A genetically targeted reporter for PET imaging of deep neuronal circuits in mammalian brains.

Positron emission tomography (PET) allows biomolecular tracking but PET monitoring of brain networks has been hampered by a lack of suitable reporters. Here, we take advantage of bacterial dihydrofolate reductase, ecDHFR, and its unique antagonist, TMP, to facilitate in vivo imaging in the brain. Peripheral administration of radiofluorinated and fluorescent TMP analogs enabled PET and intravital microscopy, respectively, of neuronal ecDHFR expression in mice. This technique can be used to the visualize neuronal circuit activity elicited by chemogenetic manipulation in the mouse hippocampus. Notably, ecDHFR-PET allows mapping of neuronal projections in non-human primate brains, demonstrating the applicability of ecDHFR-based tracking technologies for network monitoring. Finally, we demonstrate the utility of TMP analogs for PET studies of turnover and self-assembly of proteins tagged with ecDHFR mutants. These results establish opportunities for a broad spectrum of previously unattainable PET analyses of mammalian brain circuits at the molecular level.

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.

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.

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.

Multimodal sensory feedback associated with motor attempts alters BOLD responses to paralyzed hand movement in chronic stroke patients.

Electroencephalogram-based brain-computer interfaces (BCI) have been used as a potential tool for training volitional regulation of corticomuscular drive in patients who have severe hemiplegia due to stroke. However, it is unclear whether ERD observed while attempting motor execution can be regarded as a neural marker that represents M1 excitability in survivors of severe stroke. Therefore we investigated the association between ERD and the blood-oxygen-level-dependent (BOLD) fMRI signal during attempted movement of a paralyzed finger in stroke patients. Nine chronic stroke patients received BCI training for finger extension movement 1 h daily for a duration of 1 month. The sensorimotor rhythm was recorded from the sensorimotor area of the damaged hemisphere, and ongoing amplitude variations were monitored using a BCI system. Either a visual alert or the action of a motor-driven orthosis was triggered in response to ERD of the sensorimotor rhythm while patients attempted extension movements of the paralyzed fingers. Inter-subject covariance between ERD and the BOLD response in the sensorimotor areas was calculated. After BCI training, an increased ERD over the damaged hemisphere was confirmed in all participants while they attempted extension of the affected finger and this increase was associated with a BOLD response in primary sensorimotor area. Whole-brain MRI revealed that the primary sensorimotor area and supplementary motor area were activated in the damaged hemisphere after 1 month of BCI training. ERD reflects the BOLD responses of the primary motor areas in either hemisphere while patients who have severe chronic hemiplegia due to a stroke attempt an extension movement of the paralyzed fingers. One month of BCI can alter motor-related brain area activation. Combining BCI with other methods to facilitate such changes may help to implement BCI for motor rehabilitation after stroke.

Development of an ankle torque measurement device for measuring ankle torque during walking.

[Purpose] To develop a device for measuring the torque of an ankle joint during walking in order to quantify the characteristics of spasticity of the ankle and to verify the functionality of the device by testing it on the gait of an able-bodied individual and an equinovarus patient. [Subjects and Methods] An adjustable posterior strut (APS) ankle-foot orthosis (AFO) was used in which two torque sensors were mounted on the aluminum strut for measuring the anterior-posterior (AP) and medial-lateral (ML) directions. Two switches were also mounted at the heel and toe in order to detect the gait phase. An able-bodied individual and a left hemiplegic patient with equinovarus participated. They wore the device and walked on a treadmill to investigate the device's functionality. [Results] Linear relationships between the torques and the corresponding output of the torque sensors were observed. Upon the analyses of gait of an able-body subject and a hemiplegic patient, we observed toque matrices in both AP and ML directions during the gait of the both subjects. [Conclusion] We developed a device capable of measuring the torque in the AP and ML directions of ankle joints during gait.

Randomized controlled comparative study on effect of training to improve lower limb motor paralysis in convalescent patients with post-stroke hemiplegia.

[Purpose] The motor paralysis-improving effect on the hemiplegic lower limb was compared among mirror therapy, integrated volitional-control electrical stimulation, therapeutic electrical stimulation, repetitive facilitative exercises, and the standard training method in post-stroke hemiplegia patients. [Subjects and Methods] Eighty one stroke patients admitted to a convalescent rehabilitation ward were randomly allocated to the above 5 treatment groups. Each patient performed functional training of the paralytic lower limb for 20 minutes a day for 4 weeks, and changes in the lower limb function were investigated using the Stroke Impairment Assessment Set. [Results] The hip and knee joint functions did not significantly improve in the standard training control group, but significant improvements were observed after 4 weeks in the other intervention groups. Significant improvement was noted in the ankle joint function in all groups. [Conclusion] Although the results were influenced by spontaneous recovery and the standard training in the control group, the hip and knee joints were more markedly improved by the interventions in the other 4 groups of patients with moderate paralysis, compared to the control group.

How effective is the early fast treadmill gait speed training for stroke patients at the 2nd week after admission: comparison with comfortable gait speed at the 6th week.

[Purpose] The purpose of this study was to find whether a fast treadmill gait training speed is effective for the gait training of stroke patients in the early rehabilitation stage. [Subjects and Methods] Thirty-nine stroke patients were the subjects of our investigation. They walked on a treadmill with handrail supports at a fast speed (130% of their comfortable gait speed in the 2nd week). The treadmill gaits of the patients were recorded using a 3-dimensional analysis system at two and six weeks after their admissions. Intraclass Correlation Coefficients (ICC) of the temporal and spatial parameters of the two periods were statistically analyzed. [Results] For all of the patients, the ICCs of the measured parameters were greater than 0.58. In the case of patients whose gait speeds of the two periods were close, the ICC units were greater than 0.7. [Conclusion] The fast gait speed training allowed us to expose the patients to a gait speed that they were expected to acquire at a later stage of their rehabilitation. This training method was found to be beneficial for the mildly paralyzed patients.

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.

Energy analysis reveals the negative effect of delays in passive movement mirror therapy.

Wavelet transform energy analyses of the mean and standard error of the electromyogram (EMG) and electroencephalogram (EEG) of eight subjects were investigated in passive movement mirror therapies with no delay (in-phase) and with delay (out-of-phase) situations in two frequency bands of 7.81-15.62 and 15.62-31.25 Hz. It was found that the energy levels of EEG at electrode C4 in the in-phase situation were lower than those in out-of-phase situations, while the energy levels of flexor and extensor forearm muscle groups were larger. With two exceptions, this pattern could be seen in all other subjects. The difference between the in-phase (D0) and out-of-phase situations (D025 and D05) for the frequency range of 15.62-31.25 Hz was found to be significant at a significance level of 0.05 (paired t-test analysis). The respective elevation and decline of EEG and EGM with regard to the increase of the delay may indicate the necessity for synchronization of passive movement and mirror therapy.

Vessel specific imaging of glucose transfer with fluorescent glucose analogue in anesthetized mouse cortex.

The present study examined glucose transfer in the cellular scale of mouse brain microvasculature in vivo using two-photon microscopy and fluorescent glucose analogue (2-NBDG). The 2-NBDG was intravenously injected (0.04 mL/min) in the anesthetized Tie2-GFP mice in which the vascular endothelium expressed fluorescent protein. Time-lapse imaging was conducted on the cortical parenchyma, while the time-intensity change of the injected 2-NBDG was analysed in respective vascular compartments (artery, capillary, and vein). We observed that 2-NBDG signal increased monotonically in the vasculature during the period of the injection, and rapidly declined following its cessation. In tissue compartment, however, the signal intensity gradually increased even after cessation of the injection. Spatiotemporal analysis of the 2-NBDG intensity over the cross-sections of the vessels further showed distinct change of the 2-NBDG intensity across the vessel wall (endothelium), which may represents a regulation site of tissue glucose influx.

Recovery process of standing postural control in hemiplegia after stroke.

[Purpose] The aim of this study was to investigate the recovery process of standing postural control in hemiplegia after stroke. [Subjects and Methods] Thirty-four inpatients with hemiparesis after first-onset stroke were included in this study. We measured the center of pressure fluctuations during quiet standing using a force platform at 2, 4, and 6 weeks after admission. We assessed weight-bearing asymmetry, and velocity and amplitude of body sway. [Results] Weight-bearing asymmetry diminished in the first 2 weeks of observation. Velocity of body sway also decreased significantly in the first 2 weeks, though its amplitude only decreased significantly after 4 weeks of observation. [Conclusion] Amplitude of body sway requires a longer time for significant improvement than weight-bearing asymmetry and velocity of body sway. Although the loading function of the paretic lower limb improved at an early stage, attainment of optimum postural control, including management of the affected paretic lower limb, requires much time.

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.

Multi-input/output alarming system for patients with inattention caused by higher cortical function disorder.

BACKGROUND: To apply advanced methods of communication, sensing, and instrumentation technologies to make a system that can help patients suffering from hemispatial neglect caused by higher cortical function disorder. METHOD: By using several sensors and actuators, the objective was to construct a tailor-made system for each patient. The input part of the system consists of sensors, an interface and transmitters. The output part consists of a receiver, logical arithmetic, an output interface and actuators. The information from the input part is sent to the output part in a wireless manner allowing the mobility of the input and output parts. RESULTS: The system and its functionality were realized. Voice alarming and neck muscle stimuli were applied to two patients. We could verify the applicability of the system to remind the patients to put on their wheelchair's brake and raise its footrest before attempting to stand for transferring to their beds. CONCLUSION: The designed and constructed multi-input/output system can be used effectively to alarm the patients.

Age-related differences in cognitive function for interlimb coordination during split-belt walking: a pilot study.

While walking is considered a task demanding cognitive functions rather than an automatic motor task, it is still unclear whether cognitive tasks influence interlimb coordination during walking. The present study revealed that the elderly require cognitive function to control temporal interlimb coordination under difficult walking conditions. Risk of falls is thus likely to be increased in the elderly when performing attention-demanding tasks while walking.

Use of a cane for recovery from backward balance loss during treadmill walking.

PURPOSE: To study whether a cane improved balance recovery after perturbation during walking. METHOD: This study was a crossover comparison comparing the effect of walking with and without a cane for balance recovery after perturbation during treadmill walking. Five normal young volunteers participated. The velocity and acceleration of a marker sited on the seventh cerebral vertebra (C7) and vertical hand motion were measured by a motion analysis system. RESULT: When using a cane, C7 backward velocity increased by approximately 15% (413 SD 95 mm/s with cane vs. 358 SD 88 mm/s without). In addition, C7 backward acceleration increased by approximately 23% (3.2 SD 0.7 m/s(2) with cane vs. 2.6 SD 0.8 m/s(2) without) and the vertical motion of the right hand decreased (187 SD 98 mm with cane vs. 372 SD 260 mm without). Additionally, no subject was able to use a cane to broaden their base of support. CONCLUSIONS: The ability to limit trunk extension is crucial for preventing falls. Therefore, using a cane jeopardizes recovery from backward balance loss. The results encourage further research on the risk of a cane on balance recovery for the elderly population and habitual cane users.