Quantitative imaging of red blood cell velocity invivo using optical coherence Doppler tomography.
ABSTRACT: We present particle counting ultrahigh-resolution optical Doppler tomography (pc-μODT) that enables accurate imaging of red blood cell velocities (ν(RBC)) of cerebrovascular networks by detecting the Doppler phase transients induced by the passage of a RBC through a capillary. We apply pc-μODT to image the response of capillary ν(RBC) to mild hypercapnia in mouse cortex. The results show that ν(RBC) in normocapnia (ν(N) = 0.72 ± 0.15 mm/s) increased 36.1% ± 5.3% (ν(H) = 0.98 ± 0.29 mm/s) in response to hypercapnia. Due to uncorrected angle effect and low hematocrit (e.g., ∼10%), ν(RBC) directly measured by μODT were markedly underestimated (ν(N) ≈ 0.27 ± 0.03 mm/s, ν(H) ≈ 0.37± 0.05 mm/s). Nevertheless, the measured ν(RBC) increase (35.3%) matched that (36.1% ± 5.3%) by pc-μODT.
Project description:Although microvascular dysfunction accompanies cognitive decline in aging, vascular dementia, and Alzheimer's disease, tools to study microvasculature longitudinally in vivo are lacking. Here, we use Doppler optical coherence tomography (OCT) and angiography for noninvasive, longitudinal imaging of mice with chronic cerebral hypoperfusion for up to 1 month. In particular, we optimized the OCT angiography method to selectively image red blood cell (RBC)-perfused capillaries, leading to a novel way of assessing capillary supply heterogeneity in vivo. After bilateral common carotid artery stenosis (BCAS), cortical blood flow measured by Doppler OCT dropped to half of baseline throughout the imaged tissue acutely. Microscopic imaging of the capillary bed with OCT angiography further revealed local heterogeneities in cortical flow supply during hypoperfusion. The number of RBC-perfused capillaries decreased, leading to increased oxygen diffusion distances in the days immediately after BCAS. Linear regression showed that RBC-perfused capillary density declined by 0.3% for a drop in flow of 1?mL/100?g per minute, and decreases in RBC-perfused capillary density as high as 25% were observed. Taken together, these results demonstrate the existence of local supply heterogeneity at the capillary level even at nonischemic global flow levels, and demonstrate a novel imaging method to assess this heterogeneity.
Project description:To compare the microcirculatory velocity distribution in type 2 diabetic patients and nondiabetic control subjects at baseline and after local heating.The skin blood flow response to local heating (44 degrees C for 20 min) was assessed in 28 diabetic patients and 29 control subjects using a new velocity-resolved quantitative laser Doppler flowmetry technique (qLDF). The qLDF estimates erythrocyte (RBC) perfusion (velocity x concentration), in a physiologically relevant unit (grams RBC per 100 g tissue x millimeters per second) in a fixed output volume, separated into three velocity regions: v <1 mm/s, v 1-10 mm/s, and v >10 mm/s.The increased blood flow occurs in vessels with a velocity >1 mm/s. A significantly lower response in qLDF total perfusion was found in diabetic patients than in control subjects after heat provocation because of less high-velocity blood flow (v >10 mm/s). The RBC concentration in diabetic patients increased sevenfold for v between 1 and 10 mm/s, and 15-fold for v >10 mm/s, whereas no significant increase was found for v <1 mm/s. The mean velocity increased from 0.94 to 7.3 mm/s in diabetic patients and from 0.83 to 9.7 mm/s in control subjects.The perfusion increase occurs in larger shunting vessels and not as an increase in capillary flow. Baseline diabetic patient data indicated a redistribution of flow to higher velocity regions, associated with longer duration of diabetes. A lower perfusion was associated with a higher BMI and a lower toe-to-brachial systolic blood pressure ratio.
Project description:This study investigated the changes in cerebral near-infrared spectroscopy (NIRS) signals, cerebrovascular and ventilatory responses to hypoxia and CO2 during altitude exposure. At sea level (SL), after 24?hours and 5 days at 4,350?m, 11 healthy subjects were exposed to normoxia, isocapnic hypoxia, hypercapnia, and hypocapnia. The following parameters were measured: prefrontal tissue oxygenation index (TOI), oxy- (HbO2), deoxy- and total hemoglobin (HbTot) concentrations with NIRS, blood velocity in the middle cerebral artery (MCAv) with transcranial Doppler and ventilation. Smaller prefrontal deoxygenation and larger ?HbTot in response to hypoxia were observed at altitude compared with SL (day 5: ?HbO2-0.6±1.1 versus -1.8±1.3??mol/cmper mm?Hg and ?HbTot 1.4±1.3 versus 0.7±1.1??mol/cm per mm?Hg). The hypoxic MCAv and ventilatory responses were enhanced at altitude. Prefrontal oxygenation increased less in response to hypercapnia at altitude compared with SL (day 5: ?TOI 0.3±0.2 versus 0.5±0.3% mm?Hg). The hypercapnic MCAv and ventilatory responses were decreased and increased, respectively, at altitude. Hemodynamic responses to hypocapnia did not change at altitude. Short-term altitude exposure improves cerebral oxygenation in response to hypoxia but decreases it during hypercapnia. Although these changes may be relevant for conditions such as exercise or sleep at altitude, they were not associated with symptoms of acute mountain sickness.
Project description:Purpose:We aimed to observe longitudinal changes in retinal blood flow (RBF) and structural transformations in capillaries using Doppler optical coherence tomography (DOCT) and optical coherence tomography angiography (OCTA) in a feline retinal blood occlusion (RVO) model. Methods:RVO was induced by argon green laser photocoagulation (PC) in six eyes of six cats. RBF was measured at a first-order retinal artery and vein by a DOCT flowmeter, and structural changes in the capillaries around the occluded vessels (12 × 12 and 3 × 3 mm) were assessed by OCTA before (at baseline); immediately after PC; and on days 1, 4, 7, and 14 thereafter. Systemic and ocular parameters were monitored during the observation period. Results:There were no significant differences in any systemic or ocular parameters before and after PC. Arterial RBF increased significantly on day 1 (160.6 ± 8.6% vs. baseline, P < 0.001) and decreased below baseline after day 1 through 14. Venous RBF decreased immediately after PC (17.4 ± 9.6% vs. baseline, P = 0.001) and then gradually increased afterwards, but did not return to baseline. OCTA showed dilatation of retinal venules immediately after PC to day 1. Collateral vessels began to form on day 4, had matured by day 7, and were pruned on day 14, which formed as mature as normal retinal venule diameters. Conclusions:With increasing arterial RBF within 1 day after inducing RVO, venules gradually expanded to form collateral vessels, suggesting that collateral vessels originate from existing capillary networks, not neovascularization.
Project description:Impaired endothelium-dependent vasodilation has been documented in patients with sleep apnea. This impairment may result in blood flow dysregulation during apnea-induced fluctuations in arterial blood gases.To test the hypothesis that hypoxic and hypercapnic vasodilation in the forearm and cerebral circulation are impaired in patients with sleep apnea.We exposed 20 patients with moderate to severe sleep apnea and 20 control subjects, to isocapnic hypoxia and hyperoxic hypercapnia. A subset of 14 patients was restudied after treatment with continuous positive airway pressure.Cerebral flow velocity (transcranial Doppler), forearm blood flow (venous occlusion plethysmography), arterial pressure (automated sphygmomanometry), oxygen saturation (pulse oximetry), ventilation (pneumotachograph), and end-tidal oxygen and carbon dioxide tensions (expired gas analysis) were measured during three levels of hypoxia and two levels of hypercapnia. Cerebral vasodilator responses to hypoxia (-0.65 +/- 0.44 vs. -1.02 +/- 0.72 [mean +/- SD] units/% saturation; P = 0.03) and hypercapnia (2.01 +/- 0.88 vs. 2.57 +/- 0.89 units/mm Hg; P = 0.03) were smaller in patients versus control subjects. Hypoxic vasodilation in the forearm was also attenuated (-0.05 +/- 0.09 vs. -0.10 +/- 0.09 unit/% saturation; P = 0.04). Hypercapnia did not elicit forearm vasodilation in either group. Twelve weeks of continuous positive airway pressure treatment enhanced hypoxic vasodilation in the cerebral circulation (-0.83 +/- 0.32 vs. -0.46 +/- 0.29 units/% saturation; P = 0.01) and forearm (-0.19 +/- 0.15 vs. -0.02 +/- 0.08 units/% saturation; P = 0.003), and hypercapnic vasodilation in the brain showed a trend toward improvement (2.24 +/- 0.78 vs. 1.76 +/- 0.64 units/mm Hg; P = 0.06).Vasodilator responses to chemical stimuli in the cerebral circulation and the forearm are impaired in many patients with obstructive sleep apnea. Some of these impairments can be improved with continuous positive airway pressure.
Project description:We exploited two-photon microscopy and Doppler optical coherence tomography to examine the cerebral blood flow and tissue pO2 response to forced treadmill exercise in awake mice. To our knowledge, this is the first study performing both direct measure of brain tissue pO2 during acute forced exercise and underlying microvascular response at capillary and non-capillary levels. We observed that cerebral perfusion and oxygenation are enhanced during running at 5 m/min compared to rest. At faster running speeds (10 and 15 m/min), decreasing trends in arteriolar and capillary flow speed were observed, which could be due to cerebral autoregulation and constriction of arterioles in response to blood pressure increase. However, tissue pO2 was maintained, likely due to an increase in RBC linear density. Higher cerebral oxygenation at exercise levels 5-15 m/min suggests beneficial effects of exercise in situations where oxygen delivery to the brain is compromised, such as in aging, atherosclerosis and Alzheimer Disease.
Project description:The gold standard for noninvasive blood pressure (BP) measurement, the Doppler technique, does not provide systolic blood pressure (SBP) and diastolic blood pressure (DBP) and may limit therapy outcomes. To improve patient care, we tested specifically designed experimental BP (ExpBP) monitor and the Doppler technique by comparing noninvasive measures to the intraarterial (I-A) BP in 31 patients with end-stage heart failure (4 females) 2.6?±?3.4 days post-LVAD implantation (20 HeartMate II and 11 HeartWare). Bland-Altman plots revealed that the ExpBP monitor overestimated mean arterial pressure (MAP) by 1.2 (4.8) mm Hg (mean difference [standard deviation]), whereas the Doppler by 6.7 (5.8) mm Hg. The ExpBP SBP was overestimated by 0.8 (6.1) mm Hg and DBP by 1.9 (5.3) mm Hg compared with the respective I-A pressures. Both techniques achieved similar measurement reliability. In the measurement "success rate" expressed as a frequency (percent) of readable BP values per measurement attempts, Doppler accomplished 100% vs. 97%, 97%, and 94% of successful detections of MAP, SBP, and DBP provided by the ExpBP monitor. The ExpBP monitor demonstrated higher accuracy in the MAP assessment than the Doppler in addition to providing SBP and DBP in majority of subjects. Improved BP control may help to mitigate related neurologic adverse event rates.
Project description:Effects of chronic hypoxia on hemodynamic response to sensory stimulation were investigated. Using laser-Doppler flowmetry, change in cerebral blood flow (CBF) was measured in awake mice, which were housed in a hypoxic chamber (8% O₂) for 1 month. The degree of increase in CBF evoked by sensory stimulation was gradually decreased over 1 month of chronic hypoxia. No significant reduction of increase in CBF induced by hypercapnia was observed during 1 month. Voltage-sensitive dye (VSD) imaging of the somatosensory cortex showed no significant decrease in neural activation over 1 month, indicating that the reduction of increase in CBF to sensory stimulation was not caused by cerebrovascular or neural dysfunction. The simulation study showed that, when effective diffusivity for oxygen in the capillary bed (D) value increases by chronic hypoxia due to an increase in capillary blood volume, an increase in the cerebral metabolic rate of oxygen utilization during neural activation can occur without any increase in CBF. Although previous study showed no direct effects of acute hypoxia on CBF response, our finding showed that hemodynamic response to neural activation could be modified in response to a change in their balance to energy demand using chronic hypoxia experiments.
Project description:The development of memory B cells takes place in germinal centers (GC) of lymphoid follicles where antigen-driven lymphocytes undergo somatic hypermutation and affinity selection, presumably under the influence of helper T cells. However, the mechanisms that drive this complex response are not well understood. We explored the relationship between GC formation and the onset of hypermutation in response to the hapten phosphorylcholine (PC) coupled to antigenic proteins in mice bearing different frequencies of CD4+ T cells. PC-reactive GC were identified by staining frozen splenic sections with peanut agglutinin (PNA) and with monoclonal Abs against AB1-2, a dominant idiotope of T15+ anti-PC antibody. The nucleotide sequences of rearranged T15 VH1 genes were determined from polymerase chain reaction amplifications of genomic DNA from microdissected GC B cells. T15+ GC became fully developed by day 6-7 after primary immunization of euthymic mice with either PC-keyhole limpet hemocyanin (KLH) or PC-chicken gamma globulin (CGG). Yet the VH1 gene segments recovered from the primary GC as late as day 10-14 had low numbers of mutations, in contrast to responses to the haptens nitrophenyl or oxazolone that sustain high levels of hypermutation after GC formation. PC-reactive B cells proliferate in histologically typical GC for considerable periods with no or little somatic hypermutation; the signals for GC formation are independent of those for the activation of hypermutation. We then examined GC 7 d after secondary immunization with PC-KLH in euthymic mice, in nu/nu mice reconstituted with limited numbers of normal CD4+ cells before priming (CD4(+)-nu/nu) and in nu/nu mice. All of these animals develop T15+ GC after antigen priming, however, the patterns of V gene mutations in the secondary GC reflected the levels of CD4+ cells present during the primary response. VDJ sequences from secondary GC of euthymic mice were heavily mutated, but most of these mutations were shared among all related (identical VDJ joints) sequences suggesting the proliferation of mutated, memory B cells, with little de novo somatic hypermutation. In contrast, the patterns of V gene diversity in secondary GC from CD4(+)-nu/nu mice suggested that there was ongoing mutation and clonal diversification during the first week after rechallenge. The secondary GC from T cell-deficient, nu/nu mice showed little evidence for mutational and/or recombinational diversity of T15+ B cells. We conclude that the participation of CD4+ helper cells is required for full activation of the mutator in GC and takes place in a dose-dependent fashion.
Project description:This study aimed to examine the cortical microvessel diameter response to hypercapnia in misery perfusion using two-photon laser scanning microscopy (TPLSM). We evaluated whether the vascular response to hypercapnia could represent the cerebrovascular reserve. Cerebral blood flow (CBF) during normocapnia and hypercapnia was measured by laser-Doppler flowmetry through cranial windows in awake C57/BL6 mice before and at 1, 7, 14, and 28 days after unilateral common carotid artery occlusion (UCCAO). Diameters of the cortical microvessels during normocapnia and hypercapnia were also measured by TPLSM. Cerebral blood flow and the vascular response to hypercapnia were decreased after UCCAO. Before UCCAO, vasodilation during hypercapnia was found primarily in arterioles (22.9%±3.5%). At 14 days after UCCAO, arterioles, capillaries, and venules were autoregulatorily dilated by 79.5%±19.7%, 57.2%±32.3%, and 32.0%±10.8%, respectively. At the same time, the diameter response to hypercapnia in arterioles was significantly decreased to 1.9%±1.5%. A significant negative correlation was observed between autoregulatory vasodilation and the diameter response to hypercapnia in arterioles. Our findings indicate that arterioles play main roles in both autoregulatory vasodilation and hypercapnic vasodilation, and that the vascular response to hypercapnia can be used to estimate the cerebrovascular reserve.