Master of Science / Kinesiology / Timothy I. Musch / Exercise intolerance characteristic of diseases such as chronic heart failure (CHF) and diabetes is associated with reduced nitric oxide (NO) bioavailability from nitric oxide synthase (NOS), resulting in an impaired microvascular O₂ driving pressure (PO₂mv: O₂ delivery – O₂ utilization) and metabolic control. Infusions of the potent NO donor sodium nitroprusside augment NO bioavailability yet decrease mean arterial pressure (MAP) thereby reducing its potential efficacy for patient populations. To eliminate or reduce hypotensive sequellae NO₂⁻ was superfused onto the spinotrapezius muscle. It was hypothesized that local NO₂⁻ administration would elevate resting PO₂mv and slow PO₂mv kinetics (increased τ: time constant, MRT: mean response time) following the onset of muscle contractions. In 12 anesthetized male Sprague-Dawley rats, PO₂mv of the circulation-intact spinotrapezius muscle was measured by phosphorescence quenching during 180 s of electrically-induced twitch contractions (1 Hz) before and after superfusion of NaNO₂ (30 mM). NO₂⁻ superfusion elevated resting PO₂mv (CON: 28.4 ± 1.1 vs NO₂⁻: 31.6 ± 1.2 mmHg, P ≤ 0.05), τ (CON: 12.3 ± 1.2 vs NO₂⁻: 19.7 ± 2.2 s, P ≤ 0.05) and MRT (CON: 19.3 ± 1.9 vs NO₂⁻: 25.6 ± 3.3 s, P ≤ 0.05). Importantly, these effects occurred in the absence of any reduction in MAP (103 ± 4 vs 105 ± 4 mmHg, pre- and post-superfusion respectively; P ˃ 0.05). These results indicate that NO₂⁻ supplementation delivered to the muscle directly through NO₂⁻ superfusion enhances the blood-myocyte driving pressure of oxygen without compromising MAP at rest and following the onset of muscle contraction. This strategy has substantial clinical utility for a range of ischemic conditions.

Pela sua alta incidência, morbidade, mortalidade e custos ao sistema de saúde, a sepse se destaca entre as diversas indicações de internação em unidade de terapia intensiva (UTI). A disfunção da microcirculação tem papel central na gênese e manutenção da síndrome séptica, sendo um marco fisiopatológico desta síndrome. Pacientes críticos invariavelmente estão ansiosos, agitados, confusos, desconfortáveis e/ou com dor. Neste contexto, drogas sedativas são amplamente utilizadas na medicina intensiva. A dexmedetomidina, um agonista potente e altamente seletivo dos receptores alfa-2 adrenérgicos, vem conquistando espaço como o sedativo de escolha nas UTIs por seus efeitos de sedação consciente, redução da duração e incidência de delirium e preservação da ventilação espontânea. Apesar destas possíveis vantagens, a indicação de uso da dexmedetomidina na síndrome séptica ainda carece de conhecimentos sobre seus efeitos na microcirculação e perfusão orgânica. Com o intuito de caracterizar os efeitos microcirculatórios da dexmedetomidina em um modelo murino de endotoxemia que permite estudos in vivo da inflamação e disfunção da perfusão microvascular, hamsters Sírios dourados submetidos à endotoxemia induzida por administração intravenosa de lipopolissacarídeo de Escherichia coli (LPS, 1,0 mg.kg-1) foram sedados com dexmedetomidina (5,0 &#956;g.kg.h-1). A microscopia intravital da preparação experimental (câmara dorsal) permitiu a realização de uma análise quantitativa das variáveis microvasculares e do rolamento e adesão de leucócitos à parede venular. Também foram analisados os parâmetros macro-hemodinâmicos e gasométricos (arterial e venoso portal), as concentrações de lactato arterial e venoso portal, a água pulmonar total e a sobrevivência do animal. Animais não-endotoxêmicos e/ou tratados com solução salina a 0,9% serviram como controles neste experimento. O LPS aumentou o rolamento e a adesão de leucócitos à parede venular, diminuiu a densidade capilar funcional e a velocidade das hemácias nos capilares e induziu acidose metabólica. O tratamento com dexmedetomidina atenuou significativamente estas respostas patológicas (p < 0,05). A frequência de pulso dos animais foi significativamente reduzida pela droga (p < 0,05). Outros resultados não foram tão expressivos (estatisticamente ou clinicamente). Estes resultados indicam que a utilização de dexmedetomidina produz um efeito protetor sobre a microcirculação da câmara dorsal de hamsters endotoxêmicos. Efeitos anti-inflamatórios da dexmedetomidina sobre os leucócitos e o endotélio poderiam melhorar a perfusão capilar e representar o mecanismo in vivo de ação da droga na microcirculação. / Due to its high incidence, morbidity, mortality and costs to the healthcare system, sepsis stands out among the many indications for intensive care unit (ICU) admission. The microcirculatory dysfunction plays a central role in the genesis and maintenance of the septic syndrome, being a pathophysiologic milestone in this syndrome. Critically ill patients are invariably anxious, agitated, confused, uncomfortable and/or with pain. In this context, sedative drugs are widely used in intensive care medicine. Dexmedetomidine, a potent and highly selective agonist of alpha-2 adrenergic receptors, is gaining ground as the sedative of choice in ICUs due to its effects of "conscious sedation", reducing the duration and incidence of delirium and preservation of spontaneous ventilation. Despite these potential advantages, the indication of dexmedetomidine in sepsis syndrome still lacks knowledge about its effects on microcirculation and perfusion. To characterize microcirculatory actions of dexmedetomidine in an endotoxemia rodent model that allows in vivo studies of microvascular inflammation and perfusion dysfunction, endotoxemia-submitted Syrian golden hamsters, induced by intravenous Escherichia coli lipopolysaccharide (LPS, 1,0 mg.kg-1) administration, were sedated with dexmedetomidine (5,0 &#956;g.kg.h-1). Intravital microscopy of skinfold chamber preparations allowed quantitative analysis of microvascular variables and venular leukocyte rolling and adhesion. Macro-hemodynamic parameters, arterial and portal venous blood gases and lactate concentrations, pulmonary total water, and animal survival were also analyzed. Non-endotoxemic and/or normal saline treated animals served as controls in this experiment. LPS increased leukocyte rolling and adhesion, decreased functional capillary density and red blood cell velocity, and induced metabolic acidosis. Dexmedetomidine treatment significantly attenuated these pathologic responses (p < 0.05). The pulse rate was significantly reduced by the drug (p < 0.05). Other results were not as expressive (statistically or clinically). These results indicate that the use of dexmedetomidine yields a protective effect on the microcirculation of the dorsal skinfold in endotoxemic hamsters. Anti-inflammatory dexmedetomidine effects on leukocytes and the endothelium, subsequently improving capillary perfusion, could represent the in vivo mechanism of the microcirculatory action of the drug.

O objetivos deste estudo foi avaliar a influência da radiação ionizante na vitalidade do tecido pulpar mensurada através dos níveis de saturação de oxigênio (%SpO2) em pacientes com tumores malígnos intraoral ou de orofaringe, passado de 4-6 anos da radioterapia (RT). Noventa pacientes com tumores malígnos intraoral ou de orofaringe, submetidos de 4-6 anos anteriores a RT foram selecionados para este estudo. Os níveis de oxigenação e sensibilidade pulpar, avaliados através do oxímetro de pulso e do spray refrigerante TFE (tetrafluoretano), foram analisados nos dentes anteriores (superior e inferior) de cada paciente selecionado (n=693),, indiferente do quadrante e da área irradiada. Como grupo controle foram selecionados noventa pacientes saudáveis (nunca submetidos a RT) e os mesmos testes foram empregados (n=693). Todos os dentes foram considerados vitais. 100% mostraram resposta positva ao teste térmico e as médias de %SpO2 foram de 92.7% no grupo dos pacientes irradiados (SD ± 1.83) e 92.6% no grupo dos não-irradiados (SD ± 1.80), sem diferença estatística observada. Houve uma tendência de valores de %SpO2 menores em dentes caninos comparados aos incisivos, entretanto sem diferença estatística significante. Passados de 4-6 anos da RT, as %SpO2 da polpa dental estão dentro dos padrões considerados normais para uma polpa vital e podese assumir que a RT não têm influência danosa a longo prazo sobre a vitalidade do tecido pulpar, assim sugerindo que este tecido pode ser apto a retornar o fluxo sanguíneo normal após a RT. As mudanças observadas na microcirculação pulpar devido a RT parecem ser temporárias, então o tratamento endodôntico preventivo ou a extração dental em pacientes que receberam radiação ionizante são desnecessários. / The aim of this study was to evaluate the influence of radioation on pulp vitality through the measurement of pulpal oxigenation levels (%SpO2) in patients with malignant intraoral and oropharyngeal tumors at 4-6 years after radiotherapy (RT). Ninety patients with malignant tumors in the oral cavity or oropharynx, submitted to RT 4-6 years prior to the study were selected. Pulp oxygenation levels and pulp sensitivity, measured by pulse oximetry and by cold refrigerant spray TFE (tetrafluoroethane), were analyzed in the anterior teeth (upper and lower) of each patient selected (n=693), regardless of the quadrant and the irradiated area. As a control group were selected ninety healthy patients (never submitted to RT) and the same tests were performed (n=693). All teeth were considered vital. 100% showed a positive response to the thermal test and the %SpO2 rates were 92.7% in irradiated group (SD ± 1.83) and 92.6% in non-irradiated group (SD ± 1.80), without statistical difference. There was a trend for lower %SpO2 values in canine teeth compared to incisors, however it was not statistically significant. After 4-6 years of RT the dental pulp %SpO2 are within the normal range considered for a vital pulp and it can be assumed that RT did not have a long term influence on the pulp vitality, therefore suggesting that the pulp tissue may be able to recover normal blood flow after RT. The changes observed in the pulp microcirculation due to RT seems to be temporary, so preventive endodontic treatment or tooth extraction in patients who will receive RT may not be necessary.

The brain microcirculation plays a key role in cerebral physiology and neuronal activation. In the case of degenerative diseases such as Alzheimer’s, severe deterioration of the microvascular networks (e.g. vascular occlusions) limit blood flow, thus oxygen and nutrients supply, to the cortex, eventually resulting in neurons death. In addition to functional neuroimaging, modelling is a valuable tool to investigate the impact of structural variations of the microvasculature on blood flow and mass transfers. In the brain microcirculation, the capillary bed contains the smallest vessels (1-10 μm in diameter) and presents a mesh-like structure embedded in the cerebral tissue. This is the main place of molecular exchange between blood and neurons. The capillary bed is fed and drained by larger arteriolar and venular tree-like vessels (10-100 μm in diameter). For the last decades, standard network approaches have significantly advanced our understanding of blood flow, mass transport and regulation mechanisms in the human brain microcirculation. By averaging flow equations over the vascular cross-sections, such approaches yield a one-dimensional model that involves much fewer variables compared to a full three-dimensional resolution of the flow. However, because of the high density of capillaries, such approaches are still computationally limited to relatively small volumes (<100 mm3). This constraint prevents applications at clinically relevant scales, since standard imaging techniques only yield much larger volumes (∼100 cm3), with a resolution of 1-10 mm3. To get around this computational cost, we present a hybrid approach for blood flow modelling where the capillaries are replaced by a continuous medium. This substitution makes sense since the capillary bed is dense and space-filling over a cut-off length of ∼50 μm. In this continuum, blood flow is characterized by effective properties (e.g. permeability) at the scale of a much larger representative volume. Furthermore, the domain is discretized on a coarse grid using the finite volume method, inducing an important computational gain. The arteriolar and venular trees cannot be homogenized because of their quasi-fractal structure, thus the network approach is used to model blood flow in the larger vessels. The main difficulty of the hybrid approach is to develop a proper coupling model at the points where arteriolar or venular vessels are connected to the continuum. Indeed, high pressure gradients build up at capillary-scale in the vicinity of the coupling points, and must be properly described at the continuum-scale. Such multiscale coupling has never been discussed in the context of brain microcirculation. Taking inspiration from the Peaceman “well model” developed for petroleum engineering, our coupling model relies on to use analytical solutions of the pressure field in the neighbourhood of the coupling points. The resulting equations yield a single linear system to solve for both the network part and the continuum (strong coupling). The accuracy of the hybrid model is evaluated by comparison with a classical network approach, for both very simple synthetic architectures involving no more than two couplings, and more complex ones, with anatomical arteriolar and venular trees displaying a large number of couplings. We show that the present approach is very accurate, since relative pressure errors are lower than 6 %. This lays the goundwork for introducing additional levels of complexity in the future (e.g. non uniform hematocrit). In the perspective of large-scale simulations and extension to mass transport, the hybrid approach has been implemented in a C++ code designed for High Performance Computing. It has been fully parallelized using Message Passing Interface standards and specialized libraries (e.g. PETSc). Since the present work is part of a larger project involving several collaborators, special care has been taken in developing efficient coding strategies.

Purpose: The aim of this study was to develop a method of measuring changes in the skin microcirculation and skin dose for mastectomy patients in connection with the radiation treatment. The distribution of the skin dose, its dependence on the energy of the beam, field geometry and bolus material and the accuracy of the given skin dose in the treatment planning system were studied. Finally, the correlation between the given dose and the changes in skin microcirculation was evaluated. Methods: Skin dose was measured using GafChromic EBT3 films. To evaluate the impact of different energies and field geometry measurements on a PMMA phantom were done. Dose measurements were done using an anthropomorphic phantom and in patients. The measured skin doses were compered to the doses calculated using the treatment planning system. Before and after treatment, skin blood perfusion was measured using laser speckle contrast imaging. In connection with the last measurement also methyl nicotinate was used to increase the perfusion for the measurement. Results: The measurements on the PMMA-phantom indicate that a larger photon energy results in a lower dose to the skin, but a higher exit dose. Furthermore a more oblique angle results in a higher skin dose and a larger field size also results in an increased skin dose. The patient measurements showed that the skin dose was significantly different in different areas of the irradiated field. The highest dose was measured in the area in which a bolus was applied. All patients showed a significant increase in skin blood of the perfusion within the irradiated area. The comparison between the measured doses and the doses calculated using the treatment planning system shows an underestimation of the skin dose by the treatment planning system depending on the incident angle and the presence of bolus material. Conclusion: The distribution of the skin dose during breast cancer radiotherapy in mastectomy patients is heterogeneous with the highest dose in the area of the mastectomy scar, due to the presence of bolus material. A correlation can be noticed between the changed in microcirculation and the radiation dose to the skin. Estimation of the skin dose using the treatment planning system is inaccurate, but film doseimetry offers an easy-to use method to accurately measure the dose to different areas of the irradiated skin.

Radiation therapy has been shown to increase local and regional control as well as overall survival with breast cancer, but the vast majority of patients develop acute skin reactions, which are in part related to microvascular changes. These reactions vary between different skin sites. The aim of this work is to determine the absorbed dose to the skin by measurements and investigate if there is a correlation between the absorbed dose at different areas of the breast and the local changes in microcirculation in the skin after breast cancer radiotherapy. The study includes characterisation of the Gafchromic EBT3 film and Epson Perfection V600 Photo scanner which are used for absorbed dose determination. The measurements were done both on an anthropomorphic female phantom and on a patient undergoing breast cancer radiotherapy. Twenty-one pieces offilm (2x1 cm2) were placed on the surface of the breast (both for the phantom and patient) and irradiated with a prescribed dose to the target of 2.66 Gy with two opposed fields using 6 MV beam. It was observed that mainly 45-64 % of the prescribed dose was deposited at the surface, both for the phantom and patient. Using laser speckle contrast imaging and polarised light spectroscopy, the regional changes in mean blood perfusion and in mean red blood cell concentration (RBCC) at the end of the treatment with a total prescribed dose of 42.6 Gy, compared to baseline, were measured in both the treated and untreated breast of the same patient. Although marked increases in perfusion were seen in different areas of the treated breast, there was no significant correlation between the changes in perfusion and the absorbed dose at these areas. However, a statistical correlation was found between the changes in RBCC and the absorbed skin dose at the same areas. To further elucidate the relation between the changes in skin microcirculation and the absorbed radiation dose during breast cancer radiotherapy, future studies using a larger number of patients are needed.

Diameters of microvessels undergo continuous structural adaptation in response to hemodynamic and metabolic stimuli. To ensure adequate flow distribution, metabolic responses are needed to increase diameters of vessels feeding poorly perfused regions. Possible modes of metabolic control include release of signaling substances from vessel walls, from the supplied tissue and from red blood cells (RBC). Here, a theoretical model was used to compare the abilities of these metabolic control modes to provide adequate tissue oxygenation, and to generate blood flow velocities in agreement with experimental observations. Structural adaptation of vessel diameters was simulated for an observed mesenteric network structure in the rat with 576 vessel segments. For each mode of metabolic control, resulting distributions of oxygen and deviations between simulated and experimentally observed flow velocities were analyzed. It was found that wall-derived and tissue-derived growth signals released in response to low oxygen levels could ensure adequate oxygen supply, but RBC-derived signals caused inefficient oxygenation. Closest agreement between predicted and observed flow velocities was obtained with wall-derived growth signals proportional to vessel length. Adaptation in response to oxygen-independent release of a metabolic signal substance from vessel walls or the supplied tissue was also shown to be effective for ensuring tissue oxygenation due to a dilution effect if growth signal substances are released into the blood. The present results suggest that metabolic signals responsible for structural adaptation of microvessel diameters are derived from vessel walls or from perivascular tissue.

This thesis forms part of an ongoing long-term project to investigate the suitability of Doppler optical coherence tomography (OCT) as a measurement tool to investigate skin thickness and blood flow in patients with systemic sclerosis. There is a discussion of the characterisation of an electro-optic phase modulator for use in a Doppler OCT imaging system which is being built for the purpose of clinical studies. In addition to this the development of software for the same system is described. The work includes a comparison of two methods of obtaining Doppler information that were tested with the system; a phase resolved method and a correlation mapping method. Initial structural and Doppler images obtained using the system are presented. In addition to this the development of semi-automated software to measure skin thickness from both OCT and high frequency ultrasound images is discussed. The results of a study, for which this software was developed, into skin thickness measurements using both techniques in both patients with systemic sclerosis and healthy controls are presented. Both OCT and high frequency ultrasound were able to measure a statistically significant difference in epidermal thickness at multiple locations on the body. Finally, the modification of a freely available Monte Carlo simulation for light propagation in multi-layered tissue (MCML) to enable the simulation of structural and Doppler OCT images is covered. The simulation was able to extract the magnitude of the simulated flow accurately to within an order of magnitude, and after a simple filter was applied to eliminate fluctuations in the data the structure of the Doppler image closely matched what was modelled.

Hypertension, a major risk factor in the cardiovascular system, is characterized by an increase in the arterial blood pressure. High dietary sodium is linked to multiple cardiovascular disorders including hypertension. Salt sensitivity, a measure of how the blood pressure responds to salt intake is observed in more than 50% of the hypertension cases. Nitric Oxide (NO), as an endogenous vasodilator serves many important biological roles in the cardiovascular physiology including blood pressure regulation. The physiological concentrations for NO bioactivity are reported to be in 0-500 nM range. Notably, the vascular response to NO is highly regulated within a small concentration spectrum. Hence, much uncertainty surrounds how NO modulates diverse signaling mechanisms to initiate vascular relaxation and alleviate hypertension. Regulating the availability of NO in the vasculature has demonstrated vasoprotective effects. In addition, modulating the NO release by different means has proved to restore endothelial function. In this study we addressed parameters that regulated NO release in the vasculature, in physiology and pathophysiology such as salt sensitive hypertension. We showed that, in the rat mesenteric arterioles, Ca2+ induced rapid relaxation (time constants 20.8 ± 2.2 sec) followed with a much slower constriction after subsequent removal of the stimulus (time constants 104.8 ± 10.0 sec). An interesting observation was that a fourfold increase in the Ca2+ frequency improved the efficacy of arteriolar relaxation by 61.1%. Our results suggested that, Ca2+ frequency-dependent transient release of NO from the endothelium carried encoded information; which could be translated into different steady state vascular tone. Further, Agmatine, a metabolite of L-arginine, as a ligand, was observed to relax the mesenteric arterioles. These relaxations were NO-dependent and occurred via α-2 receptor activity. The observed potency of agmatine (EC50, 138.7 ± 12.1 µM; n=22), was 40 fold higher than L-arginine itself (EC50, 18.3 ± 1.3 mM; n = 5). This suggested us to propose alternative parallel mechanism for L-arginine mediated vascular relaxation via arginine decarboxylase activity. In addition, the biomechanics of rat mesentery is important in regulation of vascular tone. We developed 2D finite element models that described the vascular mechanics of rat mesentery. With an inverse estimation approach, we identified the elasticity parameters characterizing alterations in normotensive and hypertensive Dahl rats. Our efforts were towards guiding current studies that optimized cardiovascular intervention and assisted in the development of new therapeutic strategies. These observations may have significant implications towards alternatives to present methods for NO delivery as a therapeutic target. Our work shall prove to be beneficial in assisting the delivery of NO in the vasculature thus minimizing the cardiovascular risk in handling abnormalities, such as hypertension.

Le réseau vasculaire est composé non seulement des vaisseaux sanguins mais aussi des vaisseaux lymphatiques. Les premiers assurent l'approvisionnement des cellules en nutriments et oxygène et la récolte de leurs déchets métaboliques tandis que les seconds collectent les fluides, macromolécules et cellules immunitaires extravasés dans les tissus interstitiels pour les ramener à la circulation sanguine. Au cours de ces travaux de recherche, nous avons mis au point un substitut cutané reconstruit par génie tissulaire, contenant un réseau sanguin fonctionnel, exempt de toute matière exogène et potentiellement autologue. Ses applications cliniques sont particulièrement prometteuses puisque l'une des raisons majeures d'échec des greffes de peau sur les patients grands brûlés par exemple est la nécrose du greffon. Au cours d'une étude in vivo, nous avons démontré que le réseau contenu dans notre substitut cutané est fonctionnel, s'inoscule en moins de quatre jours avec le réseau vasculaire de l'hôte et qu'il apporte un avantage à la néovascularisation par rapport à un substitut non microvascularisé. Un substitut cutané microvascularisé tumoral a ensuite été développé pour étudier la biologie du mélanome. Notre modèle est particulièrement pertinent et complet pour ce type d'étude puisque les cellules cancéreuses interagissent avec les trois principaux types cellulaires du derme et de l'épidémie et évoluent au sein d'une matrice extracellulaire sécrétée par ces cellules. Ainsi, il a été vérifié que les cellules cancéreuses conservent le même type de comportement, tant au niveau du potentiel invasif que du potentiel pro-angiogénique, dans notre modèle tridimensionnel de peau qu'/« situ chez les patients. Les capillaires lymphatiques fournissent une voie privilégiée pour le processus métastatique de cancers comme les mélanomes, c'est pourquoi nous travaillons à ajouter un réseau lymphatique à notre substitut cutané microvascularisé. Jusque là, nous avons démontré que les cellules endothéliales lymphatiques s'organisent spontanément en réseau dans un substitut 3D de tissu conjonctif.