Haruki Toriumi

Reduction of TRPV1 expression in the trigeminal system by botulinum neurotoxin type-A.

Botulinum neurotoxin type-A (BoNT-A) is clinically used for patients with pain disorders and dystonia. The precise mechanism whereby BoNT-A controls pain remains elusive. Here, we studied how BoNT-A affects the expression of the transient receptor potential vanilloid subfamily member 1 (TRPV1), a cation channel critically implicated in nociception, in the trigeminal system. Histological studies revealed that subcutaneous BoNT-A injection (0.25, 0.5, or 5 ng/kg) into the face targeted the ophthalmic division of trigeminal ganglion (TG) neurons and decreased TRPV1-immunoreactive neurons in the TG and TRPV1-immunoreactive fibers in rat trigeminal terminals. Of note, TG neurons that received projections from the dura mater, a principal site of headache generation, had reduced TRPV1 expression. BoNT-A-induced cleavage of SNAP25 (synaptosomal-associated protein of 25-kDa) in the TG became obvious 2 days after BoNT-A administration and persisted for at least 14 days. Quantitative real-time RT-PCR (reverse transcription-polymerase chain reaction) data indicated that the TRPV1-decreasing effects of BoNT-A were not mediated by transcriptional downregulation. By employing a surface protein biotin-labeling assay, we demonstrated that BoNT-A inhibited TRPV1 trafficking to the plasma membrane in primary TG neurons. Moreover, Y200F-mutated TRPV1, which is incapable of trafficking to the plasma membrane, was expressed in PC12 cells by transfection, and pharmacological studies revealed that TRPV1 in the cytoplasm was more predisposed to proteasome-mediated proteolysis than plasma membrane-located TRPV1. We conclude that the mechanism by which BoNT-A reduces TRPV1 expression involves the inhibition of TRPV1 plasma membrane trafficking and proteasome-mediated degradation in the cytoplasm. This paradigm seems to explain how BoNT-A alleviates TRPV1-mediated pain. Our data reveal a likely molecular mechanism whereby BoNT-A treatment reduces TRPV1 expression in the trigeminal system and provide important clues to novel therapeutic measures for ameliorating craniofacial pain.

Distribution and origin of TRPV1 receptor-containing nerve fibers in the dura mater of rat

We examined the distribution and origin of the nerve fibers innervating the dura mater of the rat that show immunoreactivity for the TRPV1 receptor (TRPV1-IR). Nearly 70% of the nerve fibers showing TRPV1-IR in the dura mater also exhibited CGRP-IR. Using a combination of immunohistochemistry and a retrograde tracer technique, we detected tracer accumulation in 0.6% of the neurons in the trigeminal ganglion and a few neurons in the dorsal root ganglion; half of the neurons in the trigeminal ganglion were small- and medium-sized (<or=1000 microm2). Among the tracer-accumulated neurons in the trigeminal ganglion, approximately 25% exhibited TRPV1-IR. Furthermore, nearly 80% of the tracer-accumulated small- and medium-sized neurons in the trigeminal ganglion that exhibited TRPV1-IR also exhibited CGRP-IR. Our findings indicate that the TRPV1 receptor in the dura mater and sensory ganglia may contribute to the pathophysiology of migraine, providing an important clue for the development of therapeutic strategies for migraine.

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.

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.

Astrocytes and pericytes cooperatively maintain a capillary-like structure composed of endothelial cells on gel matrix

The gliovascular complex (GVC) is a structural and functional unit located at the interface between the cerebral blood flow and neural network. Even though its physiological roles have been partially clarified, the contribution of the astroglia to maintenance of the cerebral vasculature, which is tightly wired with the neuronal network, is only incompletely understood. To elucidate the role of astrocytes and pericytes in maintaining the integrity of the capillaries, we developed a new GVC model in vitro. Human brain microvascular endothelial cells (ECs) plated on a gel matrix developed a capillary-like structure (CLS). Pericytes and astrocytes migrated together and adhered to the CLS to form a GVC-like structure with pericytes between the CLS and astrocytes. Astrocytes together with pericytes suppressed CLS degradation to a greater extent than astrocytes alone. Fumagillin and suramin, angiogenesis inhibitors, suppressed GVC formation. The PDGF receptor inhibitor SU6668 suppressed pericyte/astrocyte migration and accelerated CLS degradation, whereas the VEGF receptor inhibitor SU1498 did not suppress pericyte/astrocyte migration and both types of cells maintained the CLS for a long period of time. Immunohistochemistry revealed aquaporin-4 and agrin expression at the junction of ECs and astrocytes. These results indicate the importance of both astrocytes and pericytes for maintenance of the cerebral microvasculature.