Intravital microscopic observation of the microvasculature during hemodialysis in healthy rats

Hemodialysis (HD) provides life-saving treatment for kidney failure. Patient mortality is extremely high, with cardiovascular disease (CVD) being the leading cause of death. This results from both a high underlying burden of cardiovascular disease, as well as additional physiological stress from the HD procedure itself. Clinical observations indicate that HD is associated with microvascular dysfunction (MD), underlining the need for a fundamental pathophysiological assessment of the microcirculatory consequences of HD. We therefore successfully developed an experimental small animal model, that allows for a simultaneous real-time assessment of the microvasculature. Using in-house built ultra-low surface area dialyzers and miniaturized extracorporeal circuit, we successfully dialyzed male Wistar Kyoto rats and combined this with a simultaneous intravital microscopic observation of the EDL microvasculature. Our results show that even in healthy animals, a euvolemic HD procedure can induce a significant systemic hemodynamic disturbance and induce disruption of microvascular perfusion (as evidence by a reduction in the proportion of the observed microcirculation receiving blood flow). This study, using a new small animal hemodialysis model, has allowed direct demonstration that microvascular blood flow in tissue in skeletal muscle is acutely reduced during HD, potentially in concert with other microvascular beds. It shows that preclinical small animal models can be used to further investigate HD-induced ischemic organ injury and allow rapid throughput of putative interventions directed at reducing HD-induced multi-organ ischemic injury.

Contribution of Vascular Cell Adhesion Molecule to Hemodynamics in Sickle Cell Disease

Vaso-occlusive events (VOEs) and pain crises are common clinical features of sickle cell disease (SCD) that result from sickle-shaped erythrocytes and leukocytes blocking blood flow, particularly in small vessels (Kato 2018). Activated endothelium also plays a role in the pathogenesis of VOEs in SCD. For example, VCAM-1 is expressed on blood vessels after activation by chemical and/or mechanical stimulation, which results in cytokine release. Studies have shown that patients with SCD have higher steady-state serum levels of soluble VCAM-1 compared to controls and that these levels are elevated during VOEs (White 2020). Furthermore, overexpression of these adhesion molecules on the endothelium results in prolonged adherence of white blood cells (WBCs), which has been shown to contribute to the development of VOEs and possibly cerebral vasculopathy. These findings raise the need to explore further the role of aberrant WBC- and/or RBC-endothelial interaction, mediated via VCAM-1, in the pathophysiology of cerebral microvascular hemodynamics and vasculopathy leading to cerebral microinfarcts in SCD. Therefore, we hypothesized that sickle cell mice will show greater cerebral cortical expression of VCAM-1 compared with age-matched controls and that this deposition will be associated with significant evidence of abnormal cerebral microvascular hemodynamic abnormalities. To examine the relationship between abnormalities in cerebral microvascular hemodynamics and VCAM-1 deposition in the cerebral microvasculature, we utilized a humanized sickle cell (with HbSS) and corresponding control (with HbAA). After cranial-window procedures, cortical capillaries, precapillary arterioles, and post-capillary venules were imaged using two-photon microscopy at two time points. In addition, this experiment included pre-and post-transfusion groups as we intend to study the impact of blood transfusion on hemodynamics. Using custom-written but well-validated MATLAB scripts, we analyzed line scans to identify the number and duration of rolling or adherent WBCs and RBCs, the RBC velocity in cerebral microvasculature, and the frequency and magnitude (mL/sec) of cerebral microvascular blood flow reversals. Rolling WBCs were defined as lasting two seconds or more, and adherent RBCs were defined as lasting 0.5 seconds or more. To quantify the expression of VCAM-1, we used immunohistochemistry to stain 50-micron sections of brain tissue for VCAM-1, Lectin to localize the vasculature, and Neun to localize neuronal nuclei. Images were analyzed using Phenochart and ImageJ software to examine the deposition of VCAM-1 throughout the brain tissue. As shown in Figure 1, at the first time point (baseline), we observed a significantly higher maximum RBC velocity (p<0.001) in the sickle cell mice compared to controls (figure 1a). We also found that there was significantly higher expression of VCAM-1 (p<0.001) (figure 1b) as well as significantly more leukocyte rolling (p<0.001) (figure 1c) in the sickle cell mice compared to controls. Additionally, we noted that the sickle cell mice have a significantly higher frequency of blood flow reversals (p<0.01) (figure 1d) as well as higher magnitude of microvascular blood flow reversals (p<0.001) (figure 1e) compared to controls. Interestingly, the sickle cell mice have a slightly lower average or mean capillary blood flow velocity compared to control (figure 1f), but this was not statistically significant (p=0.079). Since the mean capillary velocity is obtained as a smoothened difference between the forward flow and reversals, this decrease was surprising given the significant differences in frequency and magnitude of microvascular blood flow reversal in the sickle cell mice compared to controls (figs 1d and 1e). In conclusion, we see that the high velocity of blood flow might be a mechanical force, among other factors contributing to cerebral microvascular VCAM-1 expression in sickle cell mice. This might be responsible for the increased leukocyte-endothelial interactions and adhesion, ultimately leading to higher frequency and magnitude of cerebral microvascular blood flow reversal. Taken together, this may contribute to the observed slightly lower mean or effective microvascular forward blood flow. These pathophysiological changes might contribute to the reported higher rate of cerebral microinfarct and silent infarct in sickle cell disease. Figure 1 Figure 1. Disclosures Archer: Global Blood Therapeutics: Consultancy, Research Funding; Forma Therapeutics: Research Funding. Hyacinth: Novartis: Consultancy; Acuta Capital: Consultancy.

Green Tea Extract Concurrent with an Oral Nutritional Supplement Acutely Enhances Muscle Microvascular Blood Flow without Altering Leg Glucose Uptake in Healthy Older Adults

Postprandial macro- and microvascular blood flow and metabolic dysfunction manifest with advancing age, so vascular transmuting interventions are desirable. In this randomised, single-blind, placebo-controlled, crossover trial, we investigated the impact of the acute administration of green tea extract (GTE; containing ~500 mg epigallocatechin-3-gallate) versus placebo (CON), alongside an oral nutritional supplement (ONS), on muscle macro- and microvascular, cerebral macrovascular (via ultrasound) and leg glucose/insulin metabolic responses (via arterialised/venous blood samples) in twelve healthy older adults (42% male, 74 ± 1 y). GTE increased m. vastus lateralis microvascular blood volume (MBV) at 180 and 240 min after ONS (baseline: 1.0 vs. 180 min: 1.11 ± 0.02 vs. 240 min: 1.08 ± 0.04, both p < 0.005), with MBV significantly higher than CON at 180 min (p < 0.05). Neither the ONS nor the GTE impacted m. tibialis anterior perfusion (p > 0.05). Leg blood flow and vascular conductance increased, and vascular resistance decreased similarly in both conditions (p < 0.05). Small non-significant increases in brachial artery flow-mediated dilation were observed in the GTE only and middle cerebral artery blood flow did not change in response to GTE or CON (p > 0.05). Glucose uptake increased with the GTE only (0 min: 0.03 ± 0.01 vs. 35 min: 0.11 ± 0.02 mmol/min/leg, p = 0.007); however, glucose area under the curve and insulin kinetics were similar between conditions (p > 0.05). Acute GTE supplementation enhances MBV beyond the effects of an oral mixed meal, but this improved perfusion does not translate to increased leg muscle glucose uptake in healthy older adults.

A model of anemic tissue perfusion after blood transfusion shows critical role of endothelial response to shear stress stimuli

­­ ­Although some of the cardiovascular responses to changes in hematocrit (Hct) are not fully quantified experimentally, available information is sufficient to build a mathematical model of the consequences of treating anemia by introducing RBCs into the circulation via blood transfusion. We present such a model, which describes how the treatment of normovolemic anemia with blood transfusion impacts oxygen (O2) delivery (DO2, the product of blood O2 content and arterial blood flow) by the microcirculation. Our analysis accounts for the differential response of the endothelium to the wall shear stress (WSS) stimulus, changes in nitric oxide (NO) production due to modification of blood viscosity caused by alterations of both hematocrit (Hct) and cell free layer thickness, as well as for their combined effects on microvascular blood flow and DO2. Our model shows that transfusions of 1- and 2-unit of blood have a minimal effect on DO2 if the microcirculation is unresponsive to the WSS stimulus for NO production that causes vasodilatation increasing blood flow and DO2. Conversely, in a fully WSS responsive organism, blood transfusion significantly enhances blood flow and DO2, because increased viscosity stimulates endothelial NO production causing vasodilatation. This finding suggests that evaluation of a patients' pre-transfusion endothelial WSS responsiveness should be beneficial in determining the optimal transfusion requirements for treating anemic patients.

Six weeks of high-intensity interval training enhances contractile activity induced vascular reactivity and skeletal muscle perfusion in older adults

Impairments in muscle microvascular function are associated with the pathogenesis of sarcopenia and cardiovascular disease. High-intensity interval training (HIIT) is an intervention by which a myriad of beneficial skeletal muscle/cardiovascular adaptations have been reported across age, including capillarisation and improved endothelial function. Herein, we hypothesised that HIIT would enhance muscle microvascular blood flow and vascular reactivity to acute contractile activity in older adults, reflecting HIIT-induced vascular remodelling. In a randomised controlled-trial, twenty-five healthy older adults aged 65–85 years (mean BMI 27.0) were randomised to 6-week HIIT or a no-intervention control period of an equal duration. Measures of microvascular responses to a single bout of muscle contractions (i.e. knee extensions) were made in the m. vastus lateralis using contrast-enhanced ultrasound during a continuous intravenous infusion of Sonovue™ contrast agent, before and after the intervention period, with concomitant assessments of cardiorespiratory fitness and resting blood pressure. HIIT led to improvements in anaerobic threshold (13.2 ± 3.4 vs. 15.3 ± 3.8 ml/kg/min, P < 0.001), dynamic exercise capacity (145 ± 60 vs. 159 ± 59 W, P < 0.001) and resting (systolic) blood pressure (142 ± 15 vs. 133 ± 11 mmHg, P < 0.01). Notably, HIIT elicited significant increases in microvascular blood flow responses to acute contractile activity (1.8 ± 0.63 vs. 2.3 ± 0.8 (arbitrary contrast units (AU), P < 0.01)), with no change in any of these parameters observed in the control group. Six weeks HIIT improves skeletal muscle microvascular responsiveness to acute contractile activity in the form of active hyperaemia-induced by a single bout of resistance exercise. These findings likely reflect reports of enhanced large vessel distensibility, improved endothelial function, and muscle capillarisation following HIIT. Moreover, our findings illustrate that HIIT may be effective in mitigating deleterious alterations in muscle microvascular mediated aspects of sarcopenia.

Correlation between Perfusion Index and Left Ventricular Output in Healthy Late Preterm Infants

Aim The perfusion index (PI) is a noninvasive marker derived from photoelectric plethysmographic signals in pulse oximetry in the evaluation of peripheral perfusion. This study was aimed to determine the correlation between PI and left ventricular output (LVO) in healthy late preterm infants at 48th hour of life. Methods With new generation pulse oximeter (MASIMO Rad 7 Oximeter) pre- and post-ductal PI values were recorded from healthy late preterm babies at the 48th hour of life. PI was determined simultaneously with LVO as measured by transthoracic echocardiography. Results A total of 50 late preterm babies were included in the study. The mean gestational age of the cases was 35.4 ± 0.7 weeks and the birth weight was 2,586 ± 362 g. Mean pre- and post-ductal PI values at the postnatal 48th hour of babies' life were found to be 2.0 ± 0.9 and 1.7 ± 1.1. The mean LVO value was 438 ± 124, LVO/kg 175 ± 50. When the LVO value was normalized according to the babies' body weight, there was no statistically significant correlation between the pre- and post-ductal PI and the LVO/kg value (r <0.2, p >0.05 in both comparisons). Conclusion There was no correlation between pre- and post-ductal PI and LVO values in healthy late preterm infants. This may be due to the failure of the LVO, a systemic hemodynamic parameter, to accurately reflect microvascular blood flow due to incomplete maturation of the sympathetic nervous system involved in the regulation of peripheral tissue perfusion in preterm babies. Key Points

Identifying Elevated Risk for Future Pain Crises in Sickle-Cell Disease Using Photoplethysmogram Patterns Measured During Sleep: A Machine Learning Approach

Transient increases in peripheral vasoconstriction frequently occur in obstructive sleep apnea and periodic leg movement disorder, both of which are common in sickle cell disease (SCD). These events reduce microvascular blood flow and increase the likelihood of triggering painful vaso-occlusive crises (VOC) that are the hallmark of SCD. We recently reported a significant association between the magnitude of vasoconstriction, inferred from the finger photoplethysmogram (PPG) during sleep, and the frequency of future VOC in 212 children with SCD. In this study, we present an improved predictive model of VOC frequency by employing a two-level stacking machine learning (ML) model that incorporates detailed features extracted from the PPG signals in the same database. The first level contains seven different base ML algorithms predicting each subject's pain category based on the input PPG characteristics and other clinical information, while the second level is a meta model which uses the inputs to the first-level model along with the outputs of the base models to produce the final prediction. Model performance in predicting future VOC was significantly higher than in predicting VOC prior to each sleep study (F1-score of 0.43 vs. 0.35, p-value &lt;0.0001), consistent with our hypothesis of a causal relationship between vasoconstriction and future pain incidence, rather than past pain leading to greater propensity for vasoconstriction. The model also performed much better than our previous conventional statistical model (F1 = 0.33), as well as all other algorithms that used only the base-models for predicting VOC without the second tier meta model. The modest F1 score of the present predictive model was due in part to the relatively small database with substantial imbalance (176:36) between low-pain and high-pain subjects, as well as other factors not captured by the sleep data alone. This report represents the first attempt ever to use non-invasive finger PPG measurements during sleep and a ML-based approach to predict increased propensity for VOC crises in SCD. The promising results suggest the future possibility of embedding an improved version of this model in a low-cost wearable system to assist clinicians in managing long-term therapy for SCD patients.

Optical Brain Biopsy with a Fluorescence and Vessel Tracing Probe

BACKGROUND Accurate stereotactic biopsies of brain tumors are imperative for diagnosis and tailoring of the therapy. Repetitive needle insertions enhance risks of brain lesioning, hemorrhage, and complications due to prolonged procedure. OBJECTIVE To investigate clinical benefits of a combined 5-aminolaevulinic acid (5-ALA) fluorescence and laser Doppler flowmetry system for the detection of malignant brain tumor and blood vessels in stereotactic biopsies. METHODS Planning of targets and trajectories was followed by optical measurements in 20 patients, using the Leksell Stereotactic System and a manual insertion device. Fluorescence spectra, microvascular blood flow, and tissue grayness were recorded each millimeter along the paths. Biopsies were taken at preplanned positions. The diagnoses were compared with the fluorescence signals. The recordings were plotted against measurement positions and compared. Sites indicating a risk of hemorrhage were counted as well as the time for the procedures. RESULTS Signals were recorded along 28 trajectories, and 78 biopsies were collected. The final diagnosis showed 17 glioblastomas, 2 lymphomas, and 1 astrocytoma grade III. Fluorescence was seen along 23 of the paths with 4 having the peak of 5-ALA fluorescence 3 mm or more from the precalculated target. There was increased microcirculation in 40 of 905 measured positions. The measurement time for each trajectory was 5 to 10 min. CONCLUSION The probe provided direct feedback of increased blood flow along the trajectory and of malignant tissue in the vicinity of the target. The method can increase the precision and the safety of the biopsy procedure and reduce time.