Thesis--Ph. D., University of Hong Kong.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

In order to investigate the early ultrastructural lesions at the first sign of lameness in the oligofructose (OF) model of laminitis, the disease was induced in four horses, while another four horses were sham-treated controls. Minor lesions were detected in lamellar samples examined by light microscopy. Examination by transmission electron microscopy (TEM) showed excessive waviness, breaks and separation of portions of lamellar basement membrane (BM) in the treated horses. There was also disintegration and disappearance of hemidesmosomes (HD) and epidermal basal cell (EBC) cytoskeleton, and an increase in the distance between the EBC plasmalemma and the centre of the BM. A link was thus established between the first clinical signs of lameness and ultrastructural changes in the lamellar dermo-epidermal interface. This implied that pathogenesis was underway well before clinical signs (24 h) and that successful therapy would need to be instituted earlier than previously considered. Earlier therapy may be facilitated if delivery of efficacious drugs to the foot was achievable. A treatment modality that delivered effective concentrations of anti laminitic drugs to the target organ (the epidermal lamellae) was thus an objective of this study. Hoof lamellar tissue from five ponies treated with prolonged euglycaemic hyperinsulinaemia and four control (sham-treated) ponies were harvested and processed for TEM. Lamellae from treated ponies showed attenuation and elongation of secondary epidermal lamellae (SEL), HD number reduction and infiltration of leukocytes. Unlike carbohydrate induced laminitis in horses, there was no global separation at the lamellar dermal/epidermal interface in ponies. Two unique lamellar lesions found in this induction model was mitosis among EBCs and thickening of the BM, not normally characteristic of acute laminitis. The pathophysiology of hyperinsulinaemic laminitis remains unresolved but if insulin, delivered directly to the foot, induced laminitis several pathophysiological questions would be answered. In particular, it would emphasise the laminitogenic potential of insulin alone in the pathogenesis of laminitis. It also allows the treatment foot to be compared with the remaining three that act as internal controls. A modality that delivered drugs like insulin to the target organ (the epidermal lamellae) was needed and was an objective of this study. A microdialysis (MD) method, based on continuous sampling of the lamellar extracellular fluid (ECF), was developed to monitor lamellar drug concentrations. MD probes were implanted in the hoof lamellar tissue of six normal Standardbred horses under local anaesthesia. A bolus intravenous (IV) dose (5 mg/kg BWT) of gentamicin sulfate was injected into the jugular vein. MD and blood samples were collected at different time points during 24 h, and calibrated and analyzed using an ELISA method for gentamicin. During the first 8 h, the concentration of gentamicin was significantly higher in blood than lamellar ECF, a result that is reversed when lamellar MD is repeated during IO infusion of gentamicin. The results showed that this modestly invasive method was a useful tool to monitor changes in the lamellar ECF during drug delivery or during laminitis development. Knowledge of the anatomy and dynamics of blood circulation in the equine foot is fundamental to understand laminitis pathophysiology. Using histology, decalcification, diaphanization, computed tomography (CT), micro CT and gelatin-India Ink vascular perfusion, the normal anatomy of the dorsal part of distal phalanx (DP) and its vascular relationship to hoof lamellae was characterised. The results showed a close relationship between the distal phalangeal and lamellar circulations and raised the possibility of accessing the lamellar circulation via the DP and the possibility that IO perfusion (IOP) of the DP could deliver drugs to the lamellae. IOP of the DP with methyl methacrylate (MMA) corrosion casting material resulted in filling of the lamellar and sublamellar vascular network and incomplete filling of lamellar capillaries. Perfusion of common digital artery with a suspension of barium sulfate resulted in filling of lamellar arteries but not capillaries. Perfusion of the common digital vein resulted in filling of lamellar veins but not capillaries. Perfusion with barium sulfate partitioned veins from arteries because particle size prevented entry into capillaries. IOP with barium sulfate filled only veins revealing that vascular egress from the DP was venous. This study showed that a retrograde venous connection exists between the DP and lamellar circulations with the potential for lamellar drug delivery. Intra-arterial (IA) and IO infusion results using gelatin-India Ink were markedly improved when cadaver limbs were subjected to cyclic loading within the physiological range. Without loading lamellar capillaries failed to fill no matter what the injection pressure. Cyclic loading cadaver limbs 6 times resulted in complete lamellar capillary filling and suggested that cyclic limb loading contributed to perfusion of lamellar capillaries normally in horses. To evaluate IO delivery of drugs to hoof lamellae in the standing, conscious horse, gentamicin solution (25 mg/mL) was slowly infused (20 µL/min) through an IO bone screw. Lamellar ECF was collected via a lamellar MD probe and blood was collected from the jugular vein. Gentamicin was 50-100 times more concentrated in lamellar ECF than in blood. This study introduces a potential method for delivery of drugs into the lamellar tissue in the standing, conscious horse. Laminitis pathology occurs before clinical signs and can be induced by insulin as well as enteric OF overload. Thus therapy delivered to the target of laminitis, the hoof lamellae, has an improved chance of success if delivered promptly, safely and at high concentrations. A validated drug delivery and lamellar analysis system that achieves these criteria, was the discovery of this project and is now available to combat laminitis.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [133]-140).

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references. Also available via the Internet.

Thesis (M.S. )--University of Tennessee Health Science Center, 1999. / Title from title page screen (viewed on October 17, 2008). Research advisor: Mohammad F. Kiani. Document formatted into pages (xi, 67 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 55-67).

Thesis (PH. D.)--Michigan State University. Chemistry, 2008. / Title from PDF t.p. (viewed on Sept. 2, 2009) Includes bibliographical references. Also issued in print.

A miocardiopatia chagásica constitui uma doença prevalente e de alta morbimortalidade no Brasil. Embora seu agente causador, o Trypanosoma cruzi, já tenha sido identificado, os mecanismos fisiopatológicos e a predileção do acometimento por determinados segmentos do miocárdio ainda não foram totalmente esclarecidos. Dentre os mecanismos envolvidos nos distúrbios da microcirculação, inclui-se a hipótese na qual o acometimento preferencial das regiões apical e posterolateral do VE deva-se à presença de áreas de fluxo arterial fronteiriço (\"watershed\") entre as artérias que irrigam as paredes do VE, locais estes com maior predisposição a sofrer processo de isquemia. Ainda que plausível, esta hipótese ainda não foi testada frente a razoável variabilidade da distribuição dos vasos arteriais coronários, o que implica em variações na presença ou não de regiões de \"watershed\" em cada paciente. Objetivo: Estudar a correlação topográfica entre as regiões de \"watershed\" com as alterações da mobilidade ventricular e defeitos de perfusão miocárdica no ventrículo esquerdo. Casuística e Métodos: Realizamos um estudo descritivo com análise de 63 pacientes portadores de cardiopatia chagásica crônica que possuíam cineangiocoronariografia sem lesões obstrutivas significativas e fração de ejeção igual ou superior a 40%. Utilizando a cineangiocoronariografia definimos as regiões passíveis de \"watershed\" baseando-nos na distribuição anatômica das artérias coronárias, tendo como critérios a dominância, a extensão e os limites das artérias coronárias direita e esquerda na irrigação das paredes do VE. Em seguida verificamos se as alterações de mobilidade parietal na ventriculografia e/ou os defeitos de perfusão segmentar na cintilografia de perfusão miocárdica coincidiam com estas regiões. Utilizamos Teste Exato de Fischer para a análise estatística dos resultados. Resultados: Na ventriculografia por contraste as frequências dealterações de mobilidade para as paredes apical, inferior e posterolateral nos pacientes na presença e ausência de respectiva região de WS correspondente foram: 76,2% vs 68,3%, 46,3% vs 38,1% e 54,6% vs 42,9%. Os valores de p obtidos foram respectivamente 0,51; 0,59 e 0,69. Considerando todas as regiões onde o WS estava presente 55,6% apresentavam alteração da mobilidade, em compensação 56,5% das regiões onde o WS estava ausente também apresentavam alteração da mobilidade, com o valor de p = 1,0. Na cintilografia de perfusão miocárdica as frequências de defeitos de captação para os segmentos apicais, inferiores médio e apical, laterais e inferior basal na presença e ausência de respectiva região de WS correspondente foram: 41,2% vs 57,1%, 67,8% vs 47,2%, 50% vs 66,6% e 33,1% vs 50%. Os valores de p obtidos foram respectivamente 0,36; 0,21; 1,0 e 1,0. Considerando todas as regiões onde o WS estava presente 53,3% apresentavam defeitos de captação, em compensação 52,2% das regiões onde o WS estava ausente também apresentavam defeitos de captação com o valor de p = 1,0. Conclusão: No estudo apresentado não se comprovou que as alterações da mobilidade parietal ou os defeitos de perfusão nos segmentos do ventrículo esquerdo ocorram nas regiões de \"watershed\". Portanto nossos resultados não apoiam a hipótese de que as regiões de \"watershed\" contribua para o mecanismo de lesão miocárdica na cardiopatia chagásica crônica. / Chagasic cardiomyopathy is a prevalent disease with high morbidity and mortality in Brazil. Although its causative agent, Trypanosoma cruzi, has already been identified, pathophysiological mechanisms and the predilection for involvement of certain segments of the myocardium have not yet been fully elucidated. Among the mechanisms involved in microcirculation disorders, the hypothesis is included in which the preferential affection of the apical and posterolateral regions of the LV is due to the presence of areas of bordered arterial flow (\"watershed\") between the arteries that irrigate the walls of the LV, these places are more likely to undergo ischemia. Although plausible, this hypothesis has not yet been tested against the reasonable variability of coronary artery distribution, which implies variations in the presence or absence of watershed regions in each patient. Objective: To study the topographic correlation between watershed regions with the ventricular mobility abnormalities and myocardial perfusion defects in the left ventricle. Methods: We conducted a descriptive study with 63 patients with chronic chagasic cardiopathy who had coronary angiography without significant obstructive lesions and ejection fraction equal to or greater than 40%. Using the cineangiocoronariography, we defined watershed regions based on the anatomical distribution of the coronary arteries, taking as criteria the dominance, extension and limits of the right and left coronary arteries in the irrigation of the LV walls. Next, we verified whether the parietal mobility abnormalities in ventriculography and / or segmental perfusion defects in myocardial perfusion scintigraphy coincided with these regions. We used Fisher\'s exact test for the statistical analysis of the results. Results: In contrast ventriculography, the frequencies of mobility abnormalities for the apical, inferior and posterolateral walls in patients in the presence and absence of a corresponding WS region were: 76.2% vs 68.3%, 46.3% vs 38 , 1% and 54.6% vs 42.9%. The values of p obtained were respectively 0.51; 0.59 and 0.69. Considering all the regions where WS was present, 55.6% had mobility alterations; in contrast, 56.5% of the regions where WS was absent alsopresented mobility alterations with p value = 1.0. In myocardial perfusion scintigraphy, the frequencies of uptake defects for the apical, lower mid and apical, lateral and lower basal segments in the presence and absence of the corresponding WS region were: 41.2% vs 57.1%, 67.8 % vs 47.2%, 50% vs 66.6% and 33.1% vs 50%. The values of p obtained were respectively 0.36; 0.21; 1.0 and 1.0. Considering all the regions where the WS was present, 53.3% presented perfusional defects, in compensation 52.2% of the regions where WS was absent also had perfusional defects with p value = 1.0. Conclusion: In the presented study it was not verified that the alterations of the parietal mobility or the perfusion defects in the segments of the left ventricle occur in the watershed regions. Therefore our results do not support the hypothesis that watershed regions contribute to the mechanism of myocardial injury in chronic Chagas\' heart disease.