Effects of passive smoking on cortical spreading depolarization in male and female mice.

BACKGROUND: Patients with migraine are typically advised to avoid passive smoking because it may aggravate headaches and other health conditions. However, there is insufficient high-quality evidence on the association between passive smoking and migraine, which warrants further investigation using animal models. Therefore, using a mouse model, we examined the effect of passive smoking on susceptibility to cortical spreading depolarization (CSD), the biological basis of migraine with aura. FINDINGS: Fifty C57BL/6 mice (25 males and 25 females) were exposed for one hour to cigarette smoke or room air. Subsequently, potassium chloride (KCl) was administered under isoflurane anesthesia to induce CSD, and the CSD threshold, frequency of induction, and propagation velocity were determined. The threshold to induce CSD (median [interquartile range (IQR)]) was significantly lower in female mice (adjusted p = 0.01) in the smoking group (0.05 [0.05, 0.088]) than in the sham group (0.125 [0.1, 0.15]); however, there was no significant difference in the male mice (adjusted p = 0.77). CSD frequency or propagation velocity did not differ significantly between the two groups for either sex. CONCLUSIONS: Female mice in the smoking group showed lower CSD threshold compared to the sham group, suggesting a potential sex-specific difference in the effect of smoking on the pathogenesis of CSD and migraine with aura. This finding may contribute to the understanding of migraine pathophysiology in association with passive smoking and sex difference.

Microfluctuations in Capillary Lumens Independent of Pericyte Lining Density in the Anesthetized Mouse Cortex.

OBJECTIVE: This study aimed to examine the spatiotemporal coherence of capillary lumen fluctuations in relation to spatial variations in the pericyte lining in the cortex of anesthetized mice. METHODS: Two-photon microscopic angiography data (previously published) were reanalyzed, and spatial variations in capillary diameter fluctuations at rest and in capillary lining with vascular mural cells were measured along capillary centerlines. RESULTS: Relatively large diameters of the capillaries (5.5 µm) coincided with a dense pericyte lining, while small capillaries (4.3 µm) had a sparse pericyte lining. Temporal variations had a frequency of about 0.1 Hz with an amplitude of 0.5 µm, which were negatively correlated with pericyte lining density. Spatial frequency analysis further revealed a common pattern of spatial variations in capillary diameter and pericyte lining, but temporal variations differed. The temporal variations in capillary lumens were locally distinct from those in neighboring locations, suggesting intrinsic fluctuations independent of the pericyte lining. CONCLUSIONS: Capillary lumens in the brain exhibit slow microfluctuations that are independent of pericyte lining. These microfluctuations could affect the distribution of flowing blood cells and may be important for homogenizing their distribution in capillary networks.

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.

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.

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.

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.

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.

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.

Optical manipulation of local cerebral blood flow in the deep brain of freely moving mice.

An artificial tool for manipulating local cerebral blood flow (CBF) is necessary for understanding how CBF controls brain function. Here, we generate vascular optogenetic tools whereby smooth muscle cells and endothelial cells express optical actuators in the brain. The illumination of channelrhodopsin-2 (ChR2)-expressing mice induces a local reduction in CBF. Photoactivated adenylyl cyclase (PAC) is an optical protein that increases intracellular cyclic adenosine monophosphate (cAMP), and the illumination of PAC-expressing mice induces a local increase in CBF. We target the ventral striatum, determine the temporal kinetics of CBF change, and optimize the illumination intensity to confine the effects to the ventral striatum. We demonstrate the utility of this vascular optogenetic manipulation in freely and adaptively behaving mice and validate the task- and actuator-dependent behavioral readouts. The development of vascular optogenetic animal models will help accelerate research linking vasculature, circuits, and behavior to health and disease.

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.

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.

Differential pial and penetrating arterial responses examined by optogenetic activation of astrocytes and neurons.

A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.

Cortical spreading depolarisation-induced facial hyperalgesia, photophobia and hypomotility are ameliorated by sumatriptan and olcegepant.

Cortical spreading depolarisation (CSD), the neural mechanism underlying migraine aura, may cause headache by sensitising the trigeminal system. Photophobia, the most bothersome accompanying symptom during migraine attacks, is more prevalent in migraine with aura than in migraine without aura. Whether CSD plays a role in developing photophobia remains unknown. Moreover, migraine-induced physical hypoactivity contributes to loss of productivity. We aimed to investigate the development of trigeminal sensitisation, photophobia and locomotive abnormality after KCl-induced CSD using 86 male C57BL/6 mice. Sham-operated mice were used as controls. We confirmed the presence of trigeminal sensitisation and photophobia at 24 h after CSD. CSD-subjected mice also exhibited significantly reduced locomotive activity in both light and dark zones. Hence, the CSD-induced hypomobility was likely to be independent of photophobia. The 5-HT1B/1D agonist, sumatriptan, corrected all these CSD-induced abnormalities. Moreover, dose dependency was demonstrated in the ameliorating effect of the calcitonin gene-related peptide (CGRP) receptor antagonist, olcegepant, on these abnormalities. Sumatriptan and olcegepant improved mouse locomotion with therapeutic lags ranging from 20 to 30 min. Collectively, CSD caused trigeminal sensitisation, photophobia and hypomobility that persisted for at least 24 h by a mechanism involving the 5-HT1B/1D and CGRP activity.

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 diameter response of intraparenchymal penetrating arteries during cortical spreading depression and elimination of vasoreactivity to hypercapnia in anesthetized mice.

Cortical spreading depression (CSD) induces marked hyperemia with a transient decrease of regional cerebral blood flow (rCBF), followed by sustained oligemia. To further understand the microcirculatory mechanisms associated with CSD, we examined the temporal changes of diameter of intraparenchymal penetrating arteries during CSD. In urethane-anesthetized mice, the diameter of single penetrating arteries at three depths was measured using two-photon microscopy during passage of repeated CSD, with continuous recordings of direct current potential and rCBF. The first CSD elicited marked constriction superimposed on the upstrokes of profound dilation throughout each depth of the penetrating artery, and the vasoreaction temporally corresponded to the change of rCBF. Second or later CSD elicited marked dilation with little or no constriction phase throughout each depth, and the vasodilation also temporally corresponded to the increase of rCBF. Furthermore, the peak dilation showed good negative correlations with basal diameter and increase of rCBF. Vasodilation induced by 5% CO2 inhalation was significantly suppressed after CSD passage at any depth as well as hyperperfusion. These results may indicate that CSD-induced rCBF changes mainly reflect the diametric changes of the intraparenchymal arteries, despite the elimination of responsiveness to hypercapnia.

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.

Alterations in the threshold of the potassium concentration to evoke cortical spreading depression during the natural estrous cycle in mice.

Cortical spreading depression (CSD) has been implicated in a variety of neurological disorders. However, the relationship between serum sex hormones and susceptibility to the development of CSD in naturally estrous cycling female animals is largely unknown. The natural estrous cycle of mice consists of four stages, namely, proestrus, estrus, metestrus and diestrus. We measured the serum concentration of estradiol and progesterone in estrus and diestrus and compared the minimum potassium concentrations necessary to evoke CSD in each stage and in males. In diestrus, the minimum potassium concentration required to evoke CSD was significantly lower compared to the other three phases and male animals. The serum level of estradiol is significantly higher and serum level of progesterone is significantly lower in diestrus compared to estrus. Furthermore, when we administered an estrogen receptor antagonist, the susceptibility to the development of CSD was decreased. Conversely, the administration of a progesterone receptor antagonist increased the susceptibility to CSD. Our results demonstrated that neuronal excitability related to CSD induction differs among the natural estrous phases in mice.