Dissertation (MD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Acute renal failure still is, with the exception of cardiac deaths, the most important pathological process associated with perioperative mortality in patients operated for abdominal aortic aneurysms. The intraoperative change in renal blood flow (RBF) and glomerular function have been investigated in human and animal models, particularly over the past 15 years. Despite large variation in study populations, measurement techniques and study designs in general, a significant body of evidence has developed which suggests infrarenal aortic clamp-induced renal ischemia to be the cause of postoperative acute renal failure when this complication does occur. It is rather surprizing then that, despite some recent studies which have reported on various pharmacological interventions to prevent intraoperative renal ischemia (with variable success), very little has apparently been done to unravel the pathogenesis and exact pathophysiology of this potentially lethal complication. Although a number of investigators suggest the possibility of hormonal involvement (particularly reninangiotensin, antidiuretic hormone (ADH) and catecholamines) in the process, the exact role of these mediators have not been explored (or reported) in a structured fashion. In an initial human study, renal hemodynamics and function were measured from the preoperative period, during the intraoperative phase and at least until 4 hours after aortic unclamping. To investigate the possibility of a temporal relationship between renal changes and fluctuations in hormonal concentrations, plasma concentrations of relevant hormones were determined at every sampling period where renal parameters were measured. The decrease in RBF and glomerular filtration rate (GFR) which we demonstrated to coincide with infrarenal aortic cross clamping, is consistent with results previously published. We demonstrated persistence of the impairment of these parameters as long as 4 hours into the postoperative phase; which has previously only been reported for the period until immediately after aortic unclamping with the abdomen still open. The persistence of a depressed GFR until the time of discharge of patients is cause for concern, particularly in patients with compromised renal function prior to surgery. Of the measured hormones with a potential influence on RBF and nephron function, renin was the only mediator where changes in plasma concentrations coincided with the depression of RBF and GFR after aortic cross clamping. The design of our study did not allow us to conclude whether the concomitant increase in angiotensin II was primarily responsible for the change in renal hemodynamics, or whether the raised renin (and angiotensin) levels were stimulated by the decrease in RBF induced by another mechanism. In another patient group, we demonstrated that the combination of mannitol and dopamine provided no protection against the deleterious effects of aortic cross clamping. In fact, the high urine volumes produced under the influence of these agents (which did not correlate with RBF at the corresponding periods), is likely to prompt a false sense of security. Given the lack of any objective benefit afforded by these agents, their use in these clinical circumstances should be discouraged. The animal studies were aimed at elucidation of the exact role of angiotensin in the pathogenesis and pathophysiology of the renal changes associated with infrarenal aortic clamping, as well as the interaction of angiotensin with other modulators for which an interactive relationship had been described previously under other experimental and/or clinical circumstances. The first study showed that, although renin (and thus angiotensin) concentrations were high after aortic unclamping, the hormone had no pathogenic or pathophysiological role of significance in the observed renal changes during this period (since blocking angiotensin II activation by the prevention of renin release, or by inhibiting the conversion enzyme, did not prevent a substantial decrease in RBF or GFR during that period). Preventing angiotensin II activation did, however, prevent renal changes during aortic clamping. This beneficial effect did not establish a primary role for angiotensin during that period, since the favourable influence could also (at least partially) be explained by prevention of the permissive influence of angiotensin on other vasoconstrictors and/or other vasodilatory influences of ACE inhibition and [1- blockade which are unrelated to angiotensin. This study did indicate that (at least partially) different mechanisms are responsible for the renal changes seen during aortic clamping, and after aortic unclamping. The second study explored the role of calcium in the renal pathophysiological changes during aortic clamping and after unclamping. The protective influence effected by the administration of a Ca2 + -blocker suggest the dependence of the renal vasoconstrictive and glomerular pathophysiological process( es) on the cellular influx of Ca2 + through voltage-gated channels. It unfortunately provides no definitive insight into the primary instigators of these processes. However, it does offer a clinically useful method of preventing these changes and protecting the kidney against ischemic injury during abdominal aortic surgery. The third component of the animal studies demonstrates the importance of the protective effect of renal prostaglandins during the specific experimental (and probably also the clinical) circumstances. Again, it does not provide definitive information on the mediators responsible for the renal changes, since the deleterious effects of numerous endogenous substances have previously been shown to be counterbalanced by intrarenal synthesis of prostaglandins under various experimental and clinical circumstances. The extent of the pathophysiological and ultrastructural changes which occurred under the influence of a NSAID does, however, suggest that these drugs should not be used under these clinical circumstances. The last component of the study provides evidence that angiotensin only plays a secondary/supplementary role in the renal pathophysiological process even during aortic clamping. This may explain the contradictory evidence regarding the potential beneficial effect of ACE inhibition (on renal hemodynamics and glomerular function) during abdominal aortic surgery (Licker et al. 1996, Colson et al. 1992a). Based on our studies, ACE inhibition can not be supported for this purpose. / AFRIKAANSE OPSOMMING: Akute nierversaking is met die uitsondering van kardiale sterftes, steeds die belangrikste patologiese proses wat geassosieer is met perioperatiewe mortaliteit in pasiënte wat opereer word vir abdominale aorta aneurismes. Die intraoperatiewe veranderinge in renale bloedvloei (NBV) en glomerulêre funksie is die afgelope 15 jaar ondersoek en gerapporteer in pasiënte- sowel as diere-modelle. Ten spyte van groot variasies in studie-populasies, meettegnieke en ontwerp van studies in die algemeen, dui 'n wesenlike hoeveelheid getuienis daarop dat infrarenale klemming van die aorta renale isgemie induseer, wat die oorsaak is van postoperatiewe akute nierversaking wanneer hierdie komplikasie voorkom. Dit is verbasend dat, ten spyte van sommige onlangse studies wat rapporteer oor 'n verskeidenheid farmakologiese ingrepe om intraoperatiewe renale isgemie te voorkom (met wisselende sukses), baie min oënskynlik gedoen is om die patogenese en die presiese patofisiologie van hierdie potensieel dodelike komplikasie te ontrafel. Hoewel verskeie outeurs die moontlikheid van hormonale betrokkenheid (veral renienangiotensien, antidiuretiese hormoon en katekolamiene) in hierdie proses suggereer, is die presiese rol van hierdie mediators nog nie op 'n gestruktureerde wyse ondersoek (of rapporteer) nie. In ons aanvanklike pasiënte-studie is renale hemodinamika en -funksie gemeet vanaf die preoperatiewe periode, gedurende die intra-operatiewe fase en tot minstens vier uur na ontklemming van die aorta. Serumkonsentrasies van relevante hormone is bepaal tydens elke metingsperiode waar renale parameters gemeet is, ten einde die moontlikheid van 'n temporale verwantskap tussen renale veranderinge en variasies in hormoonkonsentrasies te ondersoek. Die vermindering in NBV en glomerulêre filtrasiespoed (GFS) wat ons aangetoon het om saam te val met infrarenale aortaklemming, stem ooreen met resultate wat tevore deur ander navorsers publiseer is. Ons het aangetoon dat die inkorting van hierdie parameters voortduur tot minstens vier uur na aorta-ontklemming. Hierdie veranderinge is tevore slegs rapporteer vir periodes tot kort na aorta-ontklemming voor sluiting van die buikwond. Die feit dat die GFS steeds verlaag is met ontslag van hierdie pasiënte, skep rede tot kommer, veral in pasiënte wat alreeds ingekorte nierfunksie het voor die chirurgiese prosedure. Van die gemete hormone wat moontlik 'n invloed sou kon uitoefen op NBV eh nefronfunksie, was renien die enigste waarvan verandering in plasmakonsentrasies saamgeval het met die onderdrukking van NBV en GFS na aortaklemming. Die ontwerp van ons studie het ons nie toegelaat om 'n besliste uitspraak te maak of die geassosieerde verhoging in angiotensien II primêr verantwoordelik was vir die verandering in renale hemodinamika, of dat die verhoogde renien (en angiotensien) bloedvlakke moontlik sekondêr stimuleer is deur die verandering in NBV wat deur 'n ander meganisme induseer is. In 'n ander pasiëntegroep het ons aangetoon dat die kombinasie van mannitol en dopamien geen beskerming verleen het teen die nadelige effekte van aorta-klemming nie. Die groot volumes uriene wat uitgeskei is onder die invloed van hierdie middels (wat nie korreleer het met NBV tydens ooreenstemmende periodes nie), het inderwaarheid 'n ontoepaslike gerustheid uitgelok. Weens die ooglopende gebrek aan objektiewe voordeel wat verleen word deur hierdie middels, behoort hulle gebruik tydens hierdie kliniese omstandighede ontmoedig te word. Die doel van die diere studies was die identifisering van die presiese rol van angiotensien in die patogenese en patofisiologie van die renale veranderinge geassosieer met infrarenale aortaklemming, sowel as die interaksie van angiotensien met ander modulators waarvoor 'n interaktiewe verwantskap voorheen beskryf is onder eksperimentele en/of kliniese omstandighede. Die eerste studie het getoon dat alhoewel renien (en dus angiotensien) konsentrasies hoog was na aorta-ontklemming, die hormone geen betekenisvolle patogenetiese of patofisiologiese rol in die waargenome renale veranderinge gedurende hierdie periode het nie (aangesien blokkade van angiotensien aktivering deur voorkoming van renien vrystelling, of deur inhibisie van angiotensien omsettingsensiem (AOE), nie 'n daling in NBV of GFS kon voorkom nie). Voorkoming van angiotensien II aktivering het egter wel renale verandering voorkom gedurende aortaklemming. Dié voordelige effek het nie 'n primêre rol vir angiotensien gedurende die periode bevestig nie, aangesien die gunstige invloed ook (ten minste gedeeltelik) verduidelik kon word deur die voorkoming van die fassiliterende invloed van angiotensien op ander vasokonstriktore en/of ander vasodilator-invloede van die onderdrukking van AOE en ïs-blokkers (wat geen verband het met angiotensien of die blokkade daarvan nie). Die studie het aangetoon dat (ten minste gedeeltelik) verskillende meganismes verantwoordelik is vir renale veranderinge wat gesien is gedurende aortaklemming en na -ontklemming. Die tweede studie het die rol van kalsium in die renale patofisiologiese veranderinge gedurende aortaklemming en na ontklemming ondersoek. Die beskermende invloed wat deur die toediening van Ca2 + -blokkers bewerkstellig is, het bevestig dat die renale vasokonstriktoriese en glomerulêre patofisiologiese prosesse afhanklik is van sellulêre influks van kalsium deur spannings-afhanklike kannale. Dit het ongelukkig geen definitiewe insig verleen ten opsigte van die primêre inisieerders van die proses nie. Dit verskaf nogtans 'n bruikbare kliniese metode om daardie veranderinge te voorkom en die niere teen isgemiese besering gedurende abdominale aorta-chirurgie te beskerm. Die derde komponent van die diere-studies demonstreer die belangrikheid van die beskermende effek van renale prostaglandiene tydens die spesifieke eksperimentele (en waarskynlik ook die kliniese) omstandighede. Weereens gee dit nie definitiewe inligting oor die bemiddelaars wat verantwoordelik is vir die renale veranderinge nie, aangesien die skadelike effekte van verskeie endogene stowwe voorheen aangetoon is om beperk of voorkom te word deur die intrarenale vrystelling van prostaglandiene. Die omvang van die patofisiologiese en ultrastrukturele veranderinge wat ontstaan het onder die invloed van nie-steroïed anti-inflammatoriese middels (wat gebruik is om prostaglandien sintese te inhibeer), dui aan dat hierdie middels vermy moet word onder soortelyke kliniese omstandighede. Die laaste komponent van die studie verskaf 'n sterk aanduiding dat angiotensien slegs 'n sekondêre/aanvullende rol speel in die renale patofisiologiese proses, selfs gedurende aortaklemming. Dit mag die weersprekende getuienis oor die potensiële voordeel van AOE onderdrukking (op renale hemodinamika en glomerulêre funksie) gedurende abdominale aortachirurgie (Licker et al. 1996, Colson et al. 1992a) verklaar. Gebaseer op ons studies, kan AOE onderdrukking nie ondersteun word vir hierdie doel nie.

Cerebral blood flow (CBF) measures are central to the investigation of ischemic strokes, spreading depressions, functional and neuronal activation. Laser Speckle Contrast Imaging (LSCI) is an optical imaging technique that has been used to obtain CBF measures in vivo at high spatial and temporal resolutions, by quantifying the localized spatial blurring of backscattered coherent light induced by blood flow. Despite being widely used for biomedical applications, LSCI's critical limitations such as its tendency to underestimate large flow changes and its inability to accurately estimate CBF through a thinned skull have not been overcome. This dissertation presents a new Multi Exposure Speckle Imaging (MESI) technique that combines a new instrument and mathematical model to overcome these limitations. Additionally, in a pilot clinical study, an adapted neurosurgical microscope was used to obtain intra-operative LSCI images of CBF in humans. The MESI instrument accurately estimates experimental constants by imaging backscattered speckles over a wide range of the camera's exposure durations. The MESI mathematical model helps account for light that has scattered from both static and moving particles. In controlled flow experiments using tissue simulating phantoms, the MESI technique was found to estimate large changes in flow accurately and the estimates of flow changes were found to be unaffected by the presence of static particles in these phantoms. In an in vivo experiment in which the middle cerebral artery in mice was occluded to induce ~100% reduction in CBF, not only was the reduction in CBF accurately estimated by the MESI technique but these estimates of CBF changes were found to be unaffected by the presence of a thinned skull. The validity of statistical models used to derive the MESI mathematical model was confirmed using in vivo dynamic light scattering (DLS) measurements of CBF in mice. The MESI technique's potential to estimate absolute values of CBF in vivo was demonstrated by comparing CBF estimates obtained using the MESI technique to DLS measurements. The MESI technique's ability to measure CBF changes quantitatively through a thinned skull makes it particularly useful in chronic and long term studies leading to the development of better, more accurate stroke models. / text

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21669号 / 医博第4475号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村井 俊哉, 教授 古川 壽亮, 教授 宮本 享 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

It is currently a well-established fact that the dynamics of interacting fluid compartments of the central nervous system (CNS) may play a role in the CNS fluid physiology and pathology of a number of neurological disorders, including neurodegenerative diseases associated with accumulation of waste products in the brain. However, the mechanisms and routes of waste clearance from the brain are still unclear. One of the main components of this interacting cerebral fluids dynamics is blood flow. In the last decades, mathematical modeling and fluid dynamics simulations have become a valuable complementary tool to experimental approaches, contributing to a deeper understanding of the circulatory physiology and pathology. However, modeling blood flow in the brain remains a challenging and demanding task, due to the high complexity of cerebral vascular networks and the difficulties that consequently arise to describe and reproduce the blood flow dynamics in these vascular districts. The first part of this work is devoted to the development of efficient numerical strategies for blood flow simulations in complex vascular networks. In cardiovascular modeling, one-dimensional (1D) and lumped-parameter (0D) models of blood flow are nowadays well-established tools to predict flow patterns, pressure wave propagation and average velocities in vascular networks, with a good balance between accuracy and computational cost. Still, the purely 1D modeling of blood flow in complex and large networks can result in computationally expensive simulations, posing the need for extremely efficient numerical methods and solvers. To address these issues, we develop a novel modeling and computational framework to construct hybrid networks of coupled 1D and 0D vessels and to perform computationally efficient and accurate blood flow simulations in such networks. Starting from a 1D model and a family of nonlinear 0D models for blood flow, with either elastic or viscoelastic tube laws, this methodology is based on (i) suitable coupling equations ensuring conservation principles; (ii) efficient numerical methods and numerical coupling strategies to solve 1D, 0D and hybrid junctions of vessels; (iii) model selection criteria to construct hybrid networks, which provide a good trade-off between accuracy in the predicted results and computational cost of the simulations. By applying the proposed hybrid network solver to very complex and large vascular networks, we show how this methodology becomes crucial to gain computational efficiency when solving networks and models where the heterogeneity of spatial and/or temporal scales is relevant, still ensuring a good level of accuracy in the predicted results. Hence, the proposed hybrid network methodology represents a first step towards a high-performance modeling and computational framework to solve highly complex networks of 1D-0D vessels, where the complexity does not only depend on the anatomical detail by which a network is described, but also on the level at which physiological mechanisms and mechanical characteristics of the cardiovascular system are modeled. Then, in the second part of the thesis, we focus on the modeling and simulation of cerebral blood flow, with emphasis on the venous side. We develop a methodology that, departing from the high-resolution MRI data obtained from a novel in-vivo microvascular imaging technique of the human brain, allows to reconstruct detailed subject-specific cerebral networks of specific vascular districts which are suitable to perform blood flow simulations. First, we extract segmentations of cerebral districts of interest in a way that the arterio-venous separation is addressed and the continuity and connectivity of the vascular structures is ensured. Equipped with these segmentations, we propose an algorithm to extract a network of vessels suitable and good enough, i.e. with the necessary properties, to perform blood flow simulations. Here, we focus on the reconstruction of detailed venous vascular networks, given that the anatomy and patho-physiology of the venous circulation is of great interest from both clinical and modeling points of view. Then, after calibration and parametrization of the MRI-reconstructed venous networks, blood flow simulations are performed to validate the proposed methodology and assess the ability of such networks to predict physiologically reasonable results in the corresponding vascular territories. From the results obtained we conclude that this work represents a proof-of-concept study that demonstrates that it is possible to extract subject-specific cerebral networks from the novel high-resolution MRI data employed, setting the basis towards the definition of an effective processing pipeline for detailed blood flow simulations from subject-specific data, to explore and quantify cerebral blood flow dynamics, with focus on venous blood drainage.

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Die Grundlage der modernen funktionellen Bildgebung des Gehirns mit der BOLD-fMRT ist die neurovaskuläre Kopplung. Sie ist in ihren Mechanismen wenig verstanden und führt zu einem komplexen Zusammenspiel von Blutfluß, Blutvolumen und Oxygenierung. Die Aufklärung der Blutflußantwort mit ihren Auswirkungen auf die Meßsignale ist für eine genaue Interpretation des BOLD-Signals kritisch. Zudem stellt sich seit einigen Jahren die Frage, ob es bei funktioneller Aktivierung aufgrund eines vermehrten neuronalen Sauerstoffverbrauchs zu einer frühen Deoxygenierung kommt. Diese könnte sich als initialer BOLD-Abfall für eine hochauflösende Bildgebung eignen. Ein Vergleich von optischen Methoden und funktioneller Magnetresonanztomographie am gleichen Stimulationsmodell kann diesen Fragen nachgehen. Wir haben die kortikale Blutflußantwort auf somatosensorische Stimulation der Ratte mit den optischen Methoden Optical Imaging und Imaging Spectroscopy sowie mit BOLD-fMRT und blutvolumengewichteter MION-fMRT gemessen. Bei der Stimulation eines einzelnen Whisker-Haares grenzte sich die entsprechende kortikale Kolumne über eine optische Abschwächung ab. Spektroskopisch zeigte sich, daß diesem Signal eine initiale Blutvolumenzunahme zugrundeliegt. Eine Lambert-Beer-Analyse, die die differentiellen Pfadlängen des Lichtes im streuenden Gewebe vernachlässigt, konnte die gemessenen Spektren nicht linear anpassen. Mit einer Annäherung errechnete sie einen artifiziellen Anstieg des Deoxy-Hb in der frühen Antwort. Die quantifizierte Lambert-Beer-Analyse unter Einschluß der differentiellen Pfadlängen konnte die gemessenen Spektren linear anpassen. Im berechneten Konzentrationsverlauf stieg Oxy-Hb zum Stimulationsbeginn an, Deoxy-Hb blieb zunächst auf dem Ruhewert und fiel dann ab. Diese Verzögerung lag im Bereich der kapillären Transitzeit. Die spektroskopisch gemessene frühe Antwort fand sich auch in der Messung der Antwort auf Vorderpfotenstimulation. Zum Vergleich wurden fMRT-Messungen an diesem Stimulationsmodell herangezogen. Die MION-fMRT erfaßte einen initialen Anstieg des plasmatischen Blutvolumens (pCBV), das BOLD-Signal delta-R2* eine verzögerte Hyperoxygenierung. Die Hyperoxygenierung im weiteren Verlauf der Blutflußantwort zeigte in Imaging Spectroscopy und fMRT einen linearen Zusammenhang mit der Dauer der Stimulation. Dabei korrelierte die delta-R2* stark mit der spektroskopisch gemessenen Deoxy-Hb-Konzentration. Auch die Antwort auf das Stimulationsende stellte sich als von der Stimulationsdauer abhängig heraus und wurde als vaskuläres Speicherphänomen interpretiert. BOLD und Deoxy-Hb zeigten beide eine Hypooxygenierung nach dem Stimulationsende. pCBV und das spektroskopisch gemessene korpuskuläre Blutvolumen, cCBV, verhielten sich nach dem Stimulationsende spiegelbildlich. Die pCBV-Zunahme bildete sich nur allmählich zurück, während das cCBV steil unter seinen Ruhewert abfiel. Im Laufe der Messung nahm das cCBV wieder zu und erreichte seinen Ruhewert zeitgleich mit dem pCBV. Eine vermehrte Volumenspeicherung als Folge venöser Streßrelaxation und eine Verschiebung des Hämatokrits aufgrund des Fahraeus-Lindquist-Effekts werden als Grund für diese Veränderungen in Betracht gezogen. Die experimentellen Daten belegen, daß optische und magnetresonanztomographische Methoden korrespondierende Signale von Oxygenierung und Blutvolumen messen. Eine frühe Deoxygenierung wurde nicht gemessen. Allerdings zeigte sich die frühe Komponente der Blutvolumenzunahme an die initiale Kapillarnetzfüllung einer kortikalen Kolumne gebunden. Ihre Detektion mit der fMRT bietet eine Perspektive auf dem Weg zu einer hochauflösenden funktionellen Bildgebung des Gehirns. / Neurovascular coupling forms the basis of modern functional brain imaging with BOLD-fMRI. Its mechanisms are poorly understood as it leads to a complex interaction of blood flow, blood volume and oxygenation. The investigation of the blood flow response with its influences on measured signals is critical for the exact interpretation of the BOLD-Signal. In addition to that, the question on whether or not an increase in oxygen consumption during functional activation leads to an early deoxygenation is not resolved yet. This early deoxygenation could cause an initial BOLD decrease suitable for high resolution imaging. A comparison of optical methods and functional magnetic resonance imaging on the same stimulation model can help to answer these questions. We have measured the cortical blood flow response on somatosensory stimulation of the rat with the optical methods Optical Imaging and Imaging Spectroscopy and with BOLD-fMRI and blood volume weighted MION-fMRI. During stimulation of a single whisker vibrissa the corresponding cortical column delineated itself as an area of increased optical attenuation. A spectroscopical analysis showed an initial blood volume increase responsible for this signal. A Lambert-Beer-Analysis that ignored the differential pathlength of light in scattering tissue could not fit the measured spectra. The result of its closest approximation showed an artificial increase of deoxy-Hb during the early response. The quantified Lambert-Beer-Analysis with inclusion of differential pathlengths succeeded in fitting the measured spectra. The calculated concentration time course showed an increase of oxy-Hb at stimulus onset with deoxy-Hb staying at baseline values and then decreasing. This delay was as long as the capillary mean transit time. The spectroscopically measured early response was also found when measuring the response to forepaw stimulation. For comparison, fMRI measurements on this stimulation model were done. MION-fMRI detected an early increase of plasmatic blood volume (pCBV), the BOLD-Signal delta-R2* a delayed hyperoxygenation. The time course of the hyperoxygenation during the blood flow response showed a linear relationship with the stimulus duration in Imaging Spectroscopy and fMRI. The delta-R2* correlated strongly with spectroscopically measured concentration changes of deoxy-Hb. In addition to that, the response on the stimulus offset was dependent on the stimulus duration. It was interpreted as a vascular storage phenomenon. Both BOLD and deoxy-Hb showed a hypooxygenation after stimulus offset. pCBV and the spectroscopically measured corpuscular blood volume, cCBV, showed mirroring signals after stimulus offset. While pCBV returned to baseline values gradually, cCBV fell below baseline values immediately. During the further measurement cCBV increased and returned to baseline values at the same time as pCBV. To explain this, an increased volume storage due to venous stress relaxation and a hematocrit shift due to the Fahraeus-Lindquist effect are taken into consideration. The experimental data proves that optical and fMRI methods measure corresponding signals of oxygenation and blood volume. An early deoxygenation was not seen. However, the early component of the blood volume increase seems to be restricted to the initial filling of the capillary net supplying a cortical column. Its detection with fMRI offers a perspective on the way to high resolution functional imaging of the brain.

Master of Science / Department of Kinesiology / Timothy I. Musch / During exercise aged individuals exhibit endothelial dysfunction and decreased levels of whole-limb blood flow (BF), both of which may be linked mechanistically to age-related increases in reactive oxygen species (ROS). Ascorbic acid (AA) reduces levels of ROS and has been shown to alleviate vascular and hyperemic dysfunction at rest (Jablonski et al., 2007) and during small muscle mass exercise in humans (Kirby et al., 2009). However, the effect of AA on vascular function and BF to individual muscles during whole-body exercise is not known. PURPOSE: To test the hypothesis that a single high-dose infusion of AA would increase BF to the hindlimb musculature of old rats at rest and during treadmill running. METHODS: 18 old (~28 months) Fischer 344 x Brown Norway rats were randomized into rest (n=9) and exercise (n=9) groups. BF to the total hindlimb and individual muscles (28 individual muscles and muscle parts) was evaluated via radiolabeled microspheres before and after intra-arterial AA administration (76 mg/kg in 3 ml heparinized saline, 30 minute infusion) at rest and during submaximal treadmill running (20m/min, 5% grade). Total antioxidant capacity (TAC) and thiobarbituric acid reactive species (TBARS) were measured before and after AA to determine the ability of this specific dose of AA to increase levels of plasma antioxidants and decrease levels of ROS, respectively. RESULTS: At rest: AA increased TAC (~37%, P<0.05) but did not change TBARS (Pre: 6.8±0.7 vs Post: 7.0±1.0 µM, P>0.05). AA decreased total hindlimb BF (Pre: 25±3 vs Post: 16±2 ml/min/100g, P<0.05) and BF to 8 of the 28 muscles that were evaluated. During exercise: TAC was increased (~35%, P<0.05) and TBARS were decreased (Pre: 9.8±2.0 vs Post: 7.0±1.0 µM, P<0.05). However, there was no effect on either total hindlimb BF (Pre: 154±14 vs Post: 162±13, P>0.05) or BF to any of the individual muscles evaluated. CONCLUSIONS: Increased TAC via AA infusion reduces hindlimb muscle BF at rest but had no effect on BF during whole-body dynamic exercise. Thus, even though TBARS decreased, there was no evidence that AA supplementation increases blood flow to the locomotor muscles of old rats during whole-body exercise.

Limb haemodynamic responses during heat-stress and the importance of local vs. central temperature-sensitive mechanisms towards their regulation remain poorly understood, both at a whole-limb level and within individual tissues (i.e. skeletal muscle and skin). The aims of this thesis were to 1) investigate the haemodynamic responses at rest to direct thermal challenges both at a local level and during progressive elevations in systemic heat stress, 2) to ascertain the contribution of local vs. systemic mechanisms towards this regulation, and 3) to investigate the same responses during single-legged small-muscle mass exercise to near maximal levels. Results from Chapters 4 and 5 characterised the haemodynamic responses during isolated cooling and heating of the arm and leg, and provided evidence of alterations in both skin and skeletal muscle blood flow controlled solely through local temperature-sensitive mechanisms. While local cooling led to modest decreases in limb blood flow due to decreases in mean blood velocity alone, increases during heating occurred as a result of an increased antegrade flow, a diminished retrograde flow, and a reduction in the potentially pro-atherogenic oscillatory shear index. In Chapter 6, whole-body heating with isolated single leg cooling displayed the continued control of limb blood flow via local thermosensitive mechanisms alone, as cooled leg blood flow remained unchanged despite significant elevations in core temperature, cardiac output, and opposing heated leg blood flow. Furthermore, elevations in heated leg V̇O2 suggested a possible metabolic contribution to the observed skeletal muscle hyperaemic response. During incremental single-legged knee-extensor exercise to near maximal levels, blood flow was determined by a combination of metabolic workload and local tissue temperatures, regardless of whether systemic heat stress was present. Chapter 7 revealed that whilst skin and muscle blood flow in the leg continued to increase in line with local temperatures to levels of severe heat stress, rapid cooling of the leg when hyperthermic resulted in a similar reverse response in muscle tissues only, as skin blood flow remained elevated despite the abolition of high skin and subcutaneous temperatures. In addition, evidence was provided that moderate levels of whole-body heat stress provided little additional benefit to anti-atherogenic shear profiles than that experienced during isolated limb heating alone. Taken together, these findings suggest that local thermosensitive mechanisms dominate limb blood flow control during direct rapid heating in humans both at rest and during small muscle mass exercise, but that underlying central mechanisms may act to maintain flow when local temperatures are reduced in the face of high core temperatures.

Distortion to hemodynamic, ischemic and metabolic stimuli during low load resistance exercise with blood flow restriction (BFR) may influence regional vascular adaptation. This thesis investigated the acute response and chronic adaptations of the peripheral vasculature to low load resistance exercise with BFR. The methodology utilised Doppler ultrasound, strain gauge plethysmography and muscle biopsy for insightful measures of the vasculature at different regions of the arterial tree. Short term (4-6 weeks) localised low load (30-40% 1RM) resistance exercise with BFR increased brachial (3.1%) and popliteal (3.3%) artery maximal diameter (in response to ischemic exercise), forearm (29%) and calf (24%) post-occlusive blood flow, and calf filtration capacity (14%). These findings indicate potential vascular remodelling at the conduit (chapters 3, 4) resistance (chapter 4) and capillary (chapter 4) level of the vascular tree. Regional, rather than systemic, factors are responsible for these adaptations as evidenced by an absent response in the contralateral control limb. Transient improvements in popliteal artery FMD% occurred at week 2 before increased maximal diameter at week 6, suggesting functional changes precede structural remodelling (chapter 4). Maximal brachial artery diameter and forearm post-occlusive blood flow returned to baseline values after a 2 week detraining period, signifying rapid structural normalisation after stimulus removal (chapter 3). Enhanced capillarity, despite low training loads, could be explained by augmentation of VEGF (~7 fold), PGC-1α (~6 fold) and eNOS (~5 fold) mRNA, and upregulation VEGFR-2 (~5 fold) and HIF-1α (~2.5 fold) mRNA with BFR (chapter 5). This indicates a targeted angiogenic response potentially mediated through enhanced metabolic, ischemic and shear stress stimuli. Large between subject variability in the level of BFR was observed during upper and lower limb cuff inflation protocols. Adipose tissue thickness and mean arterial pressure were the largest independent determinants of upper and lower limb BFR, respectively (Chapter 6). In conclusion, this thesis demonstrates that low load resistance exercise with BFR induces adaptation in the conduit, resistance and capillary vessels. The mediators of this response are likely to be the hemodynamic and chemical signals elicited by repeated bouts of BFR resistance exercise, although confirmation of these mechanisms is required. The functional significance of these adaptations is unknown and warrants further investigation.