Miyuki Unekawa

RBC velocities in single capillaries of mouse and rat brains are the same, despite 10-fold difference in body size

Employing high-speed camera laser-scanning confocal microscopy with RBC-tracking software, we previously showed that RBC velocities in intraparenchymal capillaries of rat cerebral cortex are distributed over a wide range. In the present work, we measured RBC velocities in mice, whose body weights are less than one-tenth of that of rats. In an isoflurane-anesthetized mouse, a cranial window was opened in the left temporo-parietal region. Intravenously administered FITC-labeled RBCs were automatically recognized and tracked frame-by-frame at 500fps, and the velocities of all RBCs recognized were calculated with our Matlab-domain software, KEIO-IS2. Among 15241 RBCs detected in the ROI in 21 mice, 1655 were identified as flowing in capillaries. The velocities of these RBCs ranged from 0.15 to 8.6mm/s, with a mean of 2.03+/-1.42mm/s. A frequency distribution plot showed that RBC velocities were clustered at around 1.0mm/s, tailing up to 8.6mm/s, and 59% of the RBCs in capillaries showed velocities within the range of 0.5 to 2.0mm/s. Unexpectedly, these characteristics of RBC velocities in mice were very similar to those of rats, despite differences in RBC diameter (6.0 vs. 6.5microm), body size (25 vs. 327g), heart rate (461 vs. 319bpm) and arterial blood pressure (86 vs. 84mmHg). We speculate that physical factors relating to oxygen exchange may constrain general RBC velocity in capillaries to a certain range for optimum oxygen exchange, regardless of species.

Repeated longitudinal in vivo imaging of neuro-glio-vascular unit at the peripheral boundary of ischemia in mouse cerebral cortex

Understanding the cellular events evoked at the peripheral boundary of cerebral ischemia is critical for therapeutic outcome against the insult of cerebral ischemia. The present study reports a repeated longitudinal imaging for cellular-scale changes of neuro-glia-vascular unit at the boundary of cerebral ischemia in mouse cerebral cortex in vivo. Two-photon microscopy was used to trace the longitudinal changes of cortical microvasculature and astroglia following permanent middle cerebral artery occlusion (MCAO). We found that sulforhodamine 101 (SR101), a previously-known marker of astroglia, provide a bright signal in the vessels soon after the intraperitoneal injection, and that intensity was sufficient to detect the microvasculature up to a depth of 0.8 mm. After 5-8 h from the injection of SR101, cortical astroglia was also imaged up to a depth of 0.4 mm. After 1 day from MCAO, some microvessels showed a closure of the lumen space in the occluded MCA territory, leading to a restructuring of microvascular networks up to 7 days after MCAO. At the regions of the distorted microvasculature, an increase in the number of cells labeled with SR101 was detected, which was found as due to labeled neurons. Immunohistochemical results further showed that ischemia provokes neuronal uptake of SR101, which delineate a boundary between dying and surviving cells at the peripheral zone of ischemia in vivo. Finally, reproducibility of the MCAO model was evaluated with magnetic resonance imaging (MRI) in a different animal group, which showed the consistent infarct volume at the MCA territory over the subjects.

Dually Supplied T-Junctions in Arteriolo-Arteriolar Anastomosis in Mice Key to Local Hemodynamic Homeostasis in Normal and Ischemic States?

Background and Purpose— The functional role of arteriolo-arteriolar anastomosis (AAA) between the middle cerebral artery (MCA) and anterior cerebral artery in local hemodynamics is unknown, and was investigated here. Methods— Blood flow in AAAs was examined using fluorescein isothiocyanate–labeled red blood cells (RBCs) as a flow indicator in 16 anesthetized C57BL/6J mice before and after MCA occlusion up to 7 experimental days. Results— We observed paradoxical flow in AAAs; labeled RBCs entered from both the MCA and anterior cerebral artery sides and the opposing flows met at a branching T-junction, where the flows combined and passed into a penetrating arteriole. The dually fed T-junction was not fixed in position, but functionally jumped to adjacent T-junctions in response to changing hemodynamic conditions. On MCA occlusion, RBC flow from the MCA side immediately stopped. After a period of “hesitation,” blood started to move retrogradely in one of the MCA branches toward the MCA stem. The retrograde blood flow was statistically significantly (P<0.05), serving to feed blood to other MCA branches after a lag period. In capillaries, MCA occlusion induced immediate RBC disappearance in the ischemic core and to a lesser extent in the marginal zone near AAAs. At day 3 after ischemia, we recognized the beginning of remodeling with angiogenesis centering on AAAs. Conclusions— AAAs appear to play a key role in local hemodynamic homeostasis, both in the normal state and in the development of collateral channels and revascularization during ischemia.

Automated Method for Tracking Vast Numbers of FITC-Labeled RBCs in Microvessels of Rat Brain In Vivo Using a High-Speed Confocal Microscope System

High‐speed camera investigation of rapidly moving red blood cells (RBCs) in the microvasculature has been limited by an inability to handle the vast volume of data. We have developed a novel method to analyze large numbers of RBC images captured by a high‐resolution, high‐speed camera fitted on a confocal fluorescence microscope, to determine the velocities of individual RBCs in capillaries in vivo. Fluorescein isothiocyanate (FITC)‐labeled RBCs flowing in the microvasculature of the cerebral cortex of urethane‐anesthetized Wistar rats were recorded through the skull window on video clips during specified periods at high frame rates (500 fps). Sequential frames of moving RBCs in the video clips for a specified period were analyzed offline with in‐house software (KEIO‐IS2). Images of RBCs acquired were numbered automatically in order of appearance and displayed in a two‐dimensional (2‐D) RBC tracking map. The velocities of individual RBCs were automatically computed based on the RBC displacement per frame multiplied by the frame rate (fps), and the results were displayed in a 2‐D velocity map and a 2‐D RBC number map. Single capillaries were identified by staining with FITC‐dextran. The mean capillary velocity of RBCs was evaluated as 2.05 ± 1.59 mm/second in video clips obtained at 500 fps. This method is considered to have wide potential applicability.

Oscillating neuro-capillary coupling during cortical spreading depression as observed by tracking of FITC-labeled RBCs in single capillaries.

Coupling between capillary red blood cell (RBC) movements and neuronal dysfunction during cortical spreading depression (CSD) was examined in rats by employing a high-speed camera laser-scanning confocal fluorescence microscope system in conjunction with our Matlab domain software (KEIO-IS2). Following microinjection of K(+) onto the surface of the brain, changes in electroencephalogram (EEG), DC potential and tissue optical density were all compatible with the occurrence of a transient spreading neuronal depression. RBC flow in single capillaries was not stationary. Unpredictable redistribution of RBCs at branches of capillaries was commonly observed, even though no change in diameter was apparent at the reported site of the capillary sphincter and no change of arteriolar-venule pressure difference was detected. There appeared to be a slow morphological change of astroglial endfeet. When local neurons were stunned transiently by K(+) injection, the velocity and oscillation frequency of RBCs flowing in nearby capillaries started to decrease. The flow in such capillaries was rectified, losing oscillatory components. Sluggish floating movements of RBCs in pertinent capillaries were visualized, with occasional full stops. When CSD subsided, RBC movements recovered to the original state. We postulate that neuronal depolarization blocks oscillatory signaling to local capillaries via low-shear plasma viscosity increases in the capillary channels, and a complex interaction between the RBC surface and the buffy coat on the capillary wall surface increases the capillary flow resistance. Then, when CSD subsides and oscillatory neuronal function is recovered, the normal physiological conditions are restored.

Microvascular sprouting, extension, and creation of new capillary connections with adaptation of the neighboring astrocytes in adult mouse cortex under chronic hypoxia

The present study aimed to determine the spatiotemporal dynamics of microvascular and astrocytic adaptation during hypoxia-induced cerebral angiogenesis. Adult C57BL/6J and Tie2-green fluorescent protein (GFP) mice with vascular endothelial cells expressing GFP were exposed to normobaric hypoxia for 3 weeks, whereas the three-dimensional microvessels and astrocytes were imaged repeatedly using two-photon microscopy. After 7 to14 days of hypoxia, a vessel sprout appeared from the capillaries with a bump-like head shape (mean diameter 14 μm), and stagnant blood cells were seen inside the sprout. However, no detectable changes in the astrocyte morphology were observed for this early phase of the hypoxia adaptation. More than 50% of the sprouts emerged from capillaries 60 μm away from the center penetrating arteries, which indicates that the capillary distant from the penetrating arteries is a favored site for sprouting. After 14 to 21 days of hypoxia, the sprouting vessels created a new connection with an existing capillary. In this phase, the shape of the new vessel and its blood flow were normalized, and the outside of the vessels were wrapped with numerous processes from the neighboring astrocytes. The findings indicate that hypoxia-induced cerebral angiogenesis provokes the adaptation of neighboring astrocytes, which may stabilize the blood–brain barrier in immature vessels.

Unveiling astrocytic control of cerebral blood flow with optogenetics

Cortical neural activities lead to changes in the cerebral blood flow (CBF), which involves astrocytic control of cerebrovascular tone. However, the manner in which astrocytic activity specifically leads to vasodilation or vasoconstriction is difficult to determine. Here, cortical astrocytes genetically expressing a light-sensitive cation channel, channelrhodopsin-2 (ChR2), were transcranially activated with a blue laser while the spatiotemporal changes in CBF were noninvasively monitored with laser speckle flowgraphy in the anesthetised mouse cortex. A brief photostimulation induced a fast transient increase in CBF. The average response onset time was 0.7 ± 0.7 sec at the activation foci, and this CBF increase spread widely from the irradiation spot with an apparent propagation speed of 0.8–1.1 mm/sec. The broad increase in the CBF could be due to a propagation of diffusible vasoactive signals derived from the stimulated astrocytes. Pharmacological manipulation showed that topical administration of a K+ channel inhibitor (BaCl2; 0.1–0.5 mM) significantly reduced the photostimulation-induced CBF responses, which indicates that the ChR2-evoked astrocytic activity involves K+ signalling to the vascular smooth muscle cells. These findings demonstrate a unique model for exploring the role of the astrocytes in gliovascular coupling using non-invasive, time-controlled, cell-type specific perturbations.

Sustained Decrease and Remarkable Increase in Red Blood Cell Velocity in Intraparenchymal Capillaries Associated With Potassium-Induced Cortical Spreading Depression

Please cite this paper as: Unekawa M, Tomita M, Tomita Y, Toriumi H and Suzuki N. Sustained Decrease and Remarkable Increase in Red Blood Cell Velocity in Intraparenchymal Capillaries Associated With Potassium‐Induced Cortical Spreading Depression. Microcirculation 19: 166–174, 2012.

Suppressive effect of chronic peroral topiramate on potassium-induced cortical spreading depression in rats

Objective: To evaluate the chronic effect of topiramate (TPM) on cortical spreading depression (CSD), which is thought to be related to migraine aura. Methods: Male rats (n = 30) were randomized to once-daily peroral treatment with TPM (50, 100, 200 or 600 mg/kg) or vehicle for 6 weeks. We evaluated the characteristics of CSD induced by topical application of KCl under isoflurane anesthesia and the changes in plasma level of TPM in each group. The effect of single administration of TPM on CSD was also evaluated. Results: After the final administration of TPM, when the plasma level of TPM was high, KCl-induced CSD frequency and CSD propagation velocity were dose-dependently reduced and the interval between CSD episodes was elongated, compared with the vehicle control. However, before the final administration of TPM, when the plasma level was very low, the KCl-induced CSD profile was the same as that in the vehicle control. Single administration of TPM did not alter the CSD profile. Local cerebral blood flow was not significantly altered by chronic administration of TPM. Conclusion: TPM suppressed the frequency and propagation of CSD along the cerebral cortex, and might be a candidate for relief of migraine.

High-mobility group box 1 is an important mediator of microglial activation induced by cortical spreading depression

Single episodes of cortical spreading depression (CSD) are believed to cause typical migraine aura, whereas clusters of spreading depolarizations have been observed in cerebral ischemia and subarachnoid hemorrhage. We recently demonstrated that the release of high-mobility group box 1 (HMGB1) from cortical neurons after CSD in a rodent model is dependent on the number of CSD episodes, such that only multiple CSD episodes can induce significant HMGB1 release. Here, we report that only multiple CSD inductions caused microglial hypertrophy (activation) accompanied by a greater impact on the transcription activity of the HMGB1 receptor genes, TLR2 and TLR4, while the total number of cortical microglia was not affected. Both an HMGB1-neurtalizing antibody and the HMGB1 inhibitor glycyrrhizin abrogated multiple CSD-induced microglial hypertrophy. Moreover, multiple CSD inductions failed to induce microglial hypertrophy in TLR2/4 double knockout mice. These results strongly implicate the HMGB1–TLR2/4 axis in the activation of microglia following multiple CSD inductions. Increased expression of the lysosomal acid hydrolase cathepsin D was detected in activated microglia by immunostaining, suggesting that lysosomal phagocytic activity may be enhanced in multiple CSD-activated microglia.