Striatal Blood Flow Changes by Middle Cerebral Artery Occlusion and Its Effect on Neurological Deficits in Mice.

OBJECTIVE: We attempted to record the regional cerebral blood flow (CBF) simultaneously at various regions of the cerebral cortex and the striatum during middle cerebral artery (MCA) occlusion and to evaluate neurological deficits and infarct formation. METHODS: In male C57BL/6J mice, CBF was recorded in three regions including the ipsilateral cerebral cortex and the striatum with laser Doppler flowmeters, and the origin of MCA was occluded with a monofilament suture for 15-90 min. After 48 h, neurological deficits were evaluated, and infarct was examined by triphenyltetrazolium chloride (TTC) staining. RESULTS: CBF decrease in the striatum was approximately two-thirds of the MCA-dominant region of the cortex during MCA occlusion. The characteristic CBF fluctuation because of spontaneously occurred spreading depolarization observed throughout the cortex was not found in the striatum. Ischemic foci with slight lower staining to TTC were found in the ipsilateral striatum in MCA-occluded mice for longer than 30 min (n = 54). Twenty-nine among 64 MCA-occluded mice exhibited neurological deficits even in the absence of apparent infarct with minimum staining to TTC in the cortex, and the severity of neurological deficits was not correlated with the size of the cortical infarct. CONCLUSION: Neurological deficits might be associated with the ischemic striatum rather than with cortical infarction.

Separate optogenetic manipulation of Nerve/glial antigen 2 (NG2) glia and mural cells using the NG2 promoter.

Nerve/glial antigen 2 (NG2) is a protein marker of NG2 glia and mural cells, and NG2 promoter activity is utilized to target these cells. However, the NG2 promoter cannot target NG2 glia and mural cells separately. This has been an obstacle for NG2 glia-specific manipulation. Here, we developed transgenic mice in which either cell type can be targeted using the NG2 promoter. We selected a tetracycline-controllable gene induction system for cell type-specific transgene expression, and generated NG2-tetracycline transactivator (tTA) transgenic lines. We crossed tTA lines with the tetO-ChR2 (channelrhodopsin-2)-EYFP line to characterize tTA-dependent transgene induction. We isolated two unique NG2-tTA mouse lines: one that induced ChR2-EYFP only in mural cells, likely due to the chromosomal position effect of NG2-tTA insertion, and the other that induced it in both cell types. We then applied a Cre-mediated set-subtraction strategy to the latter case and eliminated ChR2-EYFP from mural cells, resulting in NG2 glia-specific transgene induction. We further demonstrated that tTA-dependent ChR2 expression could manipulate cell function. Optogenetic mural cell activation decreased cerebral blood flow, as previously reported, indicating that tTA-mediated ChR2 expression was sufficient to impact cellular function. ChR2-mediated depolarization was observed in NG2 glia in acute hippocampal slices. In addition, ChR2-mediated depolarization of NG2 glia inhibited their proliferation but promoted their differentiation in juvenile mice. Since the tTA-tetO combination is expandable, the mural cell-specific NG2-tTA line and the NG2 glia-specific NG2-tTA line will permit us to conduct observational and manipulation studies to examine in vivo function of these cells separately.

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.

Calcitonin Gene-Related Peptide mRNA Synthesis in Trigeminal Ganglion Neurons after Cortical Spreading Depolarization.

Migraine is a debilitating neurovascular disorder characterized by recurrent headache attacks of moderate to severe intensity. Calcitonin gene-related peptide (GGRP), which is abundantly expressed in trigeminal ganglion (TG) neurons, plays a crucial role in migraine pathogenesis. Cortical spreading depolarization (CSD), the biological correlate of migraine aura, activates the trigeminovascular system. In the present study, we investigated CGRP mRNA expression in TG neurons in a CSD-based mouse migraine model. Our in situ hybridization analysis showed that CGRP mRNA expression was observed in smaller-sized neuronal populations. CSD did not significantly change the density of CGRP mRNA-synthesizing neurons in the ipsilateral TG. However, the cell sizes of CGRP mRNA-synthesizing TG neurons were significantly larger in the 48 h and 72 h post-CSD groups than in the control group. The proportions of CGRP mRNA-synthesizing TG neurons bearing cell diameters less than 14 µm became significantly less at several time points after CSD. In contrast, we found significantly greater proportions of CGRP mRNA-synthesizing TG neurons bearing cell diameters of 14-18 µm at 24 h, 48, and 72 h post-CSD. We deduce that the CSD-induced upward cell size shift in CGRP mRNA-synthesizing TG neurons might be causative of greater disease activity and/or less responsiveness to CGRP-based therapy.

Effect of Goreisan, a Japanese Traditional Medicine, on Cortical Spreading Depolarization in Mice.

Goreisan, a traditional Japanese Kampo medicine, is often used to treat headaches, including migraines; however, the underlying mechanisms remain unknown. Therefore, we investigated whether chronic treatment with Goreisan affects cortical spreading depolarization (CSD) in migraines. CSD susceptibility was assessed in male and female C57BL/6 mice by comparing CSD threshold, propagation velocity, and CSD frequency between animals treated with Goreisan for approximately 3 weeks and the corresponding controls with a potassium-induced CSD model. No significant differences were observed in CSD susceptibility between mice that were chronically treated with Goreisan and the control mice. Additionally, no significant differences were observed in other physiological parameters, including body weight, blood gases, and blood pressure. CSD susceptibility was not affected by chronic treatment with Goreisan, which suggests that the drug treats headaches via mechanisms that do not involve CSD modulation.

Sustained Effects of CGRP Blockade on Cortical Spreading Depolarization-Induced Alterations in Facial Heat Pain Threshold, Light Aversiveness, and Locomotive Activity in the Light Environment.

A migraine is clinically characterized by repeated headache attacks that entail considerable disability. Many patients with migraines experience postdrome, the symptoms of which include tiredness and photophobia. Calcitonin gene-related peptide (GGRP) is critically implicated in migraine pathogenesis. Cortical spreading depolarization (CSD), the biological correlate of migraine aura, sensitizes the trigeminovascular system. In our previous study, CSD caused hypomotility in the light zone and tendency for photophobia at 72 h, at which time trigeminal sensitization had disappeared. We proposed that this CSD-induced disease state would be useful for exploring therapeutic strategies for migraine postdrome. In the present study, we observed that the CGRP receptor antagonist, olcegepant, prevented the hypomotility in the light zone and ameliorated light tolerability at 72 h after CSD induction. Moreover, olcegepant treatment significantly elevated the threshold for facial heat pain at 72 h after CSD. Our results raise the possibility that CGRP blockade may be efficacious in improving hypoactivity in the light environment by enhancing light tolerability during migraine postdrome. Moreover, our data suggest that the CGRP pathway may lower the facial heat pain threshold even in the absence of overt trigeminal sensitization, which provides an important clue to the potential mechanism whereby CGRP blockade confers migraine prophylaxis.

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.

Characteristics of cortical spreading depression and c-Fos expression in transgenic mice having a mutation associated with familial hemiplegic migraine 2.

BACKGROUND: Cortical spreading depression is thought to be the underlying mechanism of migraine aura. In 2006, three relatives having the point mutation E700K in ATP1A2 exon 15 were diagnosed with familial hemiplegic migraine 2 characterized by complicated forms of aura. Here, we generated a transgenic mouse model having the human E700K mutation in the Atp1a2 orthologous gene. OBJECTIVE: To investigate the characteristics of cortical spreading depression in a mouse model with E700K mutation in the Atp1a2. METHODS: Cortical spreading depression was induced by applying stepwise increases of KCl concentration or electrical stimulation intensity to C57BL/6J-Tg(Atp1a2*E700K)9151Kwk mice (Tg, both sexes) and corresponding wild-type animals. Under urethane anesthesia, the responsiveness and threshold to cortical spreading depression were examined and the distribution of c-Fos expression, a neuronal activity marker, was immunohistochemically determined. RESULTS: Overall, Tg mice showed significantly faster propagation velocity (p < 0.01) and longer full-width-at-half-maximum (p < 0.01) than wild-type animals, representing a slower recovery from direct current potential deflection. The cortical spreading depression threshold tended to be lower in Tg, especially in females. c-Fos-positive cells were significantly enhanced in the ipsilateral somatosensory cortex, piriform cortex, amygdala and striatum (each p < 0.05 vs. contralateral side). Numbers of c-Fos positive cells were significantly higher in the ipsilateral amygdala of Tg, as compared with wild-type animals (p < 0.01). CONCLUSION: The effect of cortical spreading depression may be greater in E700K transgenic mice than that in wild-type animals, while the threshold for cortical spreading depression shows little change. Higher c-Fos expression in the amygdala may indicate alterations of the limbic system in Tg, suggesting an enhanced linkage between cortical spreading depression and amygdala connectivity in familial hemiplegic migraine 2 patients.

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.

Enhanced susceptibility to cortical spreading depression in two types of Na+,K+-ATPase α2 subunit-deficient mice as a model of familial hemiplegic migraine 2.

Background Patients with familial hemiplegic migraine type 2 (FHM2) have a mutated ATP1A2 gene (encoding Na+,K+-ATPase α2 subunit) and show prolonged migraine aura. Cortical spreading depression (CSD), which involves mass depolarization of neurons and astrocytes that propagates slowly through the gray matter, is profoundly related to aura. Methods In two types of Atp1a2-defective heterozygous mice, Atp1a2tm1Kwk (C-KO) and Atp1a2tm2Kwk (N-KO), the sensitivity and responsiveness to CSD were examined under urethane anesthesia. Results In both cases, heterozygotes exhibited a low threshold for induction of CSD, faster propagation rate, slower recovery from DC deflection, and profound suppression of the electroencephalogram, compared to wild-type mice. A high dose of KCl elicited repeated CSDs for a longer period, with a tendency for a greater frequency of CSD occurrence in heterozygotes. The difference of every endpoint was slightly greater in N-KO than C-KO. Change of regional cerebral blood flow in response to CSD showed no significant difference. Conclusion Heterozygotes of Atp1a2-defective mice simulating FHM2 demonstrated high susceptibility to CSD rather than cortical vasoreactivity, and these effects may differ depending upon the knockout strategy for the gene disruption. These results suggest that patients with FHM2 may exhibit high susceptibility to CSD, resulting in migraine.

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.

In vivo imaging of the mouse neurovascular unit under chronic cerebral hypoperfusion.

BACKGROUND AND PURPOSE: Proper brain function is maintained by an integrated system called the neurovascular unit (NVU) comprised cellular and acellular elements. Although the individual features of specific neurovascular components are understood, it is unknown how they respond to ischemic stress as a functional unit. Therefore, we established an in vivo imaging method and clarified the NVU response to chronic cerebral hypoperfusion. METHODS: Green mice (b-act-EGFP) with SR101 plasma labeling were used in this experiment. A closed cranial window was made over the left somatosensory cortex. To mimic chronic cerebral hypoperfusion, mice were subjected to bilateral common carotid artery stenosis operations using microcoils. In vivo real-time imaging was performed using 2-photon laser-scanning microscopy during the preoperative period, and after 1 day and 1 and 2 weeks of bilateral common carotid artery stenosis or sham operations. RESULTS: Our method allowed 3-dimensional observation of most of the components of the NVU, as well as dynamic capillary microcirculation. Under chronic cerebral hypoperfusion, we did not detect any structural changes of each cellular component in the NVU; however, impairment of microcirculation was detected over a prolonged period. In the pial small arteries and veins, rolling and adhesion of leukocyte were detected, more prominently in the latter. In the deep cortical capillaries, flow stagnation because of leukocyte plugging was frequently observed. CONCLUSIONS: We established an in vivo imaging method for real-time visualization of the NVU. It seems that under chronic cerebral hypoperfusion, leukocyte activation has a critical role in microcirculation disturbance.

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.

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.

Sustained decrease and remarkable increase in red blood cell velocity in intraparenchymal capillaries associated with potassium-induced cortical spreading depression.

OBJECTIVES: To examine changes in red blood cell (RBC) velocity in intraparenchymal capillaries of rat cerebral cortex in response to KCl-induced cortical spreading depression (CSD). METHODS: In isoflurane-anesthetized rats, the velocity of fluorescently labeled RBCs flowing in capillaries in layer I was measured with a high-speed camera laser-scanning confocal fluorescence microscope, with simultaneous monitoring of DC potential, the electroencephalogram (EEG), partial pressure of oxygen (PO(2) ), and cerebral blood flow (CBF). RESULTS: After KCl application, a transient deflection of DC potential (i.e., CSD) repeatedly appeared concomitantly with depression of EEG, and was propagated in the distal direction. PO(2) transiently decreased and CBF was slowly elevated. The frequency distribution of RBC velocity was shifted downward during CSD and was still low after the passage of CSD. When we observed RBC velocity in 38 individual capillaries, 10 capillaries exhibited slowed-down RBC during CSD and RBC velocity remained low in 2 even after the passage of CSD. On the other hand, RBCs with moderately (<3 mm/sec) or remarkably (>3 mm/sec) increased velocities were seen in 10 and 5 capillaries, respectively. CONCLUSION: CSD-induced excitation of neurons may sustainably decrease or greatly increase RBC velocity in capillaries.

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.

Close association between spreading depolarization and development of infarction under experimental ischemia in anesthetized male mice.

Clinical and experimental evidence suggests that spreading depolarizations (SD) usually occur in patients with ischemic or hemorrhagic stroke when the gray matter of the brain is affected. In this study, we evaluated spatiotemporal changes of cerebral blood flow (CBF) during middle cerebral artery (MCA) occlusion and examined the relationship between SD occurrence and cerebral infarct development. In male isoflurane-anesthetized C57BL/6J mice, CBF changes over the ipsilateral parietal bone were recorded by laser speckle flowgraphy during and after transient (45 min, n = 22) or permanent occlusion (n = 22) of the distal MCA. Infarct volume was evaluated 24 hr after the operation. Upon MCA occlusion, CBF decreased by -55.6 ± 8.5 % in the lowest CBF and linearly recovered with increasing distance from the region. At 1-10 min after onset of occlusion, SD occurred and concentrically propagated from the core region, showing a decrease of CBF in the whole observed area along with a transient hyperemia and oligemia in the normal region. SD spontaneously re-occurred and propagated around the ischemic area in 37 % of mice, accompanied with a marked decrease of CBF in the core or a marked increase of CBF in the normal region. The CBF response to SDs gradually changed from the core to the normal area, depending upon the distance from the core region. Infarction was not observed in transiently (n = 2) or permanently (n = 4) occluded mice without SD. The infarct area tended to be larger with increasing number of SDs in transiently occluded mice. In conclusion, our findings suggest that the occurrence of SD during ischemia might elicit infarct formation and/or influence infarct development.

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.

Developmental and circulatory profile of the diploic veins.

To examine the development of the diploic veins in the calvarium, FITC-dextran was injected into the tail vein. The total area of the diploic veins showed a continuous, age-dependent development. We also measured the red blood cell (RBC) velocities in the diploic veins using an in vivo imaging technique and revealed RBCs with a significantly high velocity and unidirectional characteristics at the entrance route. The route passed from the basal periosteum of the cranial bone via the dura mater and into the diploic veins. Our findings indicate the existence of communications between intra- and extra-cranial circulation.

Protracted hypomobility in the absence of trigeminal sensitization after cortical spreading depolarization: Relevance to migraine postdrome.

Migraine sufferers often exhibit photophobia and physical hypoactivity in the postdrome and interictal periods, for which no effective therapy currently exists. Cortical spreading depolarization (CSD) is a neural phenomenon underlying migraine aura. We previously reported that CSD induced trigeminal sensitization, photophobia, and hypomobility at 24 h in mice. Here, we examined the effects of CSD induction on light sensitivity and physical activity in mice at 48 h and 72 h. Trigeminal sensitization was absent at both time points. CSD-subjected mice exhibited significantly less ambulatory time in both light (P = 0.0074, the Bonferroni test) and dark (P = 0.0354, the Bonferroni test) zones than sham-operated mice at 72 h. CSD-subjected mice also exhibited a significantly shorter ambulatory distance in the light zone at 72 h than sham-operated mice (P = 0.0151, the Bonferroni test). Neurotropin® is used for the management of chronic pain disorders, mainly in Asian countries. The CSD-induced reductions in ambulatory time and distance in the light zone at 72 h were reversed by Neurotropin® at 0.27 NU/kg. Our experimental model seems to recapitulate migraine-associated clinical features observed in the postdrome and interictal periods. Moreover, Neurotropin® may be effective in ameliorating postdromal/interictal hypoactivity, especially in a light environment.