We present electrophysiological (EP) signals correlated with cellular cell activities in the adrenal cortex and medulla using an adrenal gland implantable flexible EP probe. With such a probe, we could observe the EP signals from the adrenal cortex and medulla in response to various stress stimuli, such as enhanced hormone activity with adrenocorticotropic hormone, a biomarker for chronic stress response, and an actual stress environment, like a forced swimming test. This technique could be useful to continuously monitor the elevation of cortisol level, a useful indicator of chronic stress that potentially causes various diseases.
Deep brain stimulation (DBS) of the anterior nucleus of the thalamus (ANT) has been widely used as an effective treatment for refractory temporal lobe epilepsy. Despite its promising clinical outcome, the exact mechanism of how ANT-DBS alleviates seizure severity has not been fully understood, especially at the cellular level. To assess effects of DBS, the present study examined electroencephalography (EEG) signals and locomotor behavior changes and conducted immunohistochemical analyses to examine changes in neuronal activity, number of neurons, and neurogenesis of inhibitory neurons in different hippocampal subregions. ANT-DBS alleviated seizure activity, abnormal locomotor behaviors, reduced theta-band, increased gamma-band EEG power in the interictal state, and increased the number of neurons in the dentate gyrus (DG). The number of parvalbumin- and somatostatin-expressing inhibitory neurons was recovered to the level in DG and CA1 of naïve mice. Notably, BrdU-positive inhibitory neurons were increased. In conclusion, ANT-DBS not only could reduce the number of seizures, but also could induce neuronal changes in the hippocampus, which is a key region involved in chronic epileptogenesis. Importantly, our results suggest that ANT-DBS may lead to hippocampal subregion-specific cellular recovery of GABAergic inhibitory neurons.
Understanding the neurovascular coupling (NVC) underlying hemodynamic changes in epilepsy is crucial to properly interpreting functional brain imaging signals associated with epileptic events. However, how excitatory and inhibitory neurons affect vascular responses in different epileptic states remains unknown. We conducted real-time in vivo measurements of cerebral blood flow (CBF), vessel diameter, and excitatory and inhibitory neuronal calcium signals during recurrent focal seizures. During preictal states, decreases in CBF and arteriole diameter were closely related to decreased γ-band local field potential (LFP) power, which was linked to relatively elevated excitatory and reduced inhibitory neuronal activity levels. Notably, this preictal condition was followed by a strengthened ictal event. In particular, the preictal inhibitory activity level was positively correlated with coherent oscillating activity specific to inhibitory neurons. In contrast, ictal states were characterized by elevated synchrony in excitatory neurons. Given these findings, we suggest that excitatory and inhibitory neurons differentially contribute to shaping the ictal and preictal neural states, respectively. Moreover, the preictal vascular activity, alongside with the γ-band, may reflect the relative levels of excitatory and inhibitory neuronal activity, and upcoming ictal activity. Our findings provide useful insights into how perfusion signals of different epileptic states are related in terms of NVC.
Epilepsy is a brain disorder that is characterized by unprovoked seizures and often causes postictal neurological dysfunction. Cerebral hypoperfusion in the postictal state has been considered an underlying cause of neurological dysfunction associated with epilepsy, but its underlying mechanism remains unclear. Here, we investigated the causes of postictal hypoperfusion in a 4-aminopyridine-induced mouse seizure model via electrophysiological recording, laser Doppler flowmetry (LDF) and two-photon microscopy imaging. Hypoperfusion with a 30% reduction in cerebral blood flow (CBF) during the postictal period has two contributing factors: the early hypoperfusion up to ~30 min post-seizure was mainly induced by the constriction of arterial vessels, while hypoperfusion that persisted for over an hour was due to increased capillary stalling and decreased red blood cell (RBC) flow accompanied by constriction of capillaries and venules. In this condition, increased adhesion of neutrophils to brain capillaries was found in the postictal mouse brains. When the adhesion of neutrophils was suppressed by administration of an anti-Ly6G antibody specifically targeting neutrophils, recovery of the prolonged postictal vascular changes to control levels was observed. The expression of intercellular adhesion molecule-1 (ICAM-1) proteins was specifically increased, and an antibody against leukocyte function associated antigen-1 (LFA-1) on neutrophils, which mediates adhesive interactions with ICAM-1, could resolve the prolonged postictal CBF reductions. Taken together, our findings suggest that increased neutrophil adhesion to cerebral microvessels can substantially affect CBF in the epileptic brain, especially in the postictal state. Our data revealed that augmented adhesive interaction with ICAM-1 might be the mechanism underlying increased neutrophil adhesion in the postictal state. Reducing neutrophil adhesion by inhibiting this interaction may be a therapeutic approach to prevent prolonged postictal hypoperfusion and neurological dysfunction in epilepsy.
Cerebral hypoperfusion has been proposed as a potential cause of postictal neurological dysfunction in epilepsy, but its underlying mechanism is still unclear. We show that a 30% reduction in postictal cerebral blood flow (CBF) has two contributing factors: the early hypoperfusion up to ∼30 min post-seizure was mainly induced by arteriolar constriction, while the hypoperfusion that persisted for over an hour was due to increased capillary stalling induced by neutrophil adhesion to brain capillaries, decreased red blood cell (RBC) flow accompanied by constriction of capillaries and venules, and elevated intercellular adhesion molecule-1 (ICAM-1) expression. Administration of antibodies against the neutrophil marker Ly6G and against LFA-1, which mediates adhesive interactions with ICAM-1, prevented neutrophil adhesion and recovered the prolonged CBF reductions to control levels. Our findings provide evidence that seizure-induced neutrophil adhesion to cerebral microvessels via ICAM-1 leads to prolonged postictal hypoperfusion, which may underlie neurological dysfunction in epilepsy.
Chronic stress disrupts brain homeostasis and adversely affects the cerebro-vascular system. Even though the effects of chronic stress on brain system have been extensively studied, there are few in vivo dynamic studies on the effects of chronic stress on the cerebro-vascular system. In this study, the effects of chronic stress on cerebral vasculature and BBB permeability were studied using in vivo two-photon (2p) microscopic imaging with an injection of fluorescence-conjugated dextran. Our real-time 2p imaging results showed that chronic stress reduced the vessel diameter and reconstructed vascular volume, regardless of vessel type and branching order. BBB permeability was investigated with two different size of tracers. Stressed animals exhibited a greater BBB permeability to 40-kDa dextran, but not to 70-kDa dextran, which is suggestive of weakened vascular integrity following stress. Molecular analysis revealed significantly higher VEGFa mRNA expression and a reduction in claudin-5. In summary, chronic stress decreases the size of cerebral vessels and increases BBB permeability. These results may suggest that the sustained decrease in cerebro-vascular volume due to chronic stress leads to a hypoxic condition that causes molecular changes such as VEGF and claudin-5, which eventually impairs the function of BBB.
Understanding the intricate interplay among immune responses and homeostatic cell function in Alzheimer's disease (AD) remains challenging. Here, we present a novel strategy to mitigate AD pathology by directly modulating the immune checkpoint PD-1/PD-L1 signaling pathway in the brain, where elevated levels of microglial PD-1 and astrocytic PD-L1 have been observed. We found that a single intracortical injection of anti-PD-L1 antibody facilitates the infiltration of peripheral immune cells into the brain, including IL-10-secreting Ly6C+ monocytes. Subsequently, this leads to the restoration of microglial homeostatic functions including an increase in P2RY12 expression, which enhances microglial process extension. This cascade of events following anti-PD-L1 injection is crucial for regulating abnormally hyperactive neurons and reducing amyloid-beta plaques. These findings suggest that the direct application of immune checkpoint blockade in the brain could offer a new approach to managing the delicate cell-cell interactions among neurons, glial cells, and peripheral immune cells in the AD brain.
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