Since aggressive cancers are frequently hypermetabolic with angiogenic vessels, quantification of blood flow (BF) can be vital for cancer diagnosis. Our laboratory has developed a noncontact diffuse correlation tomography (ncDCT) system for 3-D imaging of BF distribution in deep tissues (up to centimeters). The ncDCT system employs two sets of optical lenses to project source and detector fibers respectively onto the tissue surface, and applies finite element framework to model light transportation in complex tissue geometries. This thesis reports our first step to adapt the ncDCT system for 3-D imaging of BF contrasts in human breast tumors. A commercial 3-D camera was used to obtain breast surface geometry which was then converted to a solid volume mesh. An ncDCT probe scanned over a region of interest on the breast mesh surface and the measured boundary data were used for 3-D image reconstruction of BF distribution. This technique was tested with computer simulations and in 28 patients with breast tumors. Results from computer simulations suggest that relatively high accuracy can be achieved when the entire tumor was within the sensitive region of diffuse light. Image reconstruction with a priori knowledge of the tumor volume and location can significantly improve the accuracy in recovery of tumor BF contrasts. In vivo ncDCT imaging results from the majority of breast tumors showed higher BF contrasts in the tumor regions compared to the surrounding tissues. Reconstructed tumor depths and dimensions matched ultrasound imaging results when the tumors were within the sensitive region of light propagation. The results demonstrate that ncDCT system has the potential to image BF distributions in soft and vulnerable tissues without distorting tissue hemodynamics. In addition to this primary study, detector fibers with different modes (i.e., single-mode, few-mode, multimode) for photon collection were experimentally explored to improve the signal-to-noise ratio of diffuse correlation spectroscopy flow-oximeter measurements.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

We assessed potential mechanisms that may jeopardize skeletal muscle perfusion during surgery leading to adverse outcomes including muscle injury and flap hypoxia. In craniotomy patients, we observed an increase in serum lactate and creatine kinase and urine myoglobin; indicative of muscle damage. The early rise in lactate correlated with elevated BMI, suggesting that obesity caused tissue compression and muscle ischemia. In our rodent model, we investigated the effects of flap preparation and phenylephrine on muscle perfusion by assessing microvascular blood flow and tissue PO2. Phenylephrine reduced muscle blood flow by ~20%, yet increased PO2 by ~10% suggestive of decreased O2 metabolism. At baseline, muscle flap blood flow was reduced by ~50% while PO2 was severely reduced ~80% (~5 torr) suggesting that flap perfusion was attenuated and O2 metabolism was increased. Phenylephrine infusion further reduced muscle flap perfusion. These data demonstrate multiple mechanisms by which muscle perfusion is jeopardized during surgery.

We assessed potential mechanisms that may jeopardize skeletal muscle perfusion during surgery leading to adverse outcomes including muscle injury and flap hypoxia. In craniotomy patients, we observed an increase in serum lactate and creatine kinase and urine myoglobin; indicative of muscle damage. The early rise in lactate correlated with elevated BMI, suggesting that obesity caused tissue compression and muscle ischemia. In our rodent model, we investigated the effects of flap preparation and phenylephrine on muscle perfusion by assessing microvascular blood flow and tissue PO2. Phenylephrine reduced muscle blood flow by ~20%, yet increased PO2 by ~10% suggestive of decreased O2 metabolism. At baseline, muscle flap blood flow was reduced by ~50% while PO2 was severely reduced ~80% (~5 torr) suggesting that flap perfusion was attenuated and O2 metabolism was increased. Phenylephrine infusion further reduced muscle flap perfusion. These data demonstrate multiple mechanisms by which muscle perfusion is jeopardized during surgery.

Les diagnostics cliniques des maladies cardio-vasculaires sont principalement effectués à l’aide d’échographies Doppler-couleur malgré ses restrictions : mesures de vélocité dépendantes de l’angle ainsi qu’une fréquence d’images plus faible à cause de focalisation traditionnelle. Deux études, utilisant des approches différentes, adressent ces restrictions en utilisant l’imagerie à onde-plane, post-traitée avec des méthodes de délai et sommation et d’autocorrélation. L’objectif de la présente étude est de ré-implémenté ces méthodes pour analyser certains paramètres qui affecte la précision des estimations de la vélocité du flux sanguin en utilisant le Doppler vectoriel 2D. À l’aide d’expériences in vitro sur des flux paraboliques stationnaires effectuées avec un système Verasonics, l’impact de quatre paramètres sur la précision de la cartographie a été évalué : le nombre d’inclinaisons par orientation, la longueur d’ensemble pour les images à orientation unique, le nombre de cycles par pulsation, ainsi que l’angle de l’orientation pour différents flux. Les valeurs optimales sont de 7 inclinaisons par orientation, une orientation de ±15° avec 6 cycles par pulsation. La précision de la reconstruction est comparable à l’échographie Doppler conventionnelle, tout en ayant une fréquence d’image 10 à 20 fois supérieure, permettant une meilleure caractérisation des transitions rapides qui requiert une résolution temporelle élevée. / Clinical diagnosis of cardiovascular disease is dominated by colour-Doppler ultrasound despite its limitations: angle-dependent velocity measurements and low frame-rate from conventional focusing. Two studies, varying in their approach, address these limitations using plane-wave imaging, post-processed with the delay-and-sum and autocorrelation methods. The aim of this study is to re-implement these methods, investigating some parameters which affect blood velocity estimation accuracy using 2D vector-Doppler. Through in vitro experimentation on stationary parabolic flow, using a Verasonics system, four parameters were tested on mapping accuracy: number of tilts per orientation, ensemble length for single titled images, cycles per transmit pulse, and orientation angle at various flow-rates. The optimal estimates were found for 7 compounded tilts per image, oriented at ±15° with 6 cycles per pulse. Reconstruction accuracies were comparable to conventional Doppler; however, maintaining frame-rates more than 10 to 20 times faster, allowing better characterization of fast transient events requiring higher temporal resolution.

Arterial spin labeling is a completely non-invasive method for blood-flow measurement techniques. Alzheimer's disease pathology includes microvascular abnormalities in addition to practically all risk factors having a vascular component that reduces cerebral perfusion. Hemodynamic parameters of cerebral blood flow and arterial transit time were estimated via single-compartment modeling of pseudo continuous arterial spin labeling data and neurocognitive test scores (Alzheimer's disease assessment scale and mini-mental state examination) were compared between a group of healthy (N=20) and early Alzheimer's disease (N=25) subjects before and six months after the Alzheimer's subjects began treatment with cholinesterase inhibitors. The early Alzheimer's group showed improved CBF after 6 months' treatment in every Alzheimer's-prone region except the medial and lateral temporal lobes. No difference in arterial transit time was found between groups, indicating that the pathophysiological process causing hypoperfusion in Alzheimer's disease may differ from vascular dementia.

Arterial spin labeling is a completely non-invasive method for blood-flow measurement techniques. Alzheimer's disease pathology includes microvascular abnormalities in addition to practically all risk factors having a vascular component that reduces cerebral perfusion. Hemodynamic parameters of cerebral blood flow and arterial transit time were estimated via single-compartment modeling of pseudo continuous arterial spin labeling data and neurocognitive test scores (Alzheimer's disease assessment scale and mini-mental state examination) were compared between a group of healthy (N=20) and early Alzheimer's disease (N=25) subjects before and six months after the Alzheimer's subjects began treatment with cholinesterase inhibitors. The early Alzheimer's group showed improved CBF after 6 months' treatment in every Alzheimer's-prone region except the medial and lateral temporal lobes. No difference in arterial transit time was found between groups, indicating that the pathophysiological process causing hypoperfusion in Alzheimer's disease may differ from vascular dementia.

Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.

The eyes of snakes and most geckos, as well as a number of other disparate squamate taxa, are shielded beneath a layer of transparent integument referred to as the “reptilian spectacle.” Derived from the embryonic fusion of palpebral tissues, the spectacle contains a number of specializations of the skin to benefit vision while still allowing it to function as the primary barrier to the environment. For example, in nearly all species that possess it, it is markedly thinned compared to the surrounding integument and its keratinized scale is optically transparent. While the spectacle may thus seem ideally adapted to vision in allowing the eyes to be always unoccluded, it does have a few drawbacks. One such drawback is its vascularity, the implications of which are still not fully understood, but are explored herein. As no recent synthesis exists of the body of knowledge on reptilian spectacles, the first chapter of this thesis consists of a review of spectacle anatomy, physiology, adaptive significance and evolution to help put into context the following chapters that present original research. The second chapter describes the dynamics of blood flow through the spectacle vasculature of colubrid snakes, demonstrating three main points: (1) that the spectacle vasculature exhibits cycles of regular dilation and constriction, (2) that the visual perception of a threat induces vasoconstriction of its vessels, and (3) that spectacle vessels remain dilated throughout the renewal phase. The implications of these points are discussed. The third chapter describes the spectral transmittance of the shed spectacle scale, the only keratinized structure in the animal kingdom to contribute to the dioptric apparatus of the eye, as well as its thickness. Spectacle scale transmittance and thickness was found to differ dramatically between snakes and geckos and found in snakes to vary between families. The adaptive significance of the observed variation is discussed. The fourth chapter describes biochemical analyses of the shed spectacle scales of snakes and geckos and compares their composition to other scales in the integument. Spectacle scales were found to differ significantly from other scales in their keratin composition, and gecko spectacle scales in particular were found to lack ß keratin, that hard corneous protein thought to be common to all reptile scales. The concluding chapter will discuss where this research has brought the state of our knowledge on the spectacle and offers thoughts on potentially useful avenues for further research.

Nitric oxide (NO) is an important regulator of pulmonary blood flow and attenuates hypoxic pulmonary vasoconstriction (HPV). Nitric oxide is synthesized enzymatically in a number of tissues, including the lungs, and can also be generated from reduction of nitrite during hypoxia and acidosis. Inhaled nitric oxide (INO) is a selective pulmonary vasodilator, with no effects on systemic arterial blood pressure due to inactivation by hemoglobin in the blood. INO has distant effects both within the lungs and in other organs, since NO can be transported to remote tissues bound to proteins, or as more stable molecules of nitrite and nitrate. In healthy pigs, INO causes vasoconstriction and down regulation of endogenous NO production in lung regions not reached by INO, and predominantly so in hypoxic lung regions, i.e. augmentation of HPV. In this thesis, distant effects of INO in pigs with endotoxemic- and lavage-induced lung injuries were studied. INO increased the NO production in lung regions not reached by INO in endotoxemic pigs, whereas endogenous NO production was unaffected in pigs with lavage-induced injury. Metabolic and/or hypercapnic acidosis frequently occurs in critically ill patients, but whether acidosis affects the endogenous pulmonary NO production is unclear. The regional NO production and blood flow in hyperoxic and hypoxic lung regions, were studied during metabolic and hypercapnic acidosis. Neither metabolic, nor hypercapnic acidosis changed the endogenous NO production in hyperoxic or hypoxic lung regions. Metabolic acidosis potentiated HPV, whereas hypercapnic acidosis transiently attenuated HPV. In conclusion, the present thesis has demonstrated that INO in experimental sepsis increases the endogenous NO production in lung regions not reached by INO, which may cause increased shunt and poor response to INO. This distant effect is not seen in lavage injuried lungs, an experimental model with less inflammation. Acidosis does not affect the endogenous pulmonary NO production in hyperoxic or hypoxic lung regions. Whereas metabolic acidosis potentiates HPV, hypercapnic acidosis transiently attenuates HPV, due to a combination of hypercapnia-induced increase in cardiac output and a probable vasodilating effect of the CO2-molecule.