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.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Observa-se exacerbaÃÃo do processo angiogÃnico em diversas doenÃas, entre elas pode ser citada a neovascularizaÃÃo de cÃrnea. Para o tratamento dessas doenÃas, sÃo estudados diversos fÃrmacos com propriedades antiangiogÃnicas. O objetivo deste trabalho foi avaliar a eficÃcia do fÃrmaco LASSBIO-596, estruturalmente designado como um hÃbrido da talidomida, sildenafil e arilsulfonamida, na angiogÃnese corneana inflamatÃria. Dezoito coelhos foram submetidos a uma cauterizaÃÃo alcalina pontual na regiÃo superior da cÃrnea direita. Os animais foram randomicamente alocados em trÃs grupos: veÃculo, dexametasona 4,0% e LASSBio-596 1,0%. Os tratamentos foram administrados em forma de colÃrio trÃs vezes por dia durante 21 dias. AvaliaÃÃes foram realizadas nos dias 3, 6, 9, 12, 15, 18 e 21 pÃs-cauterizaÃÃo. Nesses momentos, imagens digitalizadas da cÃrnea foram capturadas de uma forma padronizada. A resposta angiogÃnica foi mensurada utilizando-se um software que foi desenvolvido especificamente para esta finalidade. Foram calculados os seguintes parÃmetros: Ãrea de neovascularizaÃÃo (AN), comprimento vascular total (CT) e nÃmero de vasos sanguÃneos (NV). A partir da AN, calculou-se a taxa de angiogÃnese (TA) em cada grupo estudado. Observou-se que a dexametasona diminuiu significativamente AN, CT e NV durante todas as avaliaÃÃes. Portanto, a dexametasona inibiu completamente a angiogÃnese inflamatÃria da cÃrnea com uma TA de -0,001  0,006 mm2/dia, que foi significativamente menor (p < 0,001) do que a observada apÃs o tratamento com o veÃculo (0,078  0,024 mm2/dia) e LASSBio-596 (0,054  0,012 mm2/dia). Apesar de o LASSBio-596 ter reduzido o processo de angiogÃnese em relaÃÃo ao veÃculo, conforme AN, CT e NV, essa diferenÃa nÃo foi estatisticamente significante. No entanto, verificou-se que a TA medida no grupo LASSBio-596 foi significativamente menor (p < 0,05) do que a observada nos animais controle, indicando um potencial efeito antiangiogÃnico. ConcluÃmos que a aplicaÃÃo tÃpica de LASSBio-596 a 1,0% tem um efeito inibitÃrio parcial sobre a angiogÃnese corneana inflamatÃria em coelhos. / There is exacerbation of the angiogenic process in various diseases, especially in corneal neovascularization. For the treatment of these diseases, several drugs were studied with antiangiogenic properties. The aim of this study was to evaluate the effect of LASSBio-596, structurally designed as a hybrid of thalidomide, sildenafil and arilsulfonamide, on inflammatory corneal angiogenesis. Eighteen rabbits were submitted to an alkaline cauterization in the right cornea. The animals were randomly allocated in three groups: vehicle, dexamethasone, and LASSBio-596 group. Drugs were administrated by eye drops three times per day for 21 days. Evaluations were performed on days 3, 6, 9, 12, 15, 18, and 21 post-cauterization. At these time points, digital images of the cornea were captured in a standard fashion. Angiogenic response was measured using software that was developed specifically for this purpose. It calculated the following parameters: neovascularization area (NA), total vascular length (TVL), and blood vessel number (BVN). It was observed that dexamethasone significantly decreased NA, TVL, and BVN during all assessments. From NA it was calculated the angiogenesis rate (AR) in each group. Therefore, dexamethasone completely inhibited the inflammatory corneal angiogenesis with angiogenesis rate (AR) of -0.001  0.006 mm2/day, which was significantly lower (p < 0.001) than that observed after treatment with vehicle (0.078  0.024 mm2/dia) and LASSBio-596 (0.054  0.012 mm2/dia). Although LASSBio-596 reduced angiogenesis in relation to vehicle, according to NA, TVL and BVN values, this difference was not statistically significant. However, it was found that the AR as measured in the LASSBio-596 group was significantly lower (p < 0.05) than that seen in control animals, indicating a potential antiangiogenic effect. We conclude that topical application of LASSBio-596 at 1.0% has a partial inhibitory effect on inflammatory corneal angiogenesis in rabbits.

Age-related macular degeneration (AMD) is the leading cause for visual impairment and blindness in the elder population of developed countries. The primary underlying cause for significant visual loss is the choroidal neovascularization (CNV). Current treatment strategies for AMD include laser photocoagulation, photodynamic therapy (PDT) and excision of neovascular membranes, but have met with limited success. In our previous studies, we demonstrated that gene delivery of angiogenesis inhibitor vasostatin (VS) attenuated the corneal neovascularization in animals. The primary objective of this study was to investigate gene delivery of vasostatin (VS) attenuated the choroidal neovascularization in animals. Retinal and visual function will be evaluated. However, systematic expression of angiogenesis inhibitor may bring adverse effects to physiological processes. The feasibility, efficiency and safety of gene delivery with systemic and local routes were evaluated. Intramuscular polymer-based gene delivery had no side effect such as virus vector and revealed the safety. Recombinant adenovirus (Ad) was used gene delivery system because of its high titer, wide host range, and transduction efficiency. Adeno-associated virus (AAV) represents highly efficient that can facilirate long-term transduction. We propose to improve the efficacy and safety of VS gene delivery, and to search for the effective delivery route and other adjuvant therapy in conjunction with VS for treatment of CNV. Recently, PDT with veteporfin is an established treatment for subfoveal CNV secondary to AMD. We tried to compare the effect and safety of standard and reduced-dose light application PDT in an animal mocel of CNV. The 180-residue VS and its 48-residue (VS48) inhibited the migration and tube formation in cultured endothelial cells. Topical VS application suppresses the progression of laser-induced CNV via angiogenesis ihhibition, as well as in VS48. VS-48 inhibited the growthof vessels in arota rings. Electroretinograms (ERG) analysis revealed that topical VS-48 application for 21 days had no effect on rat retinal functions. Topical VS-48 treatment significantly reversed the CNV-induced alterations in ERG. Transfection of pCMV3-VS into muscle cells resulted in increased production and release of exogenous VS, which specifically inhibited the proliferation of endothelial cells. Rats treated with intrmuscular injection with PVP-VS also showed a significant reduction in the CNV lesions for at least 42 days. Subconjunctival injection with Ad vector revealed no retinal toxicity in ERG. Ad-luciferase via subconjunctival injections showed ocular expression for as long as 112 days by using bioluminescence image analysis in rodent. AAV-luciferase via subconjunctival injections showed ocular expression for as long as 365 days by using bioluminescence image analysis in mice, and AAV serotype 5-luciferase even showed expression lasting for 2 years. Suppression of laser photocoagulation¡Vinduced CNV by Ad-VS was documented in rat model. Combination therapies are important as treatment options. We demonstrated that PDT could effectively attenuate CNV in a rat model, and reduced doses, worked just as well as the standard dose. In the preliminary study of PDT combined topical VS application, treatment led to CNV attenuation more than alone with PDT. The above experiments would enable us to demonstrate that the vasostatin delivery might be a promising strategy for the treatment of AMD and other retinal or ocular disorders. Furthermore, the results from animal studies might be extrapolated for future clinical application.

Medical imaging tools technologically supported by the recent advances in the areas of computer vision can provide systems that aid medical professionals to carry out their expert diagnostics and investigations more effectively and efficiently. Two medical application domains that can benefit by such tools are ophthalmology and Total Hip Replacement (THR). Although a literature review conducted within the research context of this thesis revealed a number of existing solutions these are either very much limited by their application scope, robustness or scope of the extensiveness of the functionality made available. Therefore this thesis focuses on initially investigating a number of requirements defined by leading experts in the respective specialisms and providing practical solutions, well supported by the theoretical advances of computer vision and pattern recognition. This thesis provides three novel algorithms/systems for use within image analysis in the areas of Ophthalmology and THR. The first approach uses Contourlet Transform to analyse and quantify corneal neovascularization. Experimental results are provided to prove that the proposed approach provides improved robustness in the presence of noise, non-uniform illumination and reflections, common problems that exist in captured corneal images. The second approach uses a colour based segmentation approach to segment, measure and analyse corneal ulcers using the HVS colour space. Literature review conducted within the research context of this thesis revealed that there is no such system available for analysis and measurement of corneal ulcers. Finally the thesis provides a robust approach towards detecting and analysing possible dislocations and misalignments in THR X-ray images. The algorithm uses localised histogram equalisation to enhance the quality of X-ray images first prior to using Hough Transforms and filtered back projections to locate and recognise key points of the THR x-ray images. These key points are then used to measure the possible presence of dislocations and misalignments. The thesis further highlights possible extensions and improvements to the proposed algorithms and systems.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-08-04T18:17:11Z No. of bitstreams: 2 Mestrado -Luciana Lavigne Linhares - 2016.pdf: 5586092 bytes, checksum: e3eb26a179f1a588ae5f1b655c9bde54 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-05T13:33:31Z (GMT) No. of bitstreams: 2 Mestrado -Luciana Lavigne Linhares - 2016.pdf: 5586092 bytes, checksum: e3eb26a179f1a588ae5f1b655c9bde54 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-05T13:33:31Z (GMT). No. of bitstreams: 2 Mestrado -Luciana Lavigne Linhares - 2016.pdf: 5586092 bytes, checksum: e3eb26a179f1a588ae5f1b655c9bde54 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-29 / The aim of this study was to compare the sunitinib loaded nanospheres with its free form, in the inhibition of induced corneal neovascularization, in albino rabbits, identifying the degree of inhibition of this neovascularization by measuring the area of corneal neovascularization. The eyes of eleven animals were divided into two groups. Group A (right eye of each rabbit) - sunitinib loaded nanospheres and Group B (left eye of each rabbit) - sunitinib solution. Both groups were submited to corneal alkaline burn by sodium hydroxide 1 mol/L. 12 hours after the corneal burn procedure, the group A received topical nanospheres loaded with sunitinib 0.5 mg/mL (Axon Medchem BV, Groningen, Holland), and the group B received 0.5 mg/mL of the sunitinib solution (Axon Medchem BV, Groningen, Holland). Treatment was initiated 12 hours after the surgical and was administered topically 2 times a day during 14 days. After 14 days of treatment all the rabbits were submitted to the examination of the anterior segment slit lamp examination and were photographed using the iSight camera of 8 megapixels with pixels of 1.5 μ. After euthanasia, the eyes were enucleated and sent for histopathological analysis. Neovascularization area in the upper cornea was measured in groups A and B. There was no statistically significant difference between the corneal neovascularization area in each group (p = 0.949). No changes were observed in ophthalmological clinical examination compatible with toxicity to the topical use of sunitinib loaded nanospheres and its free form. No histopathologic diferences were observed in both groups. We conclude that both the free form of sunitinib and sunitinib loaded nanospheres show no difference in inhibiting corneal neovascularization. / O objetivo deste estudo foi comparar a formulação de nanoesfera do malato de sunitinibe com a sua forma livre ou não encapsulada, na inibição de neovascularização corneana induzida em coelhos albinos, identificando o grau de inibição dessa neovascularização pela medida da área de neovasos corneanos. Os olhos de 11 (onze) animais foram divididos em 2 (dois) grupos. O grupo A (olho direito de cada coelho) e o grupo B (olho esquerdo de cada coelho). Ambos os grupos foram submetidos a queimadura corneana por hidróxido de sódio 1 mol/L. 12 horas após o procedimento de queimadura corneana, o grupo A recebeu solução tópica de nanoesfera de sunitinibe 0,5 mg/mL (Axon medchem BV, Groningen, Holanda), e o grupo B recebeu 0,5 mg/mL da forma livre do sunitinibe (Axon medchem BV, Groningen, Holanda). O tratamento foi iniciado 12 horas após o procedimento cirúrgico e foi topicamente administrado 2 vezes ao dia durante 14 dias. Após 14 dias, todos os coelhos foram submetidos ao exame de biomicroscopia do segmento anterior e fotografados usando a câmera iSight de 8 megapixels com pixels de 1,5 μ. Após eutanásia dos coelhos, os olhos foram enucleados e encaminhados para análise histopatológica. Foi realizada a mensuração da área de neovasos (em mm2) no terço superior da córnea dos grupos A e B. Não houve diferença estatisticamente significante entre as áreas de neovascularização corneana em cada grupo (p= 0,949). Não foram observadas alterações no exame clínico oftalmológico compatíveis com toxicidade tanto com a nanoesfera de sunitinibe como com a forma livre de sunitinibe. Não foram observadas diferenças histopatológicas nos dois grupos estudados. Conclui-se que tanto o sunitinibe associado à nanoesfera quanto a sua forma livre não apresentam diferença na inibição da neovascularização corneana induzida.

Objetivos: Os objetivos deste estudo foram: 1) determinar a eficácia da transferência gênica do vetor de adenovírus sorotipo 5, carreando o gene do receptor do fator de crescimento endotelial vascular (VEGF) solúvel humano (sVEGFR1) para a glândula lacrimal (GL); 2) investigar se a expressão de sVEGFR1 interfere na neovascularização da córnea (NVC), induzida por queimadura alcalina; 3) avaliar a segurança do procedimento. Métodos: Trinta e dois ratos Wistar foram submetidos à queimadura central da córnea direita com solução de hidróxido de sódio (NaOH) 1 M. Os animais foram divididos em três grupos e injetados diretamente em sua GL direita 25 ?l de vetores virais AdVEGFR1 (1x1010 pfu) (12 animais), 25 ?l do vetor AdNull (1x1010pfu) (10 animais), ou 25 ?l de solução salina (Controle). Após sete dias, a NVC foi observada e fotografada na lâmpada de fenda. A secreção lacrimal foi medida com fenol. A presença do sVEGFR1 na GL foi testada por qPCR (quantitative polymerase chain reaction) e a coloração, por imunofluorescência. O qPCR foi também utilizado para comparar o RNA mensageiro (RNAm) de ilterleucina-1beta (IL-1?), ilterleucina-6 (IL-6) e fator de necrose tumoral alfa (TNF-?) na GL e no gânglio do trigêmeo (GT). Resultados: O vetor AdVEGFR1 transfectou 83% dos ratos. O sVEGFR1 estava presente nas células acinares da GL. A NVC foi prevenida em nove de doze animais do grupo AdVEGFR1, em comparação com o grupo Ad-Null (3:10) e o grupo Controle (1:10) (p=0,0317). A secreção lacrimal e o RNAm das citocinas na GL e no GT foram semelhantes nos três grupos (p>0,05). Conclusões: A transferência gênica do vetor adenoviral para a GL demonstrou expressão local do sVEGFR1 humano, e evitou a NVC na maioria dos olhos expostos a queimaduras alcalinas, se mostrando seguro para a estrutura e função da GL. / Purpose: The aims of this study were: 1) to determine the efficacy of adenovirus vector serotype 5 (Ad) encoding human soluble VEGF receptor 1 (sVEGFR1) gene transfer to the lacrimal gland (LG); 2) to investigate whether expression of sVEGFR1 acts in corneal neovascularization (CNV), induced by alkali burn and; 3) to evaluate the safety of the procedure. Methods: AdVEGFR1viral vectors (25 ?l, 1x1010pfu) were injected in the right LG of rats and compared with AdNull vector (25 ?l, 1x1010pfu) or 25?l saline (Control) before cornea alkali burn with 1 M NaOH. After seven days, CNV was observed and photographed in the slit lamp. Tear secretion was measured with phenol red thread. The animals were tested for human VEGFR1 mRNA and protein in the LG by qPCR and immunofluorescence staining, respectively. qPCR was also used to compare the mRNA of IL-1?, IL-6, and TNF-? in LG and ipsilateral trigeminal ganglion (TG). Results: Ad-VEGFR1 transfected 83% of the rats. VEGFR1 was present in LG acinar cells. CNV was prevented in 9 of 12 animals of Ad-VEGFR1 group, compared to Ad-Null (3:10) and Control (1:10) (p=0.0317). The tear secretion and the cytokines mRNA in LG and TG were similar all three groups (p>0.05). Conclusion: Adenoviral vector gene transfer to LG as the has shown local expression of human sVEGFR1, as It prevented CNV in most of the eyes exposed to alkali burn and was safe for LG structure and function.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Um programa de computador foi desenvolvido especificamente para a segmentaÃÃo e quantificaÃÃo de angiogÃnese corneana em imagens digitais capturadas in vivo. O propÃsito foi estabelecer um mÃtodo automÃtico para quantificar, de forma rÃpida, objetiva e acurada, a progressÃo temporal da resposta angiogÃnica. Desenvolveu-se ainda um modelo de angiogÃnese corneana inflamatÃria em coelhos, induzida por uma cauterizaÃÃo alcalina pontual, onde a resposta angiogÃnica foi avaliada, conforme cinco mÃtodos manuais, nos dias 3, 6, 9, 12, 15, 18 e 21 apÃs a cauterizaÃÃo, ocasiÃes em que tambÃm foram obtidas, de maneira padronizada, imagens digitais da cÃrnea. O software à composto de cinco mÃdulos, com funÃÃes especÃficas, que identificam e segmentam automaticamente os neovasos e quantificam a resposta angiogÃnica mediante a determinaÃÃo de trÃs parÃmetros principais: Ãrea de neovascularizaÃÃo, comprimento vascular total e nÃmero de vasos sangÃÃneos. Assim, o sistema dispensa a rotulaÃÃo prÃvia da neovascularizaÃÃo, propiciando, por conseguinte, o monitoramento espacial e temporal da resposta angiogÃnica in vivo. A avaliaÃÃo da validade do mÃtodo automÃtico proposto envolveu a determinaÃÃo do grau da correlaÃÃo entre cada um dos quantificadores automÃticos e os cinco parÃmetros manuais. Compreendeu ainda a comparaÃÃo do reconhecimento automÃtico dos vasos sangÃÃneos realizado pelo software em 50 subimagens com o reconhecimento manual efetuado, consensualmente, por trÃs examinadores (padrÃo-ouro), determinando-se entÃo a sensibilidade, especificidade e o coeficiente de concordÃncia kappa. Para avaliar a confiabilidade, trÃs examinadores utilizaram o mÃtodo automÃtico para analisar 50 imagens em dois momentos. A concordÃncia intra e interexaminadores foi medida pelo coeficiente de correlaÃÃo intraclasse. Para ilustrar a utilidade do mÃtodo automÃtico, delineou-se um ensaio que avaliou o efeito dos fÃrmacos prednisona, talidomida, Ãcido acetilsalicÃlico, etoricoxib e celecoxib no modelo de angiogÃnese corneana inflamatÃria. A anÃlise dos grÃficos dos quantificadores manuais e automÃticos revelou que a evoluÃÃo temporal da resposta angiogÃnica no modelo de angiogÃnese corneana inflamatÃria seguiu um padrÃo bifÃsico: crescimento exponencial atà o dia 12 (fase de proliferaÃÃo), seguido de crescimento linear atà o dia 21 (fase de maturaÃÃo). Constatou-se uma forte correlaÃÃo linear positiva estatisticamente significante entre os quantificadores automÃticos e manuais, denotando que o mÃtodo automÃtico mensurou a resposta angiogÃnica de forma anÃloga à realizada pelo mÃtodo manual. A sensibilidade do mÃtodo automÃtico foi 85,32%, enquanto sua especificidade foi 96,37%. A concordÃncia entre o reconhecimento manual e o reconhecimento automÃtico dos vasos sangÃÃneos foi classificada pela estatÃstica kappa como quase perfeita. A concordÃncia interexaminadores foi avaliada em seis situaÃÃes, sendo classificada como excelente em quatro delas e como boa nas outras duas. A concordÃncia intra-examinador foi mensurada em nove situaÃÃes, sendo classificada como boa em apenas uma e como excelente nas demais. AlÃm disso, o sistema automÃtico foi capaz de identificar diferenÃas na intensidade do efeito antiangiogÃnico dos diferentes fÃrmacos avaliados, possibilitando graduar a atividade inibitÃria em plena (prednisona), parcial (talidomida, Ãcido acetilsalicÃlico e etoricoxib) e insuficiente (celecoxib), demonstrando, assim, o seu valor como mÃtodo para anÃlise quantitativa de angiogÃnese. Portanto, tais dados demonstram que o sistema automÃtico proposto constitui um mÃtodo de quantificaÃÃo de angiogÃnese acurado e reprodutÃvel. / A software was developed specifically for segmentation and quantification of corneal angiogenesis in digital images acquired in vivo. The purpose was to establish an automatic method to quantify the corneal progression of the neovascular response in a rapid, objective and accurate manner. It was also developed a model of inflammatory corneal angiogenesis in rabbits induced by a punctual alkaline cauterization. Angiogenic response was evaluated at days 3, 6, 9, 12, 15, 18 and 21 after cauterization. At these occasions, digital images of cornea were also captured in a standardized fashion. The software is composed of five modules, each with specific functions, which automatically identify and segment the neovessels and quantify the neovascular response through the calculation of three main parameters: neovascularization area, total vascular length and blood vessels number. Thus, the system does not require previous enhancement of the neovascularization, so that it provides the temporal and spatial monitoring of the angiogenic response in vivo. To assess the validity of the automatic method, it was determined the correlation degree between each automatic parameters and the five manual variables. It was also evaluated by comparing automatic recognition of blood vessels performed by the software on 50 subimages with the manual recognition performed by three raters by consensus (gold-standard) and then calculating the sensitivity, specificity and kappa coefficient. To assess reliability, three raters used the automatic method for analysing 50 images at two moments. The intra and inter-raters agreement was assessed by calculating intraclass correlation coefficient. To illustrate the utility of the automatic method, it was designed an assay that evaluated the effect of prednisone, thalidomide, acetylsalicylic acid, etoricoxib and celecoxib in the model of inflammatory corneal angiogenesis. The analysis of the graphics of the manual and automatic parameters showed that the temporal progression of the neovascular response in this model followed a biphasic pattern: exponential growth until day 12 (proliferation phase), followed by linear growth until day 21 (maturation phase). It was found a statistically significant positive linear correlation between automatic and manual variables, denoting that both methods measured the angiogenic response in an analogous fashion. The sensitivity of the automatic method was 85.32% and the specificity was 96.37%. The agreement between manual and automatic recognition of blood vessels was classified as almost perfect, according to kappa statistics. The inter-raters agreement was measured in six situations and it was classified as good in two of them and as excellent in the others. Among the nine situations analysed, the intra-rater agreement was classified as good in only one of them and as excellent in the others. Furthermore, the automatic system was able to detect differences in the intensity of the antiangiogenic effect of tested drugs, allowing the inhibitory activity to be graduated as full (prednisone), partial (thalidomide, acetylsalicylic acid and etoricoxib) and insufficient (celecoxib), demonstrating, thus, its value as method for quantitative analysis of angiogenesis. Therefore, such results demonstrate that the proposed automatic system constitutes an accurate and reproducible method of angiogenesis quantification.

Objetivos: Os objetivos deste estudo foram: 1) determinar a eficácia da transferência gênica do vetor de adenovírus sorotipo 5, carreando o gene do receptor do fator de crescimento endotelial vascular (VEGF) solúvel humano (sVEGFR1) para a glândula lacrimal (GL); 2) investigar se a expressão de sVEGFR1 interfere na neovascularização da córnea (NVC), induzida por queimadura alcalina; 3) avaliar a segurança do procedimento. Métodos: Trinta e dois ratos Wistar foram submetidos à queimadura central da córnea direita com solução de hidróxido de sódio (NaOH) 1 M. Os animais foram divididos em três grupos e injetados diretamente em sua GL direita 25 ?l de vetores virais AdVEGFR1 (1x1010 pfu) (12 animais), 25 ?l do vetor AdNull (1x1010pfu) (10 animais), ou 25 ?l de solução salina (Controle). Após sete dias, a NVC foi observada e fotografada na lâmpada de fenda. A secreção lacrimal foi medida com fenol. A presença do sVEGFR1 na GL foi testada por qPCR (quantitative polymerase chain reaction) e a coloração, por imunofluorescência. O qPCR foi também utilizado para comparar o RNA mensageiro (RNAm) de ilterleucina-1beta (IL-1?), ilterleucina-6 (IL-6) e fator de necrose tumoral alfa (TNF-?) na GL e no gânglio do trigêmeo (GT). Resultados: O vetor AdVEGFR1 transfectou 83% dos ratos. O sVEGFR1 estava presente nas células acinares da GL. A NVC foi prevenida em nove de doze animais do grupo AdVEGFR1, em comparação com o grupo Ad-Null (3:10) e o grupo Controle (1:10) (p=0,0317). A secreção lacrimal e o RNAm das citocinas na GL e no GT foram semelhantes nos três grupos (p>0,05). Conclusões: A transferência gênica do vetor adenoviral para a GL demonstrou expressão local do sVEGFR1 humano, e evitou a NVC na maioria dos olhos expostos a queimaduras alcalinas, se mostrando seguro para a estrutura e função da GL. / Purpose: The aims of this study were: 1) to determine the efficacy of adenovirus vector serotype 5 (Ad) encoding human soluble VEGF receptor 1 (sVEGFR1) gene transfer to the lacrimal gland (LG); 2) to investigate whether expression of sVEGFR1 acts in corneal neovascularization (CNV), induced by alkali burn and; 3) to evaluate the safety of the procedure. Methods: AdVEGFR1viral vectors (25 ?l, 1x1010pfu) were injected in the right LG of rats and compared with AdNull vector (25 ?l, 1x1010pfu) or 25?l saline (Control) before cornea alkali burn with 1 M NaOH. After seven days, CNV was observed and photographed in the slit lamp. Tear secretion was measured with phenol red thread. The animals were tested for human VEGFR1 mRNA and protein in the LG by qPCR and immunofluorescence staining, respectively. qPCR was also used to compare the mRNA of IL-1?, IL-6, and TNF-? in LG and ipsilateral trigeminal ganglion (TG). Results: Ad-VEGFR1 transfected 83% of the rats. VEGFR1 was present in LG acinar cells. CNV was prevented in 9 of 12 animals of Ad-VEGFR1 group, compared to Ad-Null (3:10) and Control (1:10) (p=0.0317). The tear secretion and the cytokines mRNA in LG and TG were similar all three groups (p>0.05). Conclusion: Adenoviral vector gene transfer to LG as the has shown local expression of human sVEGFR1, as It prevented CNV in most of the eyes exposed to alkali burn and was safe for LG structure and function.

Ocular neovascularization (NV) associated with corneal NV, ischemic retinopathies and age-related macular degeneration is a leading cause of severe vision loss. While numerous contributing factors have been identified, the potential role of the CD36 scavenger receptor has been largely overlooked notwithstanding its crucial involvement in normal retinal function. Accordingly, the central aim of this work was to elucidate the contribution and regulation of CD36 during ocular NV using the cornea as a model. / Initial work investigating the role of CD36 10 maintaining corneal avascularity, an important feature of the normal cornea, revealed that genetic ablation of CD36 elicits age-related corneal NV. Subsequent studies using a pathophysiologically relevant model of inflammatory corneal NV showed constitutive expression of CD36 in the normal cornea with marked induction in the neovascularized cornea. Importantly, activation of CD36 suppressed and induced regression of corneal NV, effects that proceeded via concerted inhibition of VEGFA, JNK-1, and cJun. / Because hypoxia is a fundamental stimulus for angiogenesis, it was pertinent to explore the role and regulation of CD36 during hypoxia. We demonstrate that CD36 expression was significantly elevated in hypoxia-exposed corneal and retinal tissue and in hypoxic retinal pigment epithelial cells. Essential contributions of hypoxia-inducible factor (HIF)-1 and reactive oxygen species were also established. Functional consequences were depicted by augmentations in CD36 phagocytic and anti-angiogenic activities. / Collectively, data disclose CD36 as an important modulator of corneal avascularity and inflammatory corneal NV; this imparts several interesting avenues for future research on the involvement of CD36 in neovascular diseases of the eye. Novel data further identify CD36 as a hypoxia and HIF-1 regulated gene thus creating a framework for future elucidation of the regulatory aspects of this receptor.