Capillary Red Blood Cell Flux Research Articles

Neuroinflammation increases oxygen extraction in a mouse model of Alzheimer’s disease

BackgroundNeuroinflammation, impaired metabolism, and hypoperfusion are fundamental pathological hallmarks of early Alzheimer’s disease (AD). Numerous studies have asserted a close association between neuroinflammation and disrupted cerebral energetics. During AD progression and other neurodegenerative disorders, a persistent state of chronic neuroinflammation reportedly exacerbates cytotoxicity and potentiates neuronal death. Here, we assessed the impact of a neuroinflammatory challenge on metabolic demand and microvascular hemodynamics in the somatosensory cortex of an AD mouse model.MethodsWe utilized in vivo 2-photon microscopy and the phosphorescent oxygen sensor Oxyphor 2P to measure partial pressure of oxygen (pO2) and capillary red blood cell flux in cortical microvessels of awake mice. Intravascular pO2 and capillary RBC flux measurements were performed in 8-month-old APPswe/PS1dE9 mice and wildtype littermates on days 0, 7, and 14 of a 14-day period of lipopolysaccharide-induced neuroinflammation.ResultsBefore the induced inflammatory challenge, AD mice demonstrated reduced metabolic demand but similar capillary red blood cell flux as their wild type counterparts. Neuroinflammation provoked significant reductions in cerebral intravascular oxygen levels and elevated oxygen extraction in both animal groups, without significantly altering red blood cell flux in capillaries.ConclusionsThis study provides evidence that neuroinflammation alters cerebral oxygen demand at the early stages of AD without substantially altering vascular oxygen supply. The results will guide our understanding of neuroinflammation’s influence on neuroimaging biomarkers for early AD diagnosis.

Differential reductions in the capillary red-blood-cell flux between retina and brain under chronic global hypoperfusion.

It has been hypothesized that abnormal microcirculation in the retina might predict the risk of ischemic damages in the brain. Direct comparison between the retinal and the cerebral microcirculation using similar animal preparation and under similar experimental conditions would help test this hypothesis. We investigated capillary red-blood-cell (RBC) flux changes under controlled conditions and bilateral-carotid-artery-stenosis (BCAS)-induced hypoperfusion, and then compared them with our previous measurements performed in the brain. We measured capillary RBC flux in mouse retina with two-photon microscopy using a fluorescence-labeled RBC-passage approach. Key physiological parameters were monitored during experiments to ensure stable physiology. We found that under the controlled conditions, capillary RBC flux in the retina was much higher than in the brain (i.e., cerebral cortical gray matter and subcortical white matter), and that BCAS induced a much larger decrease in capillary RBC flux in the retina than in the brain. We demonstrated a two-photon microscopy-based technique to efficiently measure capillary RBC flux in the retina. Since cerebral subcortical white matter often exhibits early pathological developments due to global hypoperfusion, our results suggest that retinal microcirculation may be utilized as an early marker of brain diseases involving global hypoperfusion.

Measurements of cerebral microvascular blood flow, oxygenation, and morphology in a mouse model of whole-brain irradiation-induced cognitive impairment by two-photon microscopy and optical coherence tomography: evidence for microvascular injury in the cerebral white matter.

Whole-brain irradiation (WBI, also known as whole-brain radiation therapy) is a mainstay treatment modality for patients with multiple brain metastases. It is also used as a prophylactic treatment for microscopic tumors that cannot be detected by magnetic resonance imaging. WBI induces a progressive cognitive decline in ~ 50% of the patients surviving over 6months, significantly compromising the quality of life. There is increasing preclinical evidence that radiation-induced injury to the cerebral microvasculature and accelerated neurovascular senescence plays a central role in this side effect of WBI. To better understand this side effect, male C57BL/6 mice were first subjected to a clinically relevant protocol of fractionated WBI (5Gy, two doses per week, for 4weeks). Nine months post the WBI treatment, we applied two-photon microscopy and Doppler optical coherence tomography to measure capillary red-blood-cell (RBC) flux, capillary morphology, and microvascular oxygen partial pressure (PO2) in the cerebral somatosensory cortex in the awake, head-restrained, WPI-treated mice and their age-matched controls, through a cover-glass-sealed chronic cranial window. Thanks to the extended penetration depth with the fluorophore - Alexa680, measurements of capillary blood flow properties (e.g., RBC flux, speed, and linear density) in the cerebral subcortical white matter were enabled. We found that the WBI-treated mice exhibited a significantly decreased capillary RBC flux in the white matter. WBI also caused a significant reduction in capillary diameter, as well as a large (although insignificant) reduction in segment density at the deeper cortical layers (e.g., 600-700μm), while the other morphological properties (e.g., segment length and tortuosity) were not obviously affected. In addition, we found that PO2 measured in the arterioles and venules, as well as the calculated oxygen saturation and oxygen extraction fraction, were not obviously affected by WBI. Lastly, WBI was associated with a significant increase in the erythrocyte-associated transients of PO2, while the changes of other cerebral capillary PO2 properties (e.g., capillary mean-PO2, RBC-PO2, and InterRBC-PO2) were not significant. Collectively, our findings support the notion that WBI results in persistent cerebral white matter microvascular impairment, which likely contributes to the WBI-induced brain injury and cognitive decline. Further studies are warranted to assess the WBI-induced changes in brain tissue oxygenation and malfunction of the white matter microvasculature as well.

Sex Difference in Capillary Reperfusion After Transient Middle Cerebral Artery Occlusion in Diabetic Mice.

Type 2 diabetes (DM2) exacerbates stroke injury, reduces efficacy of endovascular therapy, and worsens long-term functional outcome. Sex differences exist in stroke incidence, response to therapy, poststroke microvascular dysfunction, and functional recovery. In this study, we tested the hypotheses that poor outcome after stroke in the setting of DM2 is linked to impaired microvascular tissue reperfusion and that male and female DM2 mice exhibit different microvascular reperfusion response after transient middle cerebral artery occlusion (MCAO). Transient MCAO was induced for 60 minutes using an intraluminal filament in young adult DM2 and nondiabetic control male and female mice. Capillary flux in deep cortical layers was assessed using optical coherence tomography-based optical microangiography (OMAG), and associated regional brain infarct size was evaluated by hematoxylin and eosin staining. Compared to baseline, MCAO reduced absolute capillary red blood cell flux by 84% at 24 hours post-MCAO in male DM2 (P<0.001) but not male control mice. When normalized to pre-MCAO baseline, red blood cell flux 24 hours after stroke was 64% lower in male DM2 mice than male nondiabetic controls (P<0.01). In females, MCAO decreased capillary flux by 48% at 24 hours post-MCAO compared with baseline in DM2 (P<0.05) but not in control mice. Red blood cell flux of female DM2 mice did not differ from that of nondiabetic controls either before or 24 hours after MCAO. Furthermore, normalized capillary flux 24 hours after MCAO failed to differ between female DM2 mice and nondiabetic controls. Concomitantly, male but not female DM2 mice experienced 25% larger infarct in caudate-putamen versus respective nondiabetic controls (P<0.05). DM2 impairs capillary perfusion and exacerbates ischemic deep brain injury in male but not female young adult mice. Premenopausal females appear to be protected against DM2-related capillary dysfunction and brain injury.

GPR39 Knockout Worsens Microcirculatory Response to Experimental Stroke in a Sex-Dependent Manner.

No current treatments target microvascular reperfusion after stroke, which can contribute to poor outcomes even after successful clot retrieval. The G protein-coupled receptor GPR39 is expressed in brain peri-capillary pericytes, and has been implicated in microvascular regulation, but its role in stroke is unknown. We tested the hypothesis that GPR39 plays a protective role after stroke, in part due to preservation of microvascular perfusion. We generated GPR39 knockout (KO) mice and tested whether GPR39 gene deletion worsens capillary blood flow and exacerbates brain injury and functional deficit after focal cerebral ischemia. Stroke was induced in male and female GPR39 KO and WT littermates by 60-min middle cerebral artery occlusion (MCAO). Microvascular perfusion was assessed via capillary red blood cell (RBC) flux in deep cortical layers in vivo using optical microangiography (OMAG). Brain injury was assessed by measuring infarct size by 2,3,5-triphenyltetrazolium chloride staining at 24h or brain atrophy at 3weeks after ischemia. Pole and cylinder behavior tests were conducted to assess neurological function deficit at 1 and 3weeks post-stroke. Male but not female GPR39 KO mice exhibited larger infarcts and lower capillary RBC flux than WT controls after stroke. Male GPR39 KO mice also exhibited worse neurologic deficit at 1week post-stroke, though functional deficit disappeared in both groups by 3weeks. GPR39 deletion worsens brain injury, microvascular perfusion, and neurological function after experimental stroke. Results indicate that GPR39 plays a sex-dependent role in re-establishing microvascular flow and limiting ischemic brain damage after stroke.

Quantifying Intestinal Capillary Oxygenation Using Two‐photon Phosphorescence Lifetime Microscopy

Impaired oxygen delivery reduces the diversity of the gut microbiota1,2. Quantifying the microvascular oxygenation in the small intestines may help us better understand progression of the intestinal diseases such as inflammatory bowel disease (IBD), their relation with the changes in the gut microbiota, as well as implications to the microbiota–gut–brain axis. To start addressing these questions, we applied two-photon phosphorescence lifetime microscopy (2PLM) to quantify partial pressure of oxygen (pO2) and Red Blood Cell (RBC) flux in the capillary plexus through entire cecum wall in healthy, 2-month-old, female C57BL6 mice. Mice were anesthetized with isoflurane mixed with air (3% during surgery and 2% during imaging). The animals were breathing spontaneously through a loose mask. A small incision was made into the abdomen and the cecum was gently pulled out and positioned into a custom-made chamber with a cover glass on top. The exposed intestine was kept moist with a warmed 1x phosphate buffered saline (PBS) solution. Both core body temperature and temperature over the cecum were maintained at 37 °C. Experiments were terminated when the peristaltic movement prevented microscopic measurements. We measured pO2 and RBC flux during normoxia (n=4 mice, mean pO2 = 60 ± 6 mmHg, mean capillary RBC flux = 23 ± 8 RBCs/s) and hyperoxia (100% O2 inhalation, n=3, mean pO2= 120 ± 15 mmHg, mean capillary RBC flux = 31 ± 17 RBCs/s), as a function of depth in the cecum wall from 0 to 120 µm. Our results show expected significant increase in pO2 in response to hyperoxia, small capillary mean RBC flux values, and approximately uniform capillary pO2 across the thickness of the cecum wall during normoxia. Future steps include investigation of the capillary oxygenation and RBC flux in pathological conditions and in relation with the diversity of the gut microbiome.

Two-photon microscopic imaging of capillary red blood cell flux in mouse brain reveals vulnerability of cerebral white matter to hypoperfusion

Despite the importance of understanding the regulation of microvascular blood flow in white matter, no data on subcortical capillary blood flow parameters are available, largely due to the lack of appropriate imaging methods. To address this knowledge gap, we employed two-photon microscopy using a far-red fluorophore Alexa680 and photon-counting detection to measure capillary red blood cell (RBC) flux in both cerebral gray and white matter, in isoflurane-anesthetized mice. We have found that in control animals, baseline capillary RBC flux in the white matter was significantly higher than in the adjacent cerebral gray matter. In response to mild hypercapnia, RBC flux in the white matter exhibited significantly smaller fractional increase than in the gray matter. Finally, during global cerebral hypoperfusion, RBC flux in the white matter was reduced significantly in comparison to the controls, while RBC flux in the gray matter was preserved. Our results suggest that blood flow in the white matter may be less efficiently regulated when challenged by physiological perturbations as compared to the gray matter. Importantly, the blood flow in the white matter may be more susceptible to hypoperfusion than in the gray matter, potentially exacerbating the white matter deterioration in brain conditions involving global cerebral hypoperfusion.

Spatio-temporal dynamics of cerebral capillary segments with stalling red blood cells

Optical coherence tomography (OCT) allows label-free imaging of red blood cell (RBC) flux within capillaries with high spatio-temporal resolution. In this study, we utilized time-series OCT-angiography to demonstrate interruptions in capillary RBC flux in mouse brain in vivo. We noticed ∼7.5% of ∼200 capillaries had at least one stall in awake mice with chronic windows during a 9-min recording. At any instant, ∼0.45% of capillaries were stalled. Average stall duration was ∼15 s but could last over 1 min. Stalls were more frequent and longer lasting in acute window preparations. Further, isoflurane anesthesia in chronic preparations caused an increase in the number of stalls. In repeated imaging, the same segments had a tendency to stall again over a period of one month. In awake animals, functional stimulation decreased the observance of stalling events. Stalling segments were located distally, away from the first couple of arteriolar-side capillary branches and their average RBC and plasma velocities were lower than nonstalling capillaries within the same region. This first systematic analysis of capillary RBC stalls in the brain, enabled by rapid and continuous volumetric imaging of capillaries with OCT-angiography, will lead to future investigations of the potential role of stalling events in cerebral pathologies.

Recovery dynamics of skeletal muscle oxygen uptake during the exercise off-transient

The time course of muscle .V(O2) recovery from contractions (i.e., muscle .V(O2) off-kinetics), measured directly at the site of O(2) exchange, i.e., in the microcirculation, is unknown. Whereas biochemical models based upon creatine kinase flux rates predict slower .V(O2) off- than on-transients [Kushmerick, M.J., 1998. Comp. Biochem. Physiol. B: Biochem. Mol. Biol.], whole muscle .V(O2) data [Krustrup, et al. J. Physiol.] suggest on-off symmetry. We tested the hypothesis that the slowed recovery blood flow (Qm) kinetics profile in the spinotrapezius muscle [Ferreira et al., 2006. J. Physiol.] was associated with a slowed muscle .V(O2) recovery compared with that seen at the onset of contractions (time constant, tau approximately 23s, Behnke et al., 2002. Resp. Physiol.), i.e., on-off asymmetry. Measurements of capillary red blood cell flux and microvascular pressure of O(2) (P(O2) mv) were combined to resolve the temporal profile of muscle .V(O2) across the moderate intensity contractions-to-rest transition. Muscle .V(O2) decreased from an end-contracting value of 7.7+/-0.2 ml/100 g/min to 1.7+/-0.1 ml/100g/min at the end of the 3 min recovery period, which was not different from pre-stimulation .V(O2). Contrary to our hypothesis, muscle .V(O2) in recovery began to decrease immediately (i.e., time delay <2s) and demonstrated rapid first-order kinetics (tau, 25.5+/-2.6s) not different (i.e., symmetrical to) to those during the on-transient. This resulted in a systematic increase in microvascular P(O2) during the recovery from contractions. The slowed Qm kinetics in recovery serves to elevate the Qm/.V(O2) ratio and thus microvascular P(O2) . Whether this Qm response is obligatory to the rapid muscle .V(O2) kinetics and hence speeds the repletion of high-energy phosphates by maximizing conductive and diffusive O(2) flux is an important question that awaits resolution.

Skeletal Muscle Oxygen Uptake Dynamics during the Exercise Off‐Transient

The time course of muscle V̇O2 recovery (i.e., muscle V̇O2 kinetics) from contractions, measured at the site of O2 exchange i.e., in the microcirculation, is unknown. It is known that, in the rat spinotrapezius muscle, blood flow (Qm) kinetics are slower in recovery (time constant, t ~41 s) than at the onset of contractions (t ~17 s, Ferreira et al. J. Physiol. 2006).PurposeWe tested the hypothesis that this Qm kinetics profile was associated with a slowed m V̇O2 recovery compared with that seen at the onset of contractions (t ~23 s, Behnke et al. Resp. Physiol. 2002).MethodsMeasurements of capillary red blood cell flux and microvascular PO2 (PO2) were combined to resolve the time course of muscle V̇O2across the moderate intensity contractions‐to‐rest transition.ResultsContrary to our hypothesis, in recovery muscle V̇O2 began to decrease immediately (i.e., time delay 0 s in all but one animal) and demonstrated rapid first order‐kinetics (t 22.4 ± 2.1 s).ConclusionsThe slowed Qm kinetics in recovery serves to elevate the Qm/ V̇O2 ratio and thus microvascular PO2. Whether this Qm response is obligatory to the rapid muscle V̇O2 kinetics and hence speeds the repletion of high‐energy phosphates by maximizing conductive and diffusive O2 flux is an important question that awaits resolution. Support NIH AG031327.

The effects of aging on capillary hemodynamics in contracting rat spinotrapezius muscle

Advancing age alters the structural and functional determinants of convective and diffusive muscle oxygen (O 2) flux. However, capillary red blood cell (RBC) hemodynamics have not been investigated during contractions in muscles of old animals. Therefore, we tested the hypothesis that aging induces significant capillary hemodynamic alterations during electrically-induced contractions in the spinotrapezius muscle of old Fischer 344 × Brown Norway rats when compared to younger counterparts. The spinotrapezius muscle was observed via intravital microscopy under both resting and contracting conditions in 8 old (O: 26–30 months) and 5 young (Y: 6–8 months) animals. Capillary RBC flux ( F RBC), velocity ( V RBC), hematocrit ( H CAP), and lineal F RBC ( F RBC × capillary lineal density) were determined in all visible capillaries and fields at rest and during the steady-state of muscle contractions. At rest F RBC and V RBC were elevated in O compared to Y rats, while there was no difference in H CAP or lineal F RBC between groups. During the contracting steady-state, Δ F RBC (Y: 28.8 ± 7.7, O: − 2.9 ± 1.4 cells/s), Δ V RBC (Y: 253 ± 68, O: − 4 ± 15 μm/s), Δ H CAP (Y: 0.02 ± 0.02, O: − 0.03 ± 0.01), and the Δlineal F RBC (Y: 892 ± 255, O: − 24 ± 30 cells/s/mm) were all lower ( P < 0.05) in O compared to Y rats. These results indicate that despite maintained total perfusive and diffusive O 2 transport at rest, advancing age results in significant impairments in capillary hemodynamics during electrically-induced contractions. These alterations likely contribute to the mechanisms responsible for the reduced exercise capacity commonly found in elderly populations.

The Effects of Aging on Microcirculatory Oxygen Delivery (QO2) in Contracting Rat Spinotrapezius Muscle

Advancing age alters both the structural and functional determinants of convective muscle oxygen (O2) flux. However, capillary red blood cell (RBC) hemodynamics have not been investigated during contractions in muscles of older animals.Purpose:To test the hypothesis that, during muscle contractions, old rats exhibit capillary RBC hemodynamics that constrain convective muscle O2 flux.Methods:The spinotrapezius muscle was observed via intravital microscopy in 8 old (O: 26–30 months) and 5 young (Y: 6–8 months) Fischer 344 x Brown Norway rats. Electrodes elicited 1‐Hz (6–8 volts) contractions for 3 minutes. Capillary RBC flux (FRBC) and the lineal density of flowing capillaries (LD) were measured at rest and during contractions. QO2m was calculated as FRBC x LD.Results:At rest, FRBC was elevated (Y: 23 ± 3, O: 34 ± 3 cells/s, P &lt; 0.05) and LD was reduced (Y: 30 ± 1, O: 24 ± 1 capillaries/mm, P &lt; 0.05) in O compared to Y rats, resulting in a similar QO2m (Y: 723 ± 153, O: 773 ± 86 cells/s/mm). However, during contractions QO2m was reduced in O muscles (Y: 1615 ± 472, O: 781 ± 120 cells/s/mm, P &lt; 0.05). In contrast to Y, during contractions O muscles exhibited stoppage of flow in some capillaries where continuous flow was observed at rest.Conclusion:These data indicate that capillary convective O2 flux is severely impaired in muscles of older animals.

Effects of eccentric exercise on microcirculation and microvascular oxygen pressures in rat spinotrapezius muscle

A single bout of eccentric exercise results in muscle damage, but it is not known whether this is correlated with microcirculatory dysfunction. We tested the following hypotheses in the spinotrapezius muscle of rats either 1 (DH-1; n = 6) or 3 (DH-3; n = 6) days after a downhill run to exhaustion (90-120 min; -14 degrees grade): 1) in resting muscle, capillary hemodynamics would be impaired, and 2) at the onset of subsequent acute concentric contractions, the decrease of microvascular O(2) pressure (Pmv(o(2))), which reflects the dynamic balance between O(2) delivery and O(2) utilization, would be accelerated compared with control (Con, n = 6) rats. In contrast to Con muscles, intravital microscopy observations revealed the presence of sarcomere disruptions in DH-1 and DH-3 and increased capillary diameter in DH-3 (Con: 5.2 +/- 0.1; DH-1: 5.1 +/- 0.1; DH-3: 5.6 +/- 0.1 mum; both P < 0.05 vs. DH-3). At rest, there was a significant reduction in the percentage of capillaries that sustained continuous red blood cell (RBC) flux in both DH running groups (Con: 90.0 +/- 2.1; DH-1: 66.4 +/- 5.2; DH-3: 72.9 +/- 4.1%, both P < 0.05 vs. Con). Capillary tube hematocrit was elevated in DH-1 but reduced in DH-3 (Con: 22 +/- 2; DH-1: 28 +/- 1; DH-3: 16 +/- 1%; all P < 0.05). Although capillary RBC flux did not differ between groups (P > 0.05), RBC velocity was lower in DH-1 compared with Con (Con: 324 +/- 43; DH-1: 212 +/- 30; DH-3: 266 +/- 45 mum/s; P < 0.05 DH-1 vs. Con). Baseline Pmv(O(2)) before contractions was not different between groups (P > 0.05), but the time constant of the exponential fall to contracting Pmv(O(2)) values was accelerated in the DH running groups (Con: 14.7 +/- 1.4; DH-1: 8.9 +/- 1.4; DH-3: 8.7 +/- 1.4 s, both P < 0.05 vs. Con). These findings are consistent with the presence of substantial microvascular dysfunction after downhill eccentric running, which slows the exercise hyperemic response at the onset of contractions and reduces the Pmv(O(2)) available to drive blood-muscle O(2) delivery.

Microcirculatory effects of hypoxic and hypercapnic vasoconstriction in frog skin

The effects in frog (albino Xenopus laevis) skin of hypoxic and hypercapnic vasoconstriction on the following microcirculatory parameters were determined: capillary red blood cell flux, capillary red blood cell velocity, perfused capillary density, lineal red blood cell density, and the temporal heterogeneity of capillary red blood cell velocities. All of these parameters affect the gas exchange characteristics of respiratory organs. Measurements were made by fluorescent video microscopy of a 1.5-cm2 region of skin exposed to different gas mixtures (air, O2, N2, 5% CO2-95% air, 5% CO2-95% N2). N2 caused red blood cell flux and velocity to fall to 52 +/- 10% (P < 0.05) and 47 +/- 10% (P < 0.01), respectively, of those values during air exposure. Five percent CO2 caused capillary red blood cell flux and velocity to decrease by 51 +/- 11% (P < 0.05) and 43 +/- 11% (P < 0.01), respectively. Fluxes (P < 0.01) and velocities (P < 0.01) were also less with 5% CO2-95% N2 than with air. There were no significant differences in flux and velocity between N2, 5% CO2-95% air and 5% CO2-95% N2 (P > 0.1). There was no significant difference in flux or in velocity between O2 and air (P > 0.1). Gas composition had no significant effect on lineal red blood cell density (P > 0.35) or the density of perfused capillaries (P > 0.22). The heterogeneity of cell velocities was significantly greater with N2 than with the other gases (P < 0.01). There was no significant difference in red blood cell velocity heterogeneity between the other gases (P > 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of heterogeneous oxygen delivery on the oxygen distribution in skeletal muscle

Calculations of the oxygen distribution in resting and contracting skeletal muscle are presented, based on a mathematical model and experimental data obtained on the hamster cremaster muscle [Klitzman et al., Microvasc. Res. 25:108–131 (1983)]. The model considers a slab of tissue penetrated by a regular square array of capillaries with concurrent flow. The intracapillary resistance to oxygen transport is neglected. The capillary red blood cell flux and capillary inlet oxygen tension are assumed random variables following certain probability distributions. The sensitivity of the tissue and intracapillary PO 2 to variations of these probability distributions are investigated. The mean tissue PO 2 decreases as the dispersion of the random variables increases, provided that their mean values remain constant. Hypoxic regions appear gradually, especially in the case of contracting muscle, as the dispersion increases. The effect of the number of capillaries in the sample on the resultant distribution of oxygen is studied systematically. The calculated tissue PO 2 histograms are compared with previously reported PO 2 distributions obtained experimentally for resting and contracting skeletal muscle.