ότι η διαταραχή της ισορροπίας μεταξύ αγγειογενετικών και αντί-αγγειογενετικών παραγόντων ευθύνεται, κυρίως για την επαγωγή αγγειογένεσης. Η παρστατίνη είναι ένα πεπτίδιο 41 αμινοξέων που ελευθερώνεται μετά την πρωτεολυτική ενεργοποίηση του PAR1 υποδοχέα και περιέχει μια έντονα υδρόφοβη και μια έντονα υδρόφιλη περιοχή (αλληλουχία αμινοξέων). Πρόσφατα, αποδείχθηκε ότι η παρστατίνη είναι ένας ισχυρός αναστολέας της αγγειογένεσης. Ο σκοπός αυτής της εργασίας ήταν η ανάπτυξη ενός μοντέλου νεοαγγείωσης κερατοειδούς, για την χρήση του στην εκτίμηση της επίδρασης της παρστατίνης στην φλεγμονή και τη νεοαγγείωση του κερατοειδούς και στην διερεύνηση του λειτουργικού ρόλου της δομής της στην νεοαγγείωση. Μέθοδος: για την επαγωγή της νεοαγγείωσης του κερατοειδούς σε Sprague–Dawley ποντίκια, έγινε έγκαυμα, με στειλεούς καλυμμένους 75% με διάλυμα νιτρικού αργύρου και 25% νιτρικού καλίου (κ.β.), στο κέντρο του κερατοειδούς για 4 δευτερόλεπτα. Η παρστατίνη και τα πεπτίδια με την υδρόφοβη και την υδρόφιλη περιοχή της παρστατίνης χορηγήθηκαν υπό τον επιπεφυκότα, αμέσως μετά το χημικό έγκαυμα. Η νεοαγγείωση του κερατοειδούς εκτιμήθηκε την 7η ημέρα μετά το έγκαυμα με τη χρήση ενός ημιαυτόματου προγράμματος που αναπτύχθηκε σε MATLAB 7.5. Η πυκνότητα των νεοαγγείων και η φλεγμονώδης διήθηση επίσης εκτιμήθηκε την 7η ημέρα μετά το έγκαυμα, με ιστοπαθολογική εξέταση λεπτών τομών κερατοειδούς. Αποτελέσματα: η παρστατίνη χορηγούμενη υπό τον επιπεφυκότα ανέστειλε την νεοαγγείωση του κερατοειδούς και 200 μg ήταν η πιο αποτελεσματική δόση (αναστολή 59%). Επιπλέον η παρστατίνη ανέστειλε σημαντικά την φλεγμονώδη διήθηση του κερατοειδούς. Το πεπτίδιο που περιείχε την υδρόφοβη αλληλουχία του μορίου κατέστειλε την νεοαγγείωση με μέγιστη αναστολή 47% στην δόση των 200 μg, ενώ το πεπτίδιο που περιείχε την υδρόφιλη αλληλουχία έδειξε μέγιστη αναστολή 73% επίσης στην δόση των 200 μg. Τα αναγραμματισμένα πεπτίδια δεν έδειξαν κανένα σημαντικό αποτέλεσμα. Συμπεράσματα: η παρστατίνη και ιδιαίτερα το υδρόφιλο τμήμα της, είναι ισχυρός αναστολέας της νεοαγγείωσης του κερατοειδούς και μπορεί να αποτελέσει ισχυρή θεραπευτική στρατηγική για την αντιμετώπιση της οφθαλμικής νεοαγγείωσης / Introduction: Ocular neovascularization is the primary cause of blindness in a wide range of ocular diseases. The exact mechanism underlying the pathogenesis of ocular neovascularization is not yet well understood, but it is believed that the imbalance between angiogenic stimulating factors and angiogenic inhibitors is the major contributor to angiogenesis. Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the PAR1 receptor and contains a hydrophobic and a hydrophilic domain. Recently it was demonstrated that parstatin is a potent inhibitor of angiogenesis. The purpose of the study was to develop a model of corneal neovascularization which will be used to investigate the effect of parstatin on corneal inflammation and neovascularization and to evaluate the functional role of its structure on neovascularization. Methods: Corneal neovascularization on Sprague–Dawley rats was induced with cauterization by an applicator stick, coated with 75% silver nitrate and 25% potassium nitrate, to the center of the cornea for 4 seconds. Parstatin and peptides containing the hydrophobic and hydrophilic domains of parstatin were administrated subconjunctival after the chemical burn. Corneal neovascularization was assessed the 7th day after cauterization using a semiautomatic custom software, developed in MATLAB 7.5. Corneal microvessel density and inflammatory cell infiltration was assessed also the 7th day after the burn by histopathology examination of corneal sections. Results: Subconjunctival parstatin inhibited corneal neovascularization, with 200 μg the most effective dose (59% inhibition). In addition parstatin significantly inhibited corneal inflammatory infiltration. The peptide contained the hydrophobic domains of parstatin suppressed corneal neovascularization with maximum inhibition of 47% at 200 μg, whereas the peptide contained the hydrophilic domain exhibit maximum inhibition of 73% at 200 μg. Scrambled peptides were without any significant effect. Conclusion: Parstatin and especially its hydrophilic domain, is a potent antiangiogenic agent of corneal neovascularization and may have clinical potential in the treatment of angiogenesis-related ocular disorders.

Orientador: Silvana Artioli Schellini / Resumo: 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. / Mestre

Made available in DSpace on 2014-06-11T19:23:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2002Bitstream added on 2014-06-13T19:09:41Z : No. of bitstreams: 1 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.

The accurate detection and quantification of human diabetic somatic polyneuropathy (DSPN) are important to define at risk patients, anticipate deterioration, and assess new therapies. Current methods lack sensitivity, require expert assessment and have major shortcomings when employed to define therapeutic efficacy. In recent years, in vivo corneal confocal microscopy (IVCCM) has shown potential as a surrogate endpoint for DSPN.This study aims to investigate fundamental aspects of IVCCM such as repeatability and optimal scanning methodology and establish changes in corneal nerve morphology in relation to the severity of DSPN and regeneration in response to normalisation of hyperglycaemia. Furthermore, it aims to provide a novel fully automated image analysis algorithm for the quantification of corneal nerve morphology and establish the diagnostic ability of CCM.IVCCM shows high repeatability which is enhanced with more experienced observers. Central corneal innervation is comparable to adjacent peripheral innervation in mild diabetic neuropathy but the central cornea may be more sensitive to change. Corneal nerve loss is symmetrical and progressive with increasing neuropathic severity and corneal nerves show significant regenerative capacity following rapid normalisation of glycaemic control after simultaneous pancreas and kidney transplantation. The novel image analysis algorithm strongly correlates with human expert annotation and therefore represents a rapid, objective and repeatable means of assessing corneal nerve morphology. Automated image quantification may replace human manual assessment with high diagnostic validity for DSPN.