Acceleration of the Development of Microcirculation Embolism in the Brain due to Capillary Narrowing.

BACKGROUND: Cerebral microvascular obstruction is critically involved in recurrent stroke and decreased cerebral blood flow with age. The obstruction must occur in the capillary with a greater resistance to perfusion pressure through the microvascular networks. However, little is known about the relationship between capillary size and embolism formation. This study aimed to determine whether the capillary lumen space contributes to the development of microcirculation embolism. METHODS: To spatiotemporally manipulate capillary diameters in vivo, transgenic mice expressing the light-gated cation channel protein ChR2 (channelrhodopsin-2) in mural cells were used. The spatiotemporal changes in the regional cerebral blood flow in response to the photoactivation of ChR2 mural cells were first characterized using laser speckle flowgraphy. Capillary responses to optimized photostimulation were then examined in vivo using 2-photon microscopy. Finally, microcirculation embolism due to intravenously injected fluorescent microbeads was compared under conditions with or without photoactivation of ChR2 mural cells. RESULTS: Following transcranial photostimulation, the stimulation intensity-dependent decrease in cerebral blood flow centered at the irradiation was observed (14%-49% decreases relative to the baseline). The cerebrovascular response to photostimulation showed significant constriction of the cerebral arteries and capillaries but not of the veins. As a result of vasoconstriction, a temporal stall of red blood cell flow occurred in the capillaries of the venous sides. The 2-photon excitation of a single ChR2 pericyte demonstrated the partial shrinkage of capillaries (7% relative to the baseline) around the stimulated cell. With the intravenous injection of microbeads, the occurrence of microcirculation embolism was significantly enhanced (11% increases compared to the control) with photostimulation. CONCLUSIONS: Capillary narrowing increases the risk of developing microcirculation embolism in the venous sides of the cerebral capillaries.

Neurosurgical intraoperative ultrasonography using contrast enhanced superb microvascular imaging -vessel density and appearance time of the contrast agent.

BACKGROUND: Ultrasonography (US) provides real-time information on structures within the skull during neurosurgical operations. Superb microvascular imaging (SMI) is the latest imaging technique for detecting very low-velocity flow with minimal motion artifacts, and we have reported on this technique for intraoperative US monitoring. We combined SMI with administration of contrast agent to obtain detailed information during neurosurgical operations. MATERIALS AND METHODS: Twenty patients diagnosed with brain tumor (10 meningiomas, 5 glioblastomas, 2 hemangioblastomas, 1 schwannoma, 1 malignant lymphoma, 1 brain abscess) underwent neurosurgery under US with SMI and contrast agent techniques. Vessel density and appearance time following contrast administration were analyzed. RESULTS: Flow in numerous vessels was not visualized by SMI alone, but appeared following injection of contrast agent in all cases. Flow in tumors was drastically enhanced by contrast agent in schwannoma, hemangioblastoma and meningioma, compared to normal brain tissue. Flows in the dilated and bent vessels of glioblastoma were also enhanced, although flow in hypoechoic lymphoma remained inconspicuous. The characteristics of tumor vessels were clearly visualized and tumor borders were demonstrated by the difference between tumor flow and brain flow, by the increased tumor vessel density and decreased appearance time of contrast agent compared to normal brain vessels. CONCLUSIONS: The combination of SMI and contrast agent techniques for intraoperative US monitoring could provide innovative flow images of tumor and normal brain. The neurosurgeon obtains information about tumor flow and tumor borderline before tumor resection.

Automated capillary flow segmentation and mapping for nailfold video capillaroscopy.

OBJECTIVE: This study aimed to develop an automated image analysis method for segmentation and mapping of capillary flow dynamics captured using nailfold video capillaroscopy (NVC). Methods were applied to compare capillary flow structures and dynamics between young and middle-aged healthy controls. METHODS: NVC images were obtained in a resting state, and a region of the vessel in the image was extracted using a conventional U-Net neural network. The approximate length, diameter, and radius of the curvature were calculated automatically. Flow speed and its fluctuation over time were mapped using the Radon transform and frequency spectrum analysis from the kymograph image created along the vessel's centerline. RESULTS: The diameter of the curve segment (14.4 µm and 13.0 µm) and the interval of two straight segments (13.7 µm and 32.1 µm) of young and middle-aged subjects, respectively, were significantly different. Faster flow was observed in older subjects (0.48 mm/s) than in younger subjects (0.26 mm/s). The power spectral analysis revealed a significant correlation between the high-frequency power spectrum and the flow speed. CONCLUSIONS: The present method allows a spatiotemporal characterization of capillary morphology and flow dynamics with NVC, allowing a wide application such as large-scale health assessment.

Three-dimensional microvascular network reconstruction from in vivo images with adaptation of the regional inhomogeneity in the signal-to-noise ratio.

OBJECTIVE: Quantification of angiographic images with two-photon laser scanning fluorescence microscopy (2PLSM) relies on proper segmentation of the vascular images. However, the images contain inhomogeneities in the signal-to-noise ratio (SNR) arising from regional effects of light scattering and absorption. The present study developed a semiautomated quantification method for volume images of 2PLSM angiography by adjusting the binarization threshold according to local SNR along the vessel centerlines. METHODS: A phantom model made with fluorescent microbeads was used to incorporate a region-dependent binarization threshold. RESULTS: The recommended SNR for imaging was found to be 4.2-10.6 that provide the true size of imaged objects if the binarization threshold was fixed at 50% of SNR. However, angiographic images in the mouse cortex showed variable SNR up to 45 over the depths. To minimize the errors caused by variable SNR and a spatial extent of the imaged objects in an axial direction, the microvascular networks were three-dimensionally reconstructed based on the cross-sectional diameters measured along the vessel centerline from the XY-plane images with adapted binarization threshold. The arterial volume was relatively constant over depths of 0-500 µm, and the capillary volume (1.7% relative to the scanned volume) showed the larger volumes than the artery (0.8%) and vein (0.6%). CONCLUSIONS: The present methods allow consistent segmentation of microvasculature by adapting the local inhomogeneity in the SNR, which will be useful for quantitative comparison of the microvascular networks, such as under disease conditions where SNR in the 2PLSM images varies over space and time.

Mapping of flow velocity using spatiotemporal changes in time-intensity curves from indocyanine green videoangiography.

OBJECTIVE: The present study developed an image-based analysis method that uses indocyanine green videoangiography (ICG-VA) to measure flow velocity in the arteries and veins of the cortical surface in patients undergoing neurosurgery. METHODS: MATLAB-based code was used to correct motion artifacts in the ICG-VA and determine the time-intensity curve of the ICG. The slope of the initial increase in ICG intensity following the bolus injection was measured and normalized using the predicted input function in the imaging field. Flow velocity over a certain distance determined by the user was measured based on a time shift of the time-intensity curves along the centerline of the vessels. RESULTS: The normalized slope of ICG intensity represented the expected differences in the flow velocity among the artery (0.67 ± 0.05 s-1 ), parenchymal tissue (0.49 ± 0.10 s-1 ), and vein (0.44 ± 0.11 s-1 ). The flow velocities measured along the vessel centerline were 2.5 ± 1.1 cm/s and 1.1 ± 0.3 cm/s in the arteries (0.5 ± 0.2 mm in diameter) and veins (0.6 ± 0.2 mm in diameter), respectively. CONCLUSIONS: An image-based analysis method for ICG-VA was developed to map the expected differences in the flow velocity based on the rising slope of ICG intensity and to measure the absolute flow velocities using the flexible zone and cross-correlation methods.

Vascular permeability of skeletal muscle microvessels in rat arterial ligation model: in vivo analysis using two-photon laser scanning microscopy.

Peripheral artery disease (PAD) in the lower limb compromises oxygen supply due to arterial occlusion. Ischemic skeletal muscle is accompanied by capillary structural deformation. Therefore, using novel microscopy techniques, we tested the hypothesis that endothelial cell swelling temporally and quantitatively corresponds to enhanced microvascular permeability. Hindlimb ischemia was created in male Wistar rat's by iliac artery ligation (AL). The tibialis anterior (TA) muscle microcirculation was imaged using intravenously infused rhodamine B isothiocyanate dextran fluorescent dye via two-photon laser scanning microscopy (TPLSM) and dye extravasation at 3 and 7 days post-AL quantified to assess microvascular permeability. The TA microvascular endothelial ultrastructure was analyzed by transmission electron microscopy (TEM). Compared with control (0.40 ± 0.15 µm3 × 106), using TPLSM, the volumetrically determined interstitial leakage of fluorescent dye measured at 3 (3.0 ± 0.40 µm3 × 106) and 7 (2.5 ± 0.8 µm3 × 106) days was increased (both P < 0.05). Capillary wall thickness was also elevated at 3 (0.21 ± 0.06 µm) and 7 (0.21 ± 0.08 µm) days versus control (0.11 ± 0.03 µm, both P < 0.05). Capillary endothelial cell swelling was temporally and quantitatively associated with elevated vascular permeability in the AL model of PAD but these changes occurred in the absence of elevations in protein levels of vascular endothelial growth factor (VEGF) its receptor (VEGFR2 which decreased by AL-7 day) or matrix metalloproteinase. The temporal coherence of endothelial cell swelling and increased vascular permeability supports a common upstream mediator. TPLSM, in combination with TEM, provides a sensitive and spatially discrete technique to assess the mechanistic bases for, and efficacy of, therapeutic countermeasures to the pernicious sequelae of compromised peripheral arterial function.

Error Evaluation for Automated Diameter Measurements of Cerebral Capillaries Captured with Two-Photon Laser Scanning Fluorescence Microscopy.

Cerebral capillaries respond to changes in neural activity to maintain regional balances between energy demand and supply. However, the quantitative aspects of the capillary diameter responses and their contribution to oxygen supply to tissue remain incompletely understood. The purpose of the present study is to check if the diameters measured from large-scale angiographic image data of two-photon laser scanning fluorescent microscopy (2PLSM) are correctly determined with a custom-written MATLAB software and to investigate how the measurement errors can be reduced, such as at the junction areas of capillaries. As a result, nearly 17% of the measured locations appeared to be outliers of the automated diameter measurements, in particular arising from the junction areas where three capillary segments merged. We observed that about two-thirds of the outliers originated from the measured locations within 6 µm from the branching point. The results indicate that the capillary locations in the junction areas cause non-negligible errors in the automated diameter measurements. Considering the common site of the outliers, the present study identified that the areas within 6 µm from the branch point could be separately measured from the diameter analysis, and careful manual inspection with reference to the original images for these transition areas around the branch point is further recommended.

Time Series Tracking of Cerebral Microvascular Adaptation to Hypoxia and Hyperoxia Imaged with Repeated In Vivo Two-Photon Microscopy.

The present study describes methodological aspects of image analysis for angiographic image data with long-term two-photon microscopy acquired for the investigation of dynamic changes in the three-dimensional (3D) network structure of the capillaries (less than 8 µm in diameter) in the mouse cerebral cortex. Volume images of the identical capillaries over different periods of days up to 32 days were compared for adaptation under either chronic hypoxia (8-9% O2) or hyperoxia (40-50% O2). We observed that the median diameters of measured capillaries were 5.8, 8.4, 9.0, and 8.4 µm at 0, 1, 2, and 3 weeks during exposure to hypoxia, respectively (N = 1, n = 2193 pairs at day 0), and 5.4, 5.7, 5.4, 6.0, and 6.1 µm measured weekly up to 32 days under hyperoxia (N = 1, n = 1025 pairs at day 0). In accordance with these changes in capillary diameters, tissue space was also observed to change in a depth-dependent manner under hypoxia, but not hyperoxia. The present methods provide us with a method to quantitatively determine three-dimensional vascular and tissue morphology with the aid of a computer-assisted graphical user interface, which facilitates morphometric analysis of the cerebral microvasculature and its correlation with the adaptation of brain cells imaged simultaneously with the microvasculature.

Spatiotemporal dynamics of red blood cells in capillaries in layer I of the cerebral cortex and changes in arterial diameter during cortical spreading depression and response to hypercapnia in anesthetized mice.

OBJECTIVE: Control of red blood cell velocity in capillaries is essential to meet local neuronal metabolic requirements, although changes of capillary diameter are limited. To further understand the microcirculatory response during cortical spreading depression, we analyzed the spatiotemporal changes of red blood cell velocity in intraparenchymal capillaries. METHODS: In urethane-anesthetized Tie2-green fluorescent protein transgenic mice, the velocity of fluorescence-labeled red blood cells flowing in capillaries in layer I of the cerebral cortex was automatically measured with our Matlab domain software (KEIO-IS2) in sequential images obtained with a high-speed camera laser-scanning confocal fluorescence microscope system. RESULTS: Cortical spreading depression repeatedly increased the red blood cell velocity prior to arterial constriction/dilation. During the first cortical spreading depression, red blood cell velocity significantly decreased, and sluggishly moving or retrograde-moving red blood cells were observed, concomitantly with marked arterial constriction. The velocity subsequently returned to around the basal level, while oligemia after cortical spreading depression with slight vasoconstriction remained. After several passages of cortical spreading depression, hypercapnia-induced increase of red blood cell velocity, regional cerebral blood flow and arterial diameter were all significantly reduced, and the correlations among them became extremely weak. CONCLUSIONS: Taken together with our previous findings, these simultaneous measurements of red blood cell velocity in multiple capillaries, arterial diameter and regional cerebral blood flow support the idea that red blood cell flow might be altered independently, at least in part, from arterial regulation, that neuro-capillary coupling plays a role in rapidly meeting local neural demand.

Dynamic Flow Velocity Mapping from Fluorescent Dye Transit Times in the Brain Surface Microcirculation of Anesthetized Rats and Mice.

OBJECTIVE: This study aimed to develop a new method for mapping blood flow velocity based on the spatial evolution of fluorescent dye transit times captured with CLSFM in the cerebral microcirculation of anesthetized rodents. METHODS: The animals were anesthetized with isoflurane, and a small amount of fluorescent dye was intravenously injected to label blood plasma. The CLSFM was conducted through a closed cranial window to capture propagation of the dye in the cortical vessels. The transit time of the dye over a certain distance in a single vessel was determined with automated image analyses, and average flow velocity was mapped in each vessel. RESULTS: The average flow velocity measured in the rat pial artery and vein was 4.4 ± 1.2 and 2.4 ± 0.5 mm/sec, respectively. A similar range of flow velocity to those of the rats was observed in the mice; 4.9 ± 1.4 and 2.0 ± 0.9 mm/sec, respectively, although the vessel diameter in the mice was about half of that in the rats. CONCLUSIONS: Flow velocity in the cerebral microcirculation can be mapped based on fluorescent dye transit time measurements with conventional CLSFM in experimental animals.

Automated image analysis for diameters and branching points of cerebral penetrating arteries and veins captured with two-photon microscopy.

The present study was aimed to characterize 3-dimensional (3D) morphology of the cortical microvasculature (e.g., penetrating artery and emerging vein), using two-photon microscopy and automated analysis for their cross-sectional diameters and branching positions in the mouse cortex. We observed that both artery and vein had variable cross-sectional diameters across cortical depths. The mean diameter was similar for both artery (17 ± 5 µm) and vein (15 ± 5 µm), and there were no detectable differences over depths of 50-400 µm. On the other hand, the number of branches was slightly increased up to 400-µm depth for both the artery and vein. The mean number of branches per 0.1 mm vessel length was 1.7 ± 1.2 and 3.8 ± 1.6 for the artery and vein, respectively. This method allows for quantification of the large volume data of microvascular images captured with two-photon microscopy. This will contribute to the morphometric analysis of the cortical microvasculature in functioning brains.

Vessel specific imaging of glucose transfer with fluorescent glucose analogue in anesthetized mouse cortex.

The present study examined glucose transfer in the cellular scale of mouse brain microvasculature in vivo using two-photon microscopy and fluorescent glucose analogue (2-NBDG). The 2-NBDG was intravenously injected (0.04 mL/min) in the anesthetized Tie2-GFP mice in which the vascular endothelium expressed fluorescent protein. Time-lapse imaging was conducted on the cortical parenchyma, while the time-intensity change of the injected 2-NBDG was analysed in respective vascular compartments (artery, capillary, and vein). We observed that 2-NBDG signal increased monotonically in the vasculature during the period of the injection, and rapidly declined following its cessation. In tissue compartment, however, the signal intensity gradually increased even after cessation of the injection. Spatiotemporal analysis of the 2-NBDG intensity over the cross-sections of the vessels further showed distinct change of the 2-NBDG intensity across the vessel wall (endothelium), which may represents a regulation site of tissue glucose influx.

Serotonergic regulation of cortical neurovascular coupling and hemodynamics upon awakening from sleep in mice.

Neurovascular coupling (NVC) is the functional hyperemia of the brain responding to local neuronal activity. It is mediated by astrocytes and affected by subcortical ascending pathways in the cortex that convey information, such as sensory stimuli and the animal condition. Here, we investigate the influence of the raphe serotonergic system, a subcortical ascending arousal system in animals, on the modulation of cortical NVC and cerebral blood flow (CBF). Raphe serotonergic neurons were optogenically activated for 30 s, which immediately awakened the mice from non-rapid eye movement sleep. This caused a biphasic cortical hemodynamic change: a transient increase for a few seconds immediately after photostimulation onset, followed by a large progressive decrease during the stimulation period. Serotonergic neuron activation increased intracellular Ca2+ levels in cortical pyramidal neurons and astrocytes, demonstrating its effect on the NVC components. Pharmacological inhibition of cortical neuronal firing activity and astrocyte metabolic activity had small hypovolemic effects on serotonin-induced biphasic CBF changes, while blocking 5-HT1B receptors expressed primarily in cerebral vasculature attenuated the decreasing CBF phase. This suggests that serotonergic neuron activation leading to animal awakening could allow the NVC to exert a hyperemic function during a biphasic CBF response, with a predominant decrease in the cortex.

Potassium-induced cortical spreading depression bilaterally suppresses the electroencephalogram but only ipsilaterally affects red blood cell velocity in intraparenchymal capillaries.

Cortical spreading depression (CSD) is a repetitive, propagating profile of mass depolarization of neuronal and glial cells, followed by sustained suppression of spontaneous neuronal activity. We have reported a long-lasting suppressive effect on red blood cell (RBC) velocities in intraparenchymal capillaries. Here, to test the hypothesis that the prolonged decrease of RBC velocity in capillaries is due to suppression of neuronal activity, we measured CSD-elicited changes in the electroencephalogram (EEG) as an index of neuronal activity. In isoflurane-anesthetized rats, DC potential, EEG, partial pressure of oxygen (PO2), and cerebral blood flow (CBF) were simultaneously recorded in the temporo-parietal region. The velocities of fluorescently labeled RBCs were evaluated by high-speed camera laser scanning confocal fluorescence microscopy with our original software, KEIO-IS2. Transient deflection of DC potential and PO2 and increase of CBF were repeatedly detected only in the ipsilateral hemisphere following topical KCl application. On the other hand, the relative spectral power of EEG was reduced bilaterally, showing the lowest value at 5 min after KCl application, when the other parameters had already returned to the baseline after the passage of CSD. Mean RBC velocity in capillaries was slightly but significantly reduced during and after passage of CSD in the ipsilateral hemisphere but did not change in the contralateral hemisphere in the same rats. We suggest that mass depolarization of neuronal and glial cells might transiently decelerate RBCs in nearby capillaries, but the sustained reduction of ipsilateral RBC velocity might be a result of the prolonged effect of CSD, not of neuronal suppression alone.

Dynamic two-photon imaging of cerebral microcirculation using fluorescently labeled red blood cells and plasma.

To explore the spatiotemporal dynamics of red blood cells (RBCs) and plasma flow in three-dimensional (3D) microvascular networks of the cerebral cortex, we performed two-photon microscopic imaging of the cortical microvasculature in genetically engineered rats in which the RBCs endogenously express green fluorescent protein (GFP). Water-soluble quantum dots (Qdots) were injected intravenously into the animals to label the plasma, and concurrent imaging was performed for GFP-RBCs and Qdot plasma. The RBC and plasma distributions were compared between resting state and forepaw stimulation-induced neural activation. The RBC and plasma images showed detectable signals up to a depth of 0.4 and 0.6 mm from the cortical surface, respectively. A thicker plasma layer (2-5 µm) was seen in venous vessels relative to the arterial vessels. In response to neural activation, the RBCs were redistributed among the parenchymal capillary networks. In addition, individual capillaries showed a variable ratio of RBC and plasma distributions before and after activation, indicative of dynamic changes of hematocrit in single capillaries. These results demonstrate that this transgenic animal model may be useful in further investigating the mechanism that controls dynamic RBC flow in single capillaries and among multiple capillary networks of the cerebral microcirculation.

3D analysis of intracortical microvasculature during chronic hypoxia in mouse brains.

The purpose of this study is to determine when and where the brain microvasculature changes its network in response to chronic hypoxia. To identify the hypoxia-induced structural adaptation, we longitudinally imaged cortical microvasculature at the same location within a mouse somatosensory cortex with two-photon microscopy repeatedly for up to 1 month during continuous exposure to hypoxia (either 8 or 10% oxygen conditions). The two-photon microscopy approach made it possible to track a 3D pathway from a cortical surface arteriole to a venule up to a depth of 0.8 mm from the cortical surface. The network pathway was then divided into individual vessel segments at the branches, and their diameters and lengths were measured. We observed 3-11 vessel segments between the penetrating arteriole and the emerging vein over the depths of 20-460 µm within the 3D reconstructed image (0.46 × 0.46 × 0.80 mm(3)). The average length of the individual capillaries (<7 µm in diameter) was 67 ± 46 µm, which was not influenced by hypoxia. In contrast, 1.4 ± 0.3 and 1.2 ± 0.2 fold increases of the capillary diameter were observed 1 week after exposure to 8 % and 10% hypoxia, respectively. At 3 weeks from the exposure, the capillary diameter reached 8.5 ± 1.9 and 6.7 ± 1.8 µm in 8% and 10 % hypoxic conditions, respectively, which accounted for the 1.8 ± 0.5 and 1.4 ± 0.3 fold increases relative to those of the prehypoxic condition. The vasodilation of penetrating arterioles (1.4 ± 0.2 and 1.2 ± 0.2 fold increases) and emerging veins (1.3 ± 0.2 and 1.3 ± 0.2 fold increases) showed relatively small diameter changes compared with the parenchymal capillaries. These findings indicate that parenchymal capillaries are the major site responding to the oxygen environment during chronic hypoxia.

Hypoxia-induced cerebral angiogenesis in mouse cortex with two-photon microscopy.

To better understand cellular interactions of the cerebral angiogenesis induced by hypoxia, a spatiotemporal dynamics of cortical microvascular restructuring during an exposure to continuous hypoxia was characterized with in vivo two-photon microscopy in mouse cortex. The mice were prepared with a closed cranial window over the sensory-motor cortex and housed in 8-9 % oxygen room for 2-4 weeks. Before beginning the hypoxic exposure, two-photon imaging of cortical microvasculature was performed, and the follow-up imaging was conducted weekly in the identical locations. We observed that 1-2 weeks after the onset of hypoxic exposure, a sprouting of new vessels appeared from the existing capillaries. An average emergence rate of the new vessel was 15 vessels per unit volume (mm(3)). The highest emergence rate was found in the cortical depths of 100-200 µm, indicating no spatial uniformity among the cortical layers. Further, a leakage of fluorescent dye (sulforhodamine 101) injected into the bloodstream was not detected, suggesting that the blood-brain barrier (BBB) was maintained. Future studies are needed to elucidate the roles of perivascular cells (e.g., pericyte, microglia, and astroglia) in a process of this hypoxia-induced angiogenesis, such as sprouting, growth, and merger with the existing capillary networks, while maintaining the BBB.

Measuring the vascular diameter of brain surface and parenchymal arteries in awake mouse.

The present study reports a semiautomatic image analysis method for measuring the spatiotemporal dynamics of the vessel dilation that was fluorescently imaged with either confocal or two-photon microscope. With this method, arterial dilation induced by whisker stimulation was compared between cortical surface and parenchymal tissue in the vibrissae area of somatosensory cortex in awake Tie2-GFP mice in which the vascular endothelium had genetically expressed green fluorescent protein. We observed that a mean arterial diameter during a pre-stimulus baseline state was 39 ± 7, 19 ± 1, 16 ± 4, 17 ± 4, and 14 ± 3 µm at depths of 0, 100, 200, 300, and 400 µm, respectively. The stimulation-evoked dilation induced by mechanical whisker deflection (10 Hz for 5 s) was 3.4 ± 0.8, 1.8 ± 0.8, 1.8 ± 0.9, 1.6 ± 0.9, and 1.5 ± 0.6 µm at each depth, respectively. Consequently, no significant differences were observed for the vessel dilation rate between the cortical surface and parenchymal arteries: 8.8 %, 9.9 %, 10.9 %, 9.2 %, and 10.3 % relative to their baseline diameters, respectively. These preliminary results demonstrate that the present method is useful to further investigate the quantitative relationships between the spatiotemporally varying arterial tone and the associated blood flow changes in the parenchymal microcirculation to reveal the regulatory mechanism of the cerebral blood flow.

Spatiotemporal analysis of blood plasma and blood cell flow fluctuations of cerebral microcirculation in anesthetized rats.

Cerebral hemodynamics fluctuates spontaneously over broad frequency ranges. However, its spatiotemporal coherence of flow oscillations in cerebral microcirculation remains incompletely understood. The objective of this study was to characterize the spatiotemporal fluctuations of red blood cells (RBCs) and plasma flow in the rat cerebral microcirculation by simultaneously imaging their dynamic behaviors. Comparisons of changes in cross-section diameters between RBC and plasma flow showed dissociations in penetrating arterioles. The results indicate that vasomotion has the least effect on the lateral movement of circulating RBCs, resulting in variable changes in plasma layer thickness. Parenchymal capillaries exhibited slow fluctuations in RBC velocity (0.1 to 0.3 Hz), regardless of capillary diameter fluctuations (<0.1 Hz). Temporal fluctuations and the velocity of RBCs decreased significantly at divergent capillary bifurcations. The results indicate that a transit of RBCs generates flow resistance in the capillaries and that slow velocity fluctuations of the RBCs are subject to a number of bifurcations. In conclusion, the high-frequency oscillation of the blood flow is filtered at the bifurcation through the capillary networks. Therefore, a number of bifurcations in the cerebral microcirculation may contribute to the power of low-frequency oscillations.

Capillary responses to functional and pathological activations rely on the capillary states at rest.

Brain capillaries play a crucial role in maintaining cellular viability and thus preventing neurodegeneration. The aim of this study was to characterize the brain capillary morphology at rest and during neural activation based on a big data analysis from three-dimensional microangiography. Neurovascular responses were measured using a genetic calcium sensor expressed in neurons and microangiography with two-photon microscopy, while neural acivity was modulated by stimulation of contralateral whiskers or by a seizure evoked by kainic acid. For whisker stimulation, 84% of the capillary sites showed no detectable diameter change. The remaining 10% and 6% were dilated and constricted, respectively. Significant differences were observed for capillaries in the diameter at rest between the locations of dilation and constriction. Even the seizures resulted in 44% of the capillaries having no detectable change in diameter, while 56% of the capillaries dilated. The extent of dilation was dependent on the diameter at rest. In conclusion, big data analysis on brain capillary morphology has identified at least two types of capillary states: capillaries with diameters that are relatively large at rest and stable over time regardless of neural activity and capillaries whose diameters are relatively small at rest and vary according to neural activity.