Selective Disruption of Inhibitory Synapses Leading to Neuronal Hyperexcitability at an Early Stage of Tau Pathogenesis in a Mouse Model.

Synaptic dysfunction provoking dysregulated cortical neural circuits is currently hypothesized as a key pathophysiological process underlying clinical manifestations in Alzheimer's disease and related neurodegenerative tauopathies. Here, we conducted PET along with postmortem assays to investigate time course changes of excitatory and inhibitory synaptic constituents in an rTg4510 mouse model of tauopathy, which develops tau pathologies leading to noticeable brain atrophy at 5-6 months of age. Both male and female mice were analyzed in this study. We observed that radiosignals derived from [11C]flumazenil, a tracer for benzodiazepine receptor, in rTg4510 mice were significantly lower than the levels in nontransgenic littermates at 2-3 months of age. In contrast, retentions of (E)-[11C]ABP688, a tracer for mGluR5, were unaltered relative to controls at 2 months of age but then gradually declined with aging in parallel with progressive brain atrophy. Biochemical and immunohistochemical assessment of postmortem brain tissues demonstrated that inhibitory, but not excitatory, synaptic constituents selectively diminished without overt loss of somas of GABAergic interneurons in the neocortex and hippocampus of rTg4510 mice at 2 months of age, which was concurrent with enhanced immunoreactivity of cFos, a well-characterized immediate early gene, suggesting that impaired inhibitory neurotransmission may cause hyperexcitability of cortical circuits. Our findings indicate that tau-induced disruption of the inhibitory synapse may be a critical trigger of progressive neurodegeneration, resulting in massive neuronal loss, and PET assessments of inhibitory versus excitatory synapses potentially offer in vivo indices for hyperexcitability and excitotoxicity early in the etiologic pathway of neurodegenerative tauopathies.SIGNIFICANCE STATEMENT In this study, we examined the in vivo status of excitatory and inhibitory synapses in the brain of the rTg4510 tauopathy mouse model by PET imaging with (E)-[11C]ABP688 and [11C]flumazenil, respectively. We identified inhibitory synapse as being significantly dysregulated before brain atrophy at 2 months of age, while excitatory synapse stayed relatively intact at this stage. In line with this observation, postmortem assessment of brain tissues demonstrated selective attenuation of inhibitory synaptic constituents accompanied by the upregulation of cFos before the formation of tau pathology in the forebrain at young ages. Our findings indicate that selective degeneration of inhibitory synapse with hyperexcitability in the cortical circuit constitutes the critical early pathophysiology of tauopathy.

Inhibitory effects of caffeine on gustatory plasticity in the nematode Caenorhabditis elegans.

The effects of caffeine on salt chemotaxis learning were investigated using the nematode Caenorhabditis elegans. To estimate the degree of salt chemotaxis learning, nematodes were placed in a mixed solution of NaCl and caffeine, and then the chemotaxis index of NaCl was obtained from the nematodes placed on agar medium after pre-exposure to caffeine concentrations of 0.01, 0.1, 0.3, and 1.0%. Locomotor activity and preference behavior for caffeine were also estimated under these caffeine conditions. Nematodes pre-exposed to 0.3% caffeine showed inhibition of salt chemotaxis learning. Additional experiments indicated that nematodes showed a preference response to the middle concentration of caffeine (0.1%), with preference behavior declining in the 0.3% caffeine condition. Stable locomotor activity was observed under 0.01-0.3% caffeine conditions. These results suggest that salt chemotaxis learning with 0.3% caffeine is useful for investigating the effects of caffeine on learning in nematodes.

Vasodilation Mechanism of Cerebral Microvessels Induced by Neural Activation under High Baseline Cerebral Blood Flow Level Results from Hypercapnia in Awake Mice.

OBJECTIVE: We investigated the effects of the baseline CBF level at resting state on neurovascular coupling. METHODS: Diameters of arterioles, capillaries, and venulas in awake mouse brain were measured by a two-photon microscope. Vasodilation in each of the cerebral vessels was caused by three experimental conditions: (1) sensory stimulation, (2) 5% CO2 inhalation (hypercapnia), (3) simultaneous exposure to sensory stimulation and 5% CO2 inhalation. CBF and CBV were also measured by a microscope and a CCD camera. RESULTS: Increases in CBF and CBV were observed under all experimental conditions. After the increases in CBF and CBV due to hypercapnia, additional increases in CBF and CBV occurred during sensory stimulation. Diameter changes in arterioles were significantly larger than those in capillaries and venulas under both sensory stimulation and 5% CO2 inhalation. Additional vasodilation from sensory stimulation was observed under hypercapnia. The diameter change in each vessel type during sensory stimulation was maintained under simultaneous exposure to sensory stimulation and hypercapnia. CONCLUSIONS: The diameter change of cerebral vessels during neural activation is reproducible regardless of whether baseline CBF has increased or not. Our finding directly demonstrates the concept of uncoupling between energy consumption and energy supply during cortical activation.

Chronic nicotine exposure augments gustatory plasticity in Caenorhabditis elegans: involvement of dopamine signaling.

The chemotaxis of wild-type NaCl-conditioned nematodes exposed to 100 mM NaCl, maintained on a growth medium containing 0.3 mM nicotine from first larva to young adult (YA) hermaphrodite, was significantly weaker than the chemotaxis of those maintained on a medium without nicotine. The result indicates that chronic nicotine exposure augments gustatory plasticity. The gustatory plasticity was also augmented when tph-1 mutants, with a defect in serotonin biosynthesis, were maintained on a medium containing nicotine until the YA stage. Chronic nicotine exposure did not augment gustatory plasticity in bas-1 mutants, which had defects in both serotonin and dopamine biosynthesis, and in cat-2 mutants, which had a defect in dopamine biosynthesis. However, augmentation of gustatory plasticity was observed when bas-1 and cat-2 mutants were maintained on a growth medium containing nicotine along with dopamine, suggesting that dopamine signaling is involved in the augmentation of gustatory plasticity due to chronic nicotine exposure.

Role of the central nervous system in cell non-autonomous signaling mechanisms of aging and longevity in mammals.

Multiple organs orchestrate the maintenance of proper physiological function in organisms throughout their lifetimes. Recent studies have uncovered that aging and longevity are regulated by cell non-autonomous signaling mechanisms in several organisms. In the brain, particularly in the hypothalamus, aging and longevity are regulated by such cell non-autonomous signaling mechanisms. Several hypothalamic neurons have been identified as regulators of mammalian longevity, and manipulating them promotes lifespan extension or shortens the lifespan in rodent models. The hypothalamic structure and function are evolutionally highly conserved across species. Thus, elucidation of hypothalamic function during the aging process will shed some light on the mechanisms of aging and longevity and, thereby benefiting to human health.

Evaluation of cellular activity in response to sleep deprivation by a comprehensive analysis of the whole mouse brain.

Sleep deprivation (SD) causes several adverse functional outcomes, and understanding the associated processes can improve quality of life. Although the effects of SD on neuronal activity in several brain regions have been identified, a comprehensive evaluation of the whole brain is still lacking. Hence, we performed SD using two different methods, gentle handling and a dedicated chamber, in targeted recombination in active populations 2 (TRAP2) mice crossed with Rosa-ZsGreen reporter mice and visualized cellular activity in the whole brain. Using the semi-automated post-imaging analysis tool Slice Histology Alignment, Registration, and Cell Quantification (SHARCQ), the number of activated cells was quantified. From the analysis of 14 brain regions, cellular activity was significantly increased in the olfactory areas and decreased in the medulla by the two SD methods. From the analysis of the further subdivided 348 regions, cellular activity was significantly increased in the vascular organ of the lamina terminalis, lateral hypothalamic area, parabigeminal nucleus, ventral tegmental area, and magnocellular reticular nucleus, and decreased in the anterior part of the basolateral amygdalar nucleus, nucleus accumbens, septohippocampal nucleus, reticular nucleus of the thalamus, preoptic part of the periventricular hypothalamic nucleus, ventromedial preoptic nucleus, rostral linear nucleus raphe, facial motor nucleus, vestibular nuclei, and some fiber tracts (oculomotor nerve, genu of corpus callosum, and rubrospinal tract) by the two SD methods. Two subdivided regions of the striatum (caudoputamen and other striatum), epithalamus, vascular organ of the lamina terminalis, anteroventral preoptic nucleus, superior colliculus optic layer, medial terminal nucleus of the accessory optic tract, pontine gray, and fiber tracts (medial lemniscus, columns of the fornix, brachium of the inferior colliculus, and mammillary peduncle) were differentially affected by the two SD methods. Most brain regions detected from these analyses have been reported to be involved in regulating sleep/wake regulatory circuits. Moreover, the results from the connectivity analysis indicated that the connectivity of cellular activity among brain regions was altered by SD. Together, such a comprehensive analysis of the whole brain is useful for understanding the mechanisms by which SD and/or sleep disruption affects brain function.

Selective dysfunction of fast-spiking inhibitory interneurons and disruption of perineuronal nets in a tauopathy mouse model.

In Alzheimer's disease (AD), network hyperexcitability is frequently observed and associated with subsequent cognitive impairment. Dysfunction of inhibitory interneurons (INs) is thought to be one of the key biological mechanisms of hyperexcitability. However, it is still unknown how INs are functionally affected in tau pathology, which is a major pathology in AD. To clarify this, we evaluated the neuronal activity of cortical INs in 6-month-old rTg4510 mice, a model of tauopathy. Calcium imaging with mDlx enhancer-driven labeling revealed that neuronal activity in INs was decreased in rTg4510 mice. In the patch clamp recording, the firing properties of fast-spiking INs were altered so as to reduce their activity in rTg4510 mice. In parallel with microglial activation, perineuronal nets around parvalbumin-positive INs were partially disrupted in rTg4510 mice. Taken together, our data indicate that the excitability of cortical fast-spiking INs is decreased, possibly because of the disruption of perineuronal nets.

Distribution of intraperitoneally administered deuterium-labeled water in aquaporin-4-knockout mouse brain after middle cerebral artery occlusion.

Introduction: As the movement of water in the brain is known to be involved in neural activity and various brain pathologies, the ability to assess water dynamics in the brain will be important for the understanding of brain function and the diagnosis and treatment of brain diseases. Aquaporin-4 (AQP4) is a membrane channel protein that is highly expressed in brain astrocytes and is important for the movement of water molecules in the brain. Methods: In this study, we investigated the contribution of AQP4 to brain water dynamics by administering deuterium-labeled water (D2O) intraperitoneally to wild-type and AQP4 knockout (AQP4-ko) mice that had undergone surgical occlusion of the middle cerebral artery (MCA). Water dynamics in the infarct region and on either side of the anterior cerebral artery (ACA) was monitored with proton-density-weighted imaging (PDWI) performed on a 7T animal MRI. Results: D2O caused a negative signal change quickly after administration. The AQP4-ko mice showed a delay of the time-to-minimum in both the contralateral and ipsilateral ACA regions compared to wild-type mice. Also, only the AQP4- ko mice showed a delay of the time-to-minimum in the ipsilateral ACA region compared to the contralateral side. In only the wild-type mice, the signal minimum in the ipsilateral ACA region was higher than that in the contralateral ACA region. In the infarct region, the signal attenuation was slower for the AQP4-ko mice in comparison to the wild-type mice. Discussion: These results suggest that AQP4 loss affects water dynamics in the ACA region not only in the infarct region. Dynamic PDWI after D2O administration may be a useful tool for showing the effects of AQP4 in vivo.

MRS-measured glutamate versus GABA reflects excitatory versus inhibitory neural activities in awake mice.

To assess if magnetic resonance spectroscopy (MRS)-measured Glutamate (Glu) and GABA reflect excitatory and inhibitory neural activities, respectively, we conducted MRS measurements along with two-photon mesoscopic imaging of calcium signals in excitatory and inhibitory neurons of living, unanesthetized mice. For monitoring stimulus-driven activations of a brain region, MRS signals and mesoscopic neural activities were measured during two consecutive sessions of 15-min prolonged sensory stimulations. In the first session, putative excitatory neuronal activities were increased, while inhibitory neuronal activities remained at the baseline level. In the second half, while excitatory neuronal activities remained elevated, inhibitory neuronal activities were significantly enhanced. We assessed regional neurochemical statuses by measuring MRS signals, which were overall in accordance with the neural activities, and neuronal activities and neurochemical statuses in a mouse model of Dravet syndrome under resting condition. Mesoscopic assessments showed that activities of inhibitory neurons in the cortex were diminished relative to wild-type mice in contrast to spared activities of excitatory neurons. Consistent with these observations, the Dravet model exhibited lower concentrations of GABA than wild-type controls. Collectively, the current investigations demonstrate that MRS-measured Glu and GABA can reflect spontaneous and stimulated activities of neurons producing and releasing these neurotransmitters in an awake condition.

Exploring cell membrane water exchange in aquaporin-4-deficient ischemic mouse brain using diffusion-weighted MRI.

BACKGROUND: Aquaporin-4 is a membrane channel protein that is highly expressed in brain astrocytes and facilitates the transport of water molecules. It has been suggested that suppression of aquaporin-4 function may be an effective treatment for reducing cellular edema after cerebral infarction. It is therefore important to develop clinically applicable measurement systems to evaluate and better understand the effects of aquaporin-4 suppression on the living body. METHODS: Animal models of focal cerebral ischemia were created by surgically occluding the middle cerebral artery of wild-type and aquaporin-4 knockout mice, after which multi-b-value multi-diffusion-time diffusion-weighted imaging measurements were performed. Data were analyzed with both the apparent diffusion coefficient (ADC) model and a compartmental water-exchange model. RESULTS: ADCs were estimated for five different b value ranges. The ADC of aquaporin-4 knockout mice in the contralateral region was significantly higher than that of wild-type mice for each range. In contrast, aquaporin-4 knockout mice had significantly lower ADC than wild-type mice in ischemic tissue for each b-value range. Genotype-dependent differences in the ADC were particularly significant for the lowest ranges in normal tissue and for the highest ranges in ischemic tissue. The ADCs measured at different diffusion times were significantly different for both genotypes. Fitting of the water-exchange model to the ischemic region data found that the water-exchange time in aquaporin-4 knockout mice was approximately 2.5 times longer than that in wild-type mice. CONCLUSIONS: Multi-b-value multi-diffusion-time diffusion-weighted imaging may be useful for in vivo research and clinical diagnosis of aquaporin-4-related diseases.

Neurodegenerative processes accelerated by protein malnutrition and decelerated by essential amino acids in a tauopathy mouse model.

Protein malnutrition is epidemiologically suggested as a potential risk factor for senile dementia, although molecular mechanisms linking dietary proteins and amino acids to neurodegeneration remain unknown. Here, we show that a low-protein diet resulted in down-regulated expression of synaptic components and a modest acceleration of brain atrophy in mice modeling neurodegenerative tauopathies. Notably, these abnormal phenotypes were robustly rescued by the administration of seven selected essential amino acids. The up-regulation of inflammation-associated gene expression and progressive brain atrophy in the tauopathy model were profoundly suppressed by treatment with these essential amino acids without modifications of tau depositions. Moreover, the levels of kynurenine, an initiator of a pathway inducing neuroinflammatory gliosis and neurotoxicity in the brain, were lowered by treatment through inhibition of kynurenine uptake in the brain. Our findings highlight the importance of specific amino acids as systemic mediators of brain homeostasis against neurodegenerative processes.

The Utility of Applying Various Image Preprocessing Strategies to Reduce the Ambiguity in Deep Learning-based Clinical Image Diagnosis.

PURPOSE: A general problem of machine-learning algorithms based on the convolutional neural network (CNN) technique is that the reason for the output judgement is unclear. The purpose of this study was to introduce a strategy that may facilitate better understanding of how and why a specific judgement was made by the algorithm. The strategy is to preprocess the input image data in different ways to highlight the most important aspects of the images for reaching the output judgement. MATERIALS AND METHODS: T2-weighted brain image series falling into two age-ranges were used. Classifying each series into one of the two age-ranges was the given task for the CNN model. The images from each series were preprocessed in five different ways to generate five different image sets: (1) subimages from the inner area of the brain, (2) subimages from the periphery of the brain, (3-5) subimages of brain parenchyma, gray matter area, and white matter area, respectively, extracted from the subimages of (2). The CNN model was trained and tested in five different ways using one of these image sets. The network architecture and all the parameters for training and testing remained unchanged. RESULTS: The judgement accuracy achieved by training was different when the image set used for training was different. Some of the differences was statistically significant. The judgement accuracy decreased significantly when either extra-parenchymal or gray matter area was removed from the periphery of the brain (P < 0.05). CONCLUSION: The proposed strategy may help visualize what features of the images were important for the algorithm to reach correct judgement, helping humans to understand how and why a particular judgement was made by a CNN.

Deschloroclozapine, a potent and selective chemogenetic actuator enables rapid neuronal and behavioral modulations in mice and monkeys.

The chemogenetic technology designer receptors exclusively activated by designer drugs (DREADDs) afford remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities and potential off-target effects of CNO represent areas for improvement. Here, we provide a new high-affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 µg per kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 µg per kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents a potent, selective, metabolically stable and fast-acting DREADD agonist with utility in both mice and nonhuman primates for a variety of applications.

Water Diffusion in the Brain of Chronic Hypoperfusion Model Mice: A Study Considering the Effect of Blood Flow.

PURPOSE: Chronic cerebral hypoperfusion model mice were created by unilateral common carotid artery occlusion (UCCAO) surgery, which does not cause cerebral infarction, but which does cause long-term reduction in cerebral blood flow (CBF) to the occluded side. Cognitive dysfunction in this mouse model has been demonstrated in behavioral experiments, but neuron density change was not found in a previous positron emission tomography (PET) study. As a next step, in this study we investigated the injury of neuronal fibers in chronic cerebral hypoperfusion model mice using diffusion tensor imaging (DTI). METHODS: In diffusion-weighted imaging (DWI), not only the diffusion of water but also the capillary flow in the voxel, i.e., intravoxel incoherent motion (IVIM), contributes to the signal. Thus, we used DTI to evaluate DWI signal changes in the brains of chronic hypoperfusion model mice at 4 weeks after UCCAO while monitoring the possible influence of CBF change using arterial spin-labeling (ASL) MRI. RESULTS: Simple t-tests indicated that there were significant differences in CBF between the control and occluded sides of the brain, but there was no significant difference for the mean diffusivity (MD) or fractional anisotropy (FA). However, as Pearson correlation analysis showed that MD was strongly correlated with CBF, analysis-of-covariance (ANCOVA) was then performed using CBF as a covariate and a significant difference in MD between the contra- and ipsilateral sides was found. Performing a similar procedure for the FA found no significant differences. CONCLUSION: The results suggest the injury of neuronal fibers due to chronic hypoperfusion. It is also suggested that CBF-related signal changes should be considered when DWI-based information is used for pathological diagnosis.

Imaging of Neuronal Activity in Awake Mice by Measurements of Flavoprotein Autofluorescence Corrected for Cerebral Blood Flow.

Green fluorescence imaging (e.g., flavoprotein autofluorescence imaging, FAI) can be used to measure neuronal activity and oxygen metabolism in living brains without expressing fluorescence proteins. It is useful for understanding the mechanism of various brain functions and their abnormalities in age-related brain diseases. However, hemoglobin in cerebral blood vessels absorbs green fluorescence, hampering accurate assessments of brain function in animal models with cerebral blood vessel dysfunctions and subsequent cerebral blood flow (CBF) alterations. In the present study, we developed a new method to correct FAI signals for hemoglobin-dependent green fluorescence reductions by simultaneous measurements of green fluorescence and intrinsic optical signals. Intrinsic optical imaging enabled evaluations of light absorption and scatters by hemoglobin, which could then be applied to corrections of green fluorescence intensities. Using this method, enhanced flavoprotein autofluorescence by sensory stimuli was successfully detected in the brains of awake mice, despite increases of CBF, and hemoglobin interference. Moreover, flavoprotein autofluorescence could be properly quantified in a resting state and during sensory stimulation by a CO2 inhalation challenge, which modified vascular responses without overtly affecting neuronal activities. The flavoprotein autofluorescence signal data obtained here were in good agreement with the previous findings from a condition with drug-induced blockade of cerebral vasodilation, justifying the current assaying methodology. Application of this technology to studies on animal models of brain diseases with possible changes of CBF, including age-related neurological disorders, would provide better understanding of the mechanisms of neurovascular coupling in pathological circumstances.

Changes in effective diffusivity for oxygen during neural activation and deactivation estimated from capillary diameter measured by two-photon laser microscope.

The relation between cerebral blood flow (CBF) and cerebral oxygen extraction fraction (OEF) can be expressed using the effective diffusivity for oxygen in the capillary bed (D) as OEF = 1 - exp(-D/CBF). The D value is proportional to the microvessel blood volume. In this study, changes in D during neural activation and deactivation were estimated from changes in capillary and arteriole diameter measured by two-photon microscopy in awake mice. Capillary and arteriole vessel diameter in the somatosensory cortex and cerebellum were measured under neural activation (sensory stimulation) and neural deactivation [crossed cerebellar diaschisis (CCD)], respectively. Percentage changes in D during sensory stimulation and CCD were 10.3 ± 7.3 and -17.5 ± 5.3 % for capillary diameter of <6 µm, respectively. These values were closest to the percentage changes in D calculated from previously reported human positron emission tomography data. This may indicate that thinner capillaries might play the greatest role in oxygen transport from blood to brain tissue.

Short-term nicotine exposure induces long-lasting modulation of gustatory plasticity in Caenorhabditis elegans.

Nicotine administration induces many effects on animal behavior. In wild-type Caenorhabditis elegans, gustatory plasticity results in reduced chemotaxis toward NaCl of otherwise attractive concentrations after pre-exposure to 100 mM NaCl in the absence of food. However, acute nicotine administration during a 15 min pre-exposure period inhibits gustatory plasticity, whereas chronic nicotine administration during worm development facilitates the plasticity. To investigate the relationship between the duration of nicotine administration and its effects, we exposed worms to nicotine for various periods during development. The modulatory effect of nicotine on gustatory plasticity was gradually switched from inhibition to facilitation with increased duration of nicotine administration. Moreover, inhibition of plasticity was sustained after relatively short-term chronic administration, with effects lasting for 45 h after the removal of nicotine. Similar to the acute inhibitory effect after 15 min nicotine pre-exposure, the inhibitory effect after short-term chronic administration was dependent on the nicotinic acetylcholine receptor subunit genes lev-1 and unc-29, and genes involved in serotonin biosynthesis bas-1 and tph-1. The impaired inhibition in bas-1 and tph-1mutants was recovered by exogenous serotonin, demonstrating that serotonin plays an important role in the long-lasting inhibitory effects of short-term chronic nicotine exposure.

Food Search Strategy Changes in Caenorhabditis elegans under Chronic Starvation Conditions.

Starvation is a primary threat to survival in nature. This study investigated the effects of starvation on animal behavior and neural function using a nematode model. Nematodes exhibit chemotactic responses to various compounds, including diacetyl produced by food bacteria. Locomotion, chemotactic behavior, and olfactory adaptation were measured following chronic starvation. Our results revealed a starvation-dependent reduction in locomotor activity. Chemotaxis response to the odorant diacetyl was attenuated after 2-38 hr of starvation. However, chemotactic behavior increased significantly after 48 hr of starvation compared with that after 38 hr of starvation, suggesting that food search behavior was enhanced after 48 hr of starvation. Inhibition of diacetyl adaptation was observed in the nematodes after 48 hr of starvation. However, exogenous exposure to serotonin during 48 hr of starvation caused the inhibition of diacetyl adaptation to be attenuated in following 24 hr period of normal feeding.Therefore, the inhibitory effects of starvation on olfactory adaptation may reduce chemotaxis response to the odorant diacetyl in a manner mediated by serotonin.

Long-term effects of cerebral hypoperfusion on neural density and function using misery perfusion animal model.

We investigated the chronic effects of cerebral hypoperfusion on neuronal density and functional hyperemia using our misery perfusion mouse model under unilateral common carotid artery occlusion (UCCAO). Neuronal density evaluated 28 days after UCCAO using [(11)C]flumazenil-PET and histology indicated no neurologic deficit in the hippocampus and neocortex. CBF response to sensory stimulation was assessed using laser-Doppler flowmetry. Percentage changes in CBF response of the ipsilateral hemisphere to UCCAO were 18.4 ± 3.0%, 6.9 ± 2.8%, 6.8 ± 2.3% and 4.9 ± 2.4% before, and 7, 14 and 28 days after UCCAO, respectively. Statistical significance was found at 7, 14 and 28 days after UCCAO (P < 0.01). Contrary to our previous finding (Tajima et al. 2014) showing recovered CBF response to hypercapnia on 28 days after UCCAO using the same model, functional hyperemia was sustained and became worse 28 days after UCCAO.