Changes in microvascular hematocrit during post-occlusive reactive hyperemia: descriptions and mechanisms

Doctor of PhilosophyDepartment of Anatomy and PhysiologyThomas J. BarstowThe primary aim of this dissertation was to describe the changes in microvascular hematocrit, as total[hemoglobin+myoglobin] (T[Hb+Mb] measured with near-infrared spectroscopy (NIRS), during post-occlusive reactive hyperemia (PORH). Mechanisms of reactive hyperemia within skeletal muscle were also explored. The investigation detailed in Chapter 2 of this dissertation found that the differing time courses of the kinetic responses of both oxy- and deoxy[Hb+Mb], are related to changes in T[Hb+Mb]. We also determined that adipose tissue thickness had no effect on a purely temporal analysis of NIRS data. In Chapter 3 we observed that brachial artery reactive hyperemia preceded changes in T[Hb+Mb] during reactive hyperemia. Assuming that myoglobin remained constant, we posited that changes in T[Hb+Mb] must reflect alterations in red blood cell concentration in the microvasculature, i.e., microvascular hematocrit. In Chapter 4 comparisons were made between brachial artery blood flow, cutaneous and skeletal muscle flux and T[Hb+Mb]. The conduit artery response was faster than the microvascular responses in all tissues. Within skeletal muscle, time to peak and the time constant for the on-kinetics were faster in T[Hb+Mb] compoared with intramuscular flux as measured with intramuscular laser-Doppler. We observed no differences in temporal responses between cutaneous and intramuscular measures and suggested that in a purely temporal analysis the cutaneous microvasculature could serve as an analog for the skeletal muscle microvasculature. Finally, in Chapter 5 we found that prostaglandin inhibition with ibuprofen altered the initial T[Hb+Mb] response during PORH without impacting cutaneous flux or brachial artery blood flow. Chapter 5 also discussed that the addition of a wrist cuff to our standard instrumentation prevented the accumulation of T[Hb+Mb] during the occlusion period

Functional Evaluation of the Peripheral Vasculature Using Magnetic Resonance Imaging

Akin to cardiac stress testing, functional integrity of the peripheral vasculature can be interrogated by measuring the response to a stimulus. Recent reports suggest that the reactive hyperemia response, the physiologic reaction following induced ischemia, is associated with disease presence, correlated with disease severity, and may be a sensitive biomarker of pre-clinical disease. In this dissertation, an innovative, interleaved magnetic resonance imaging method is developed, termed Perfusion, Intravascular Venous Oxygen saturation, and T2* (PIVOT), which simultaneously measures microvascular perfusion, venous oxygen saturation (SvO2), and the blood-oxygen-level dependent (BOLD) signal. PIVOT is first applied in healthy subjects to demonstrate its ability to measure reactive hyperemia response dynamics. Next, reactive hyperemia perfusion is compared between the more temporally efficient pulsed arterial spin labeling (PASL) used in PIVOT and the more recently developed and preferred method for the brain, pseudo-continuous ASL (pCASL). Assessment of the impact of blood flow variability throughout the ischemia-reperfusion paradigm on pCASL perfusion quantification is investigated. Then, both PASL and pCASL sequences are used to measure reactive hyperemia perfusion in healthy subjects. No significant differences were detected between perfusion measured with PASL or pCASL despite different labeling strategies, temporal resolutions, and perfusion quantification models. Subsequently, PIVOT is combined with a velocity-encoded dual-echo GRE to create an interleaved three-slice sequence that provides quantification of bulk blood flow in the arteries and veins in addition to the traditional PIVOT measures. This new sequence, termed Velocity and PIVOT (vPIVOT) is used to investigate the relationship of blood flow in the macro- and microvasculature and muscle oxygen consumption during the transition from exercise to rest. Finally, PIVOT is applied clinically in a cohort of patients with varying degrees of severity of peripheral artery disease. Increasing disease severity was correlated with a prolongation of the hyperemic response time, measured as a lengthening of time to peak perfusion, SvO2 washout time, and time to peak T2*. In addition, peak perfusion and SvO2 upslope were significantly different between patients with PAD and healthy controls. These results suggest the potential for PIVOT to evaluate disease severity and may present a tool to assess response to therapeutic intervention

Body mass index is associated with microvascular endothelial dysfunction in patients with treated metabolic risk factors and suspected coronary artery disease

Background--Obesity is key feature of the metabolic syndrome and is associated with high cardiovascular morbidity and mortality. Obesity is associated with macrovascular endothelial dysfunction, a determinant of outcome in patients with coronary artery disease. Here, we compared the influence of obesity on microvascular endothelial function to that of established cardiovascular risk factors such as diabetes mellitus, hypertension, hypercholesterolemia, and smoking in patients with suspected coronary artery disease. Methods and Results--Endothelial function was assessed during postocclusive reactive hyperemia of the brachial artery and downstream microvascular beds in 108 patients who were scheduled for coronary angiography. In all patients, microvascular vasodilation was assessed using peripheral arterial tonometry; laser Doppler flowmetry and digital thermal monitoring were performed. Body mass index was significantly associated with decreased endothelium-dependent vasodilatation measured with peripheral arterial tonometry (r=0.23, P=0.02), laser Doppler flowmetry (r=0.30, P < 0.01), and digital thermal monitoring (r=0.30, P < 0.01). In contrast, hypertension, hypercholesterolemia, and smoking had no influence on microvascular vasodilatation. Especially in diabetic patients, endothelial function was not significantly reduced (control versus diabetes mellitus, mean±SEM or median [interquartile range], peripheral arterial tonometry: 1.90±0.20 versus 1.67±0.20, P=0.19, laser Doppler flowmetry: 728% [interquartile range, 427-1110] v

Peripheral artery disease and resistance training: improving performance and health.

Peripheral artery disease (PAD) is a pathology of lower limbs affecting about 20% of people older than 70 years. Intermittent claudication (IC) is the most frequent symptom of PAD. IC is pain experienced during walking, typically in the calf. This limits the walking ability of patients and reduces social role, physical activity and quality of life. The main therapy for IC is supervised exercise. Whereas the primary role of supervised exercise in treatment of IC is well established there are some doubt about the best typology of exercise. Usually training includes only walking and aerobics exercises, but some elements suggested that for this patients adding strength exercises may be useful

Factors predisposing to arterial disease in children with chronic renal failure

Premature atherosclerotic disease is a major cause of morbidity and mortality in chronic renal failure (CRF). Hypertension, hyperlipidaemia, abnormalities of nitric oxide (NO) biochemistry, drugs and the uraemic milieu are possible causes. Endothelial dysfunction is a key early event in atherogenesis. In this thesis, a non-invasive technique of high- resolution ultrasound has been used to assess the physiology of endothelial function in the brachial artery of children with stable CRF but without other risk factors of atherosclerosis. Lipid subfractions and nitric oxide (NO) biochemistry were also studied in the children. NO metabolites and endogenous NO synthetase (eNOS) inhibitors were measured as an assessment of endothelial NO metabolism. Brachial artery dilatation to flow (FMD) was reduced in CRF children when compared to matched controls for age and vessel diameter. In contrast, response to glyceryltrinitrate (GTN) was similar in both groups. Antibodies against oxidised LDL (ox-LDL) were high in CRF. Endogenous NOS inhibitors were high in CRF, and intermediate NO metabolites were low. This study shows that endothelium dependent dilatation of the brachial artery is impaired in children with CRF who do not have co-existing risk factors for atherosclerosis. This may represent early evidence of atherogenic vascular disease. In the second half of the thesis, the effect of enteral feeds on lipid levels in children with CRF were studied. This is important because the anorexia of CRF is frequently managed with high carbhydrate and high fat enternal feeds, which might predispose to atherogenic blood lipid profiles. Plasma lipid sub-fractions were, therefore, measured in children with CRF whose diet was either managed conservatively or by enteral feeding. Overall, TGs were high, TC was at the upper limit of normal, and LDL, HDL, apoprotein A1 (apo A1), A2 (apo A2) and B (apo B), and lipoprotein (a) (Lp (a)) were within the normal range. There was an inverse correlation between TGs and GFR. There were no differences in the levels of TC, TGs, LDL, HDL, apo A1, apo A2 or Lp (a) between tube fed and non-tube fed children. We conclude that enteral feeding does not enhance hyperlipidaemia

Oleuropein effects on rat and human microcirculation

The aim of the present study was to investigate oleuropein effects on microvascular responses. First, we investigated the in vivo effects of oleuropein on rat pial microcirculation submitted to hypoperfusion-reperfusion injury. Therefore, we studied acute microvascular responses such as arteriolar vasodilation, permeability increase, leukocyte adhesion and capillary perfusion, by fluorescence microscopy. The working hypothesis was that this polyphenol may induce nitric oxide (NO) release from endothelial cells and consequently protect cerebral blood flow distribution and cerebral tissue. Rat cerebral cortical eNOS protein levels were evaluated as well as the impact of oxidative stress induced by hypopefusion and reperfusion on brain tissue, utilizing DCFH-DA. The second part of the study was aimed to evaluate oleuropein effects on skin microvascular blood flow oscillations of hyperlipidemic obese patients, by laser Doppler flowmetry (LDF). Therefore, hyperlipidemic obese females were administered with a hypocaloric and hypolipidic diet plus oleuropein for three months. These data were compared with the response of hyperlipidemic obese patients administered with hypocaloric and hypolipidic diet. Under baseline conditions and at the end of the study, nutritional status and lipid profile were evaluated as well as skin blood flow oscillations and reactive hyperemia by LDF. The results of the experimental study in rats indicate that oleuropein significantly improved in vivo microvascular responses after hypoperfusion-reperfusion injury. In particular, 20 mg/Kg b.w. of oleuropein induced a dilation by 28 ±2% of baseline (p < 0.01 vs. hypoperfused group) in order 3 arterioles and significantly reduced microvascular leakage (NGL: 0.13 ± 0.03; p < 0.01 vs. hypoperfused group) as well as leukocyte adhesion on venular walls (2.0 ± 0.5/100 µm v.l./30 sec; p < 0.01 vs. hypoperfused group), at the end of reperfusion. Moreover, this polyphenol was able to preserve capillary perfusion at the end of reperfusion (-26.0±4.5% of baseline; p<0.01 vs. hypoperfused group). These responses were associated to the increased eNOS expression in cortex and in striatum of treated animals. Oleuropein was also able to reduce neuronal damage and ROS production at the end of reperfusion, compared with hypoperfused animals. On the other hand, the results of the clinical study revealed that three months of hypocaloric and hypolipidic diet associated to oleuropein significantly improved nutritional status and lipid profile of hyperlipidemic obese patients. Total and LDL cholesterol, indeed, decreased by 15.0±1.2 and 16.5±1.3%, respectively, in patients treated with diet (OD group), and by 21.3±1.5 and 21.2±1.4%, respectively, in subjects treated with diet plus oleuropein (OL group). Moreover, laser Doppler measurements showed an increase in skin perfusion, compared to baseline conditions and control group (+25.6±1.4% of baseline), while the spectral analysis of skin blood flow oscillations revealed an increase in the NO-dependent and myogenic-related frequency components. Furthermore, PORH response improved in oleuropein-treated group, compared to controls. In conclusion, oleuropein appeared able to protect rat pial microcirculation from hypoperfusion-reperfusion injury increasing nitric oxide release from endothelial cells, reducing oxidative stress and, consequently, preserving pial blood flow distribution. Interestingly, this polyphenol showed beneficial effects also in humans; three months of hypocaloric and hypolipidic diet plus oleuropein increased smooth muscle cell functions and microvascular responses in hyperlipidemic obese patients, improving tissue perfusion

Chronic passive heat therapy as a novel means of improving vascular function in sedentary humans

Cardiovascular disease is the leading cause of death in the developed world. The majority of cardiovascular diseases are characterized by disorders of the arteries, predominantly caused by endothelial dysfunction and arterial stiffening. Passive heat stress results in elevations in core temperature (inducing heat shock protein expression) and changes in cardiovascular hemodynamics, such as increased cardiac output and shear stress, that are similar to exercise. Thus, repeated passive heat stress (“heat therapy”) may provide an alternative means of improving cardiovascular health, particularly for patients with limited exercise tolerance and/or capabilities. Therefore, the goal of this dissertation was to perform integrative studies to determine the effects of heat therapy on vascular function and the associated cellular pathways in young, sedentary humans. Twenty subjects were assigned to participate in 8 weeks (4-5x/week) of heat therapy (N=10; immersion in a 40.5°C bath sufficient to maintain rectal temperature ≥38.5°C for 60 min/session) or thermoneutral water immersion (N=10; sham). As discussed in Chapter V, we found that heat therapy improved numerous well-established biomarkers of conduit vessel/macrovascular function, including flow-mediated dilation (a measure of endothelial function), arterial stiffness, intima media thickness, and blood pressure. Heat therapy also improved microvascular function, as discussed in Chapter VI, measured as improved cutaneous thermal hyperemia and nitric oxide-dependent dilation (the difference between microdialysis sites receiving Lactated Ringer’s [control] and nitric oxide synthase inhibition). No changes were observed in any variables in sham subjects. In Chapter VII, we showed that both direct cellular heating and serum collected from human subjects following heat therapy improved nitric oxide bioavailability and angiogenesis in cultured endothelial cells, providing potential mechanisms by which heat therapy improves vascular function in vivo. Therefore, the studies described herein provide comprehensive evidence that passive heat therapy improves vascular health and insight into the mechanisms involved. Our data presented in Chapters IV-VII, combined with pilot data we conducted in spinal cord injured individuals (Chapter VIII), strongly indicate that passive heat therapy could be used as a simple and effective tool to improve cardiovascular health in a variety of patient populations. This dissertation includes published and unpublished co-authored material

Effects of resistance training in peripheral artery disease treatment

L\u2019arteriopatia periferica aterosclerotica degli arti inferiori (PAD) \ue8 una patologia che colpisce circa il 20% dei soggetti al di sopra dei 70 anni. La claudicatio inter-mittens (IC), che ne \ue8 il principale sintomo, \ue8 un dolore che si presenta tipicamente ai polpacci durante il cammino. Questo sintomo limita la capacit\ue0 di camminare dei pazienti influendo negativamente sul loro ruolo sociale, sul livello di attivit\ue0 fisica, e sulla qualit\ue0 di vita. La terapia principale per l\u2019IC \ue8 l\u2019esercizio fisico su-pervisionato. Tuttavia se \ue8 chiaro il ruolo che quest\u2019ultimo svolge nell\u2019incrementare la capacit\ue0 di marcia dei pazienti, non \ue8 altrettanto chiara la tipologia ottimale di esercizio da proporre. Il trattamento abitualmente utilizzato include esclusivamente cammino ed esercizi aerobici, vi sono tuttavia le basi per suppore che i pazienti potrebbero beneficiare dell\u2019aggiunta di esercizi anaerobici di forza al loro protocollo di allenamento.Peripheral artery disease (PAD) is a pathology of lower limbs affecting about 20% of people older than 70 years. Intermittent claudication (IC) is the most frequent symptom of PAD. IC is pain experienced during walking, typically in the calf. This limits the walking ability of patients and reduces social role, physical activity and quality of life. The main therapy for IC is supervised exercise. Whereas the primary role of supervised exercise in treatment of IC is well established there are some doubt about the best typology of exercise. Usually training includes only walking and aerobics exercises, but some elements suggested that for this patients adding strength exercises may be useful

A PRE AND POST EXERCISE COMPARISON OF THREE ASSESSMENT TOOLS COMMONLY EMPLOYED TO ASSESS VASCULAR FUNCTION

Background: Endothelial dysfunction (ED) is one of the earliest subclinical indicators of impaired cardiovascular health and several non-invasive tools have been developed to evaluate vascular function, including strain gauge plethysmography (SGP), brachial artery flow-mediated dilation (FMD) via ultrasound, and peripheral artery tonometry (PAT). While these tools have extensively been studied during a resting condition, the responses following acute exercise are not as well characterized. Purpose: The purpose of this study was to compare the pre- and post-exercise vascular function values obtained with SGP, FMD, and PAT. Relationships among the primary outcome variables obtained with each assessment tool were also evaluated. Methods: Vascular function was assessed in 17 sedentary, apparently healthy male subjects (24±4 yrs; 24.5±3.2 kg/m2) at rest and following an acute submaximal exercise bout with SGP, FMD, and PAT. Results: During rest, post-occlusion reactive hyperemia resulted in significant (p\u3c0.05) increases in forearm blood flow (FBF; 2.13±1.03 vs 6.35 ± 2.90 mL/min/100 mL tissue) and area under the curve (AUC; 226.77 ± 111.20 vs 588.22 ±283.33 mL/min/100 mL) as determined by SGP. Brachial artery diameter (BAD) as assessed with FMD was increased by 5.3% (p\u3c0.05). Resting reactive hyperemia index (RHI) as assessed by PAT was observed to be 1.73±0.34. Significant exercise-induced increases (p\u3c0.05) were observed in baseline and post-occlusion FBF and baseline AUC values utilizing SGP. Additionally, FMD baseline blood velocity was significantly increased (91.8±11.1 vs 108.0±17.1 cm/sec, p\u3c0.05) and the PAT augmentation index (AI) was significantly more negative (-8.8 ±9.4 vs -18.9±8.4%, p\u3c0.05) after exercise. There were no significant correlations observed among the primary outcome measures obtained from each assessment technique. There was, however, a moderate correlation between pre-exercise vascular reactivity as assessed by SGP and change in blood velocity as assessed by FMD (r= 0.566, p= 0.035). Conclusions: The addition of an exercise stress to vascular function assessment may offer greater insight into the health of the vasculature. This initial study was undertaken to further evaluate the pre- to post-exercise responses obtained using three commonly employed vascular function assessment techniques in healthy individuals. Additional research as to the value of the addition of an exercise stress to vascular function assessment in individuals with traditional cardiovascular disease risk factors or known cardiovascular disease is warranted