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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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.

Assignment (MPhil)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The mechanisms of blood flow (BF) control to skeletal muscle during dynamic exercise are still not clearly understood. The paraplegic subject (P) has reduced sympathetic innervation to the lower limbs. The current study was designed to focus on the contribution of neural control, specifically the sympathetic nervous system (SNS), as part of the central vascular mechanism to skeletal muscle BF during dynamic exercise. Aims: We studied BF parameters in P vs. able-bodied subjects (AB) to determine whether the paraplegic can serve as a model for assessing the contribution of the SNS to changes in active vs. inactive muscle BF during exercise. Further questions addressed include: the influence of level of fitness on resting and exercise BF, how lesion level affects BF control in the paraplegic, the 'muscle pump' theory and its hypothesized role in exercise hyperemia and whether blood pooling occurs in the legs of paraplegics. Method: Noninvasive duplex Doppler studies of the large conduit arteries (brachial and common femoral) were performed on 10 elite paraplegic athletes (EP), 10 sedentary paraplegics (SP) en 10 sedentary able-bodied subjects (AB). The paraplegic groups were further subdivided by lesion level with T6 being the critical level. Tests were carried out at rest and after 2 bouts of arm ergometer exercise: a maximal incremental test and 3 minutes at 75% of maximal. Diameter, mean velocity, pulsatile index and blood flow were measured/calculated. Results: Resting heart rate was significantly higher in the paraplegic groups (EP = 80 bpm ± 10, SP = 83 bpm ± 12) vs. the AB group (69 bpm ± 7), p < 0.05. Resting diameter in the common femoral artery (CFA) was similar in EP (5.93 mm ± 1.54) and SP (6.52 mm ± 0.95), but significantly lower than in AB (7.87 mm ± 1.38), p < 0.05. Similar resting pulsatile index (PI) in the CFA were contrary to that previously reported, casting doubt on venous blood pooling theories. Post-exercise values need to be interpreted with caution in view of the large resting differences in CFA diameter. Percentage change values are therefore more appropriate. These differences were not statistically significant, but may suggest interesting trends. Large variability existed for most resting and post-exercise values. Conclusion: The paraplegic subject is an ideal model for the study of the influence of the SNS on blood supply to exercising skeletal muscle. The difference in CFA diameter at rest in the paraplegic vs. the AB group confirms previous results and is probably due to structural/non-physiological changes. Our observation that the BA and CFA diameters in EP and SP subjects do not differ significantly at rest, suggests that training does not have a spillover vasomotor effect on lower limb conduit arteries in paraplegia. Similar BF and PI values post-exercise in the SP and AB groups challenge the muscle pump theory. The SNS has an important role in the control of skeletal muscle blood flow - both at rest (vascular tone) and during exercise (redistribution). Suggestions for future research are made. / AFRIKAANSE OPSOMMING: Die meganismes betrokke by die beheer van bloedvloei (BV) gedurende dinamiese oefening is nog onduidelik. Die parapleeg (P) het verminderde simpatiese innervasie na die onderste ledemate. Die huidige studie fokus op die bydrae van die simpatiese senuwee sisteem (SSS), as deel van die sentrale vaskulêre meganisme, tot skeletale spier BV tydens dinamiese oefening. Doelstellings: Ons het BV parameters in P vs. nie-gestremde proefpersone (kontrole) bestudeer om vas te stelof die parapleeg as model gebruik kan word om die bydrae van die SSS tot veranderings in die BV in aktiewe- en onaktiewe spiere gedurende oefening, te ondersoek. Verdere aspekte wat ondersoek is, sluit in: die invloed van tiksheidvlak ten opsigte van rustende en oefenings BV, of die verlammingsvlak by die parapleeg BV kontrole beïnvloed, die 'spierpomp-teorie' en sy hipotetiese rol in oefeninghiperremie, asook die vraag of bloedsaamstorting in die bene van parapleë plaasvind. Metode: Nie-indringende duplex Doppler studies van die groot geleidingsarteries (bragiaal [BA] en gemene femoral [CFA]) is by 10 elite paraplegiese atlete (EP), 10 sedentêre parapleë (SP) en 10 sedentêre nie-gestremde proefpersone (AB) uitgevoer. Die paraplegiese proefpersone is verder onderverdeel deur die vlak van T6 as kritiese verlammingsvlak te gebruik. Toetse is tydens rus en na 2 arm-ergometer oefeningsessies uitgevoer: een maksimale inkrementeie toets en een van 75% van maksimum intensiteit. Deursnit, gemiddelde vloeispoed, pulsatiewe indeks en bloedvloei is gemeet en/of bereken. Resultate: Rustende hartspoed was beduidend hoër in die paraplegiese groepe (EP = 80 slaelminuut ± 10 en SP = 83 slm ± 12) vs. die AB groep (69 slm ± 7), p < 0.05. Rustende deursnit in die gemene femorale arterie (CFA) was dieselfde in EP (5.93 mm ± 1.54) en SP (6.52 mm ± 0.95), maar beduidend laer as in AB (7.87 mm ± 1.38), p < 0.05. Die feit dat rustende pulsatiewe indeks (PI) in die CFA dieselfde in albei groepe was, laat twyfelontstaan oor die veneuse bloedopdammings teorieë soos weergegee in die literatuur. Na-oefeningswaardes moet omsigtig evalueer word met inagneming van die groot rustende verskille in CFA deursnit. Persentasieverskilwaardes is dus meer toepaslik. Hierdie veskille was nie statisties beduidend nie, maar suggereer interessante tendense. Groot variasie het voorgekom vir beide rustende en na-oefenings waardes. Gevolgtrekking: Die parapleeg is 'n ideale model vir studies om die invloed van die SSS op bloedvloei aan aktiewe skeletale spier te bestudeer. Die verskil in rustende CFA deursnit in die parapleeg vs. die AB groep bevestig vorige resultate en is waarskynlik te wyte aan strukturele, nie-funksionele veranderinge. Ons bevindinge dat die BA en CFA deursneë nie beduidend verskil in die SP en EP groep gedurende rus nie, dui daarop dat gereëlde oefening nie 'n oorloop vasomotor effek op die onderste ledemate in die parapleeg het nie. Die feit dat daar geen verskil aangetoon kon word tussen BV en PI waardes na-oefening in die SP en AB groepe, betwis die spierpomp teorie. Die studie toon dat die SSS 'n belangrike rol in die beheer van skeletale spier bloedvloei speel - beide met rus (vaskulêre tonus) en gedurende oefening (herdistribusie). Voorstelle vir toekomstige navorsing word gemaak.

The hormone glucagon-like peptide-2 (GLP-2) is important in the regulation of intestinal growth and blood flow in nonruminant animals. However, no research reports the existence of GLP-2 in ruminants. Therefore, this dissertation examined the existence of GLP-2 and its receptor, their response to physiological stimuli, and its ability to induce gastrointestinal growth and intestinal blood flow in ruminants. Experiments 1 and 2 established the gastrointestinal distribution of mRNA for proglucagon (the GLP-2 precursor) and the GLP-2 receptor. Furthermore, these experiments determined the effects of changing dietary energy intake on plasma GLP-2 concentrations and proglucagon and GLP-2 receptor mRNA expression. Experiment 3 examined the effect of exogenous bovine GLP-2 on splanchnic blood flow, splanchnic nutrient flux, and gastrointestinal growth. This research shows that ruminants possess a functional GLP-2 signaling system that responds to nutrient ingestion. Based on observed receptor distribution and growth changes with GLP-2 treatment, GLP-2 targets the small intestine and does not affect forestomach or large intestinal growth. Increases in ileal proglucagon mRNA expression and plasma GLP-2 with increasing energy intake demonstrate that GLP-2 responds to physiologic changes in nutrient intake and can be relevant to feeding practices. Furthermore, observed increases in small intestinal growth and blood flow with GLP-2 suggest that it could substantially affect the capacity of the gastrointestinal tract for nutrient absorption. Modification of GLP-2 through diet could allow for improvements in nutrient utilization and animal productivity. This research also has important implications for use of GLP-2 for human disease therapy as the observed downregulation in the blood flow response to 10-d GLP-2 administration has never been reported in any species prior to this dissertation. This research systematically characterized and evaluated the potential role of GLP-2 in the control of gastrointestinal growth and splanchnic blood flow in ruminants. While it extends the knowledge of hormonal control of the gastrointestinal tract in ruminants, it also adds crucial information to the larger body of work investigating the actions of GLP- 2. This dissertation research has contributed to the groundwork necessary to enable the use of GLP-2 in improving the health and productivity of a diverse group of mammalian species.

Ergot alkaloids present in endophyte-infected (E+) tall fescue are thought to be the causative agent of fescue toxicosis, a syndrome affecting cattle in the eastern United States. Many of the observed signs of fescue toxicosis are thought to be attributed to peripheral vasoconstriction; however, there are data indicating that ergot alkaloids can alter blood flow to the gut. An experiment was conducted using right ruminal artery and vein collected from heifers shortly after slaughter. Vessels were mounted in a multi-myograph to determine the vasoconstrictive potentials of ergot alkaloids present in E+ tall fescue. Results indicated ergot alkaloids have the potential to induce vasoconstriction of foregut vasculature. A second experiment was conducted to determine if the additional ergot alkaloids present in E+ tall fescue increase the vasoconstrictive response above that of ergovaline. Results indicated that ergovaline is the main alkaloid responsible for vasoconstriction in bovine vessels. A third study was performed to determine the effect of ergot alkaloids on ruminal epithelial blood flow in the washed rumen of steers exposed to E+ or endophyte-free (E-) tall fescue seed. Steers were dosed with seed followed by a washed rumen experiment with differing levels of ergovaline incubated in the rumen. Results indicated that E+ tall fescue seed treatment reduced ruminal epithelial blood flow. Additionally, incubating ergovaline in the rumen during the washed rumen further decreased epithelial blood flow and volatile fatty acid (VFA) absorption. A final study was conducted to determine the acute effects of ergot alkaloids on isolated rumen epithelial absorptive and barrier functions and the potential for ruminal ergovaline absorption. Results indicate that acute exposure to ergot alkaloids does not alter the absorptive or barrier function of rumen epithelium and ergovaline is absorbed from the rumen. Data from this series of experiments have shown that ergot alkaloids from E+ tall fescue can induce vasoconstriction of blood vessels in the foregut of cattle, reduce blood flow to the rumen epithelium, and decrease VFA absorption. The decrease in nutrient absorption could contribute to the observed symptoms of fescue toxicosis, including depressed growth rates and general unthriftiness.