Modulating neuroinflammation through electroacupuncture: Mechanistic insights and pharmacological synergies.

Testing of a hypothesis for osmotic opening of the blood-brain barrier.

The blood-brain barrier (BBB) is an important structure for maintaining environmental stability in the central nervous system (CNS). Our previous study showed that specific parameters of electroacupuncture (EA) at the head points Shuigou (GV26) and Baihui (GV20) can open the BBB; however, the mechanism by which stimulation of body surface acupuncture points on the head results in peripheral stimulation and affects the status of the central BBB and the neuronal excitatory changes has not been elucidated. We used laser spectroscopy, the In Vivo Imaging System (IVIS), immunofluorescence and immunoblotting to verified the role of the trigeminal nerve in BBB opening during EA, and we applied the central N-methyl-D-aspartate (NMDA) receptors blocker MK-801 to verify the mediating role of NMDA receptors in EA-induced BBB opening. Next, electroencephalogram (EEG) and in vivo calcium imaging techniques were applied to verify the possible electrical patterns of BBB opening promoted by different intensities of EA stimulation. The results showed that the trigeminal nerve plays an important role in the alteration of BBB permeability promoted by EA stimulation of the head acupoints. Brain NMDA receptors play a mediating role in promoting BBB permeability during EA of the trigeminal nerve, which may affect the expression of the TJ protein occludin, and thus alter BBB permeability. The analysis of the electrical mechanism showed that there was no significant change in the rhythm of local field potentials (LFP) in different brain regions across frequency bands immediately after EA of the trigeminal nerve at different intensities. However, the local primary somatosensory (S1BF) area corresponding to the trigeminal nerve showed a transient reduction in the delta rhythm of LFP with no change in the high-frequency band, and the action potential (spike) with short inter spike interval (ISI) varied with EA intensity. Meanwhile, EA of the trigeminal nerve resulted in rhythmic changes in calcium waves in the S1BF region, which were influenced by different EA intensities. This study provides a research perspective and a technical approach to further explore the mechanism of EA-induced BBB opening and its potential clinical applications.

To observe the effect of electroacupuncture (EA) on blood-brain barrier (BBB) permeability and proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) in the hippocampus of vascular dementia (VD) rats, so as to explore the mechanism of EA on treatment of VD. SD male rats were randomly divided into sham operation, model, and EA groups, with 15 rats in each group. The VD rat model was established by permanently occlusion of the bilateral middle cerebral artery. Rats of the EA group received EA at "Baihui" (GV20), "Dazhui" (GV14), and bilateral "Shenshu"(BL23) for 30 min, 6 days a week for a total of 4 weeks. Morris water maze test was used to assess the cognitive function of rats. Evans blue staining was used to detect the BBB permeability, transmission electron microscopy and ELISA were used to detect the ultrastructure of BBB and the contents of hippocampal IL-1β and IL-18, respectively. Following modeling, compared with the sham operation group, the mean escape latency of model group was significantly prolonged (P<0.01), the times of crossing the platform were significantly decreased (P<0.01), the content of Evans blue, and the contents of IL-1β and IL-18 in hippocampus were increased (P<0.01). After the intervention, comparison between the model and EA groups showed that the average escape latency of rats in EA group was significantly shortened (P<0.01), the times of crossing the platform were increased (P<0.05), the content of Evans blue, and the contents of IL-1β and IL-18 in hippocampus were significantly decreased (P<0.01). The ultrastructure of BBB was moderately damaged in the model group, which was evidenced by blurred endothelial cell membrane structure, obviously dropsical astrocyte foot process, and decreased tight junctions. The ultrastructure of BBB was slightly damaged and astrocyte foot had no obvious edema in the EA group. EA can significantly improve the learning and memory ability of VD rats and improve the BBB permeability, which may be related to its effect in inhibiting the expression of IL-1β and IL-18 in the hippocampus.

Traumatic brain injury (TBI) is a major health and socioeconomic problem that affects all societies. Consciousness disorder is a common complication after TBI while there is still no effective treatment currently. The aim of this study was to investigate the protective effect of electro-acupuncture (EA) on cognitive recovery for patients with mild TBI. A total of 83 patients with initial Glasgow coma scale score higher than 12 points were assigned into this study. Then patients were randomly divided into 2 groups: EA group and control group (group C). Patients in group EA received EA treatment at Neiguan and Shuigou for 2 weeks. At 0 minute before EA treatment (T1), 0 minute after EA treatment (T2), and 8 weeks after EA treatment (T3), level of neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), hypoxia inducible factor-1α (HIF-1α), and malondialdehyde were tested by enzyme-linked immunosorbent assay. The score of Montreal Cognitive Function Assessment (MoCA) and mini-mental state examination (MMSE) as well as cerebral oxygen saturation (rSO2) were detected at the same time. Compared with the baseline at T1, the level of NSE, GFAP, HIF-1α, MDA, and rSO2 decreased, and the score of MoCA and MMSE increased in the 2 groups were significantly increased at T2-3 (P < .05). Compared with group C, the level of NSE, GFAP, HIF-1α, MDA, and rSO2 decreased, and the score of MoCA and MMSE increased were significantly increased at T2-3 in group EA; the difference were statistically significant (P < .05). EA treatment could improve the cognitive recovery for patients with mild TBI and the potential mechanism may be related to improving cerebral hypoxia and alleviating brain injury.

Objective: This study investigates the effects of electroacupuncture (EA) preconditioning on blood–brain barrier (BBB) integrity and matrix metalloproteinase-9 (MMP-9) expression in subsequent ischemic hemisphere.Methods: Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. Animals were randomly divided into four groups: normal, sham-operated, MCAO and EA groups. In EA group, rats received electroacupuncture stimuli at the Baihui acupoint (GV 20) 30 minutes/day for 5 days. Twenty-four hours after last treatment, the MCAO was performed. The brain water content and BBB permeability were measured 24 hours after MCAO. MMP-9 expression and activity were measured at 6, 12 and 24 hours after MCAO.Results: The results showed that the brain water content of ischemic hemisphere was lower in EA group (81.45 ± 1.09%) compared with MCAO group (83.98 ± 1.30%; p<0.05). Similarly, the Evans blue content in EA group (4.90 ± 1.77 μg/g) was lower compared with MCAO group (9.41 ± 2.87 μg/g; p<0.05). The protein expression and enzyme activity of MMP-9 increased and reached maximum at 24 hours after reperfusion. However, the protein expression was lower in EA group at 12 and 24 hours after reperfusion (p<0.01, versus MCAO group), and enzyme activity was lower in EA group only at 24 hours (p<0.01, versus MCAO group).Discussion: EA preconditioning could attenuate brain edema and BBB disruption caused by subsequent cerebral ischemia. EA preconditioning could decrease MMP-9 expression and activity, which may be an important mechanism of cerebral ischemic tolerance.

Background. Recombinant tissue plasminogen activator (rtPA) is the only recommended pharmacological treatment for acute ischemic stroke, but it has a restricted therapeutic time window. When administered at time points greater than 4.5 h after stroke onset, rtPA disrupts the blood-brain barrier (BBB), which leads to serious brain edema and hemorrhagic transformation. Electroacupuncture (EA) exerts a neuroprotective effect on cerebral ischemia; however, researchers have not clearly determined whether EA increases the safety of thrombolysis and extends the therapeutic time window of rtPA administration following ischemic stroke. Objective. The present study was conducted to test the hypothesis that EA extends the therapeutic time window of rtPA for ischemic stroke in a male rat model of embolic stroke. Methods. SD rats were randomly divided into the sham operation group, model group, rtPA group, EA+rtPA group, and rtPA+MEK1/2 inhibitor group. An injection of rtPA was administered 6 h after ischemia. Rats were treated with EA at the Shuigou (GV26) and Neiguan (PC6) acupoints at 2 h after ischemia. Neurological function, infarct volume, BBB permeability, brain edema, and hemorrhagic transformation were assessed at 24 h after ischemia. Western blotting and immunofluorescence staining were performed to detect the levels of proteins involved in the ERK1/2 signaling pathway (MEK1/2 and ERK1/2), tight junction proteins (Claudin5 and ZO-1), and MMP9 in the ischemic penumbra at 24 h after stroke. Results. Delayed rtPA treatment aggravated hemorrhagic transformation and brain edema. However, treatment with EA plus rtPA significantly improved neurological function and reduced the infarct volume, hemorrhagic transformation, brain edema, and EB leakage in rats compared with rtPA alone. EA increased the levels of tight junction proteins, inhibited the activation of the ERK1/2 signaling pathway, and reduced MMP9 overexpression induced by delayed rtPA thrombolysis. Conclusions. EA potentially represents an effective adjunct method to increase the safety of thrombolytic therapy and extend the therapeutic time window of rtPA administration following ischemic stroke. This neuroprotective effect may be mediated by the inhibition of the ERK1/2-MMP9 pathway and alleviation of the destruction of the BBB.

The blood-brain barrier (BBB) plays an important role in both the physiological state and pharmacological state of the brain. Transiently enhancing the permeability of the BBB may allow the use of more types of medications for neuropsychiatric diseases. Our previous research revealed that electroacupuncture (EA) stimulation at certain parameters can enhance the permeability of the BBB in Sprague-Dawley rats, but this phenomenon is not well characterized. We propose that specific frequency EA can transiently open the BBB and may be related to the change of tight junctions (TJ). To find the best EA frequency among commonly used frequencies, preliminarily explore the mechanism, we detected BBB permeability by measuring the intensity of Evans Blue and 20 kDa FITC-dextran fluorescence in the cerebral cortex. Then, we used a laser spectrometer, immunofluorescence, western blotting, and transmission electron microscopy to detect the mechanism of BBB opening. Finally, measured brain water content, AQP4, GFAP, Iba1, and used the DeadEndTM Fluorometric TUNEL System to clear whether the stimulation caused obvious negative effects. The results show that EA stimulation at 2/100 Hz maximally increased BBB permeability, and the BBB closed within 12 h after EA stimulation was removed. EA stimulation increased blood perfusion, c-fos levels, and Substance P expression in the cerebral cortex, decreased ZO-1 and occludin levels and induced ultrastructural changes in TJ morphology. EA stimulation at specific parameters did not cause brain edema, activation of glial cells, or cell apoptosis. This study shows that EA stimulation induces a reversible, frequency-dependent alteration of BBB permeability and proposes a hypothetical mechanism of BBB opening related to vasodilation and TJ disruption. Transiently enhancing the permeability of the BBB with EA at specific parameters may be a new strategies for delivering therapeutics to the central nervous system. Further study of this technology is needed.

The presence of the Blood–Brain barrier (BBB) makes it difficult for therapeutic agents to reach the brain to treat brain diseases. As an emerging treatment, electroacupuncture (EA)-mediated BBB opening can temporarily increase the permeability of the brain blood vessels to increase the concentration of the therapeutic agent in the brain parenchyma, which offers great clinical benefits. Recent studies have suggested that BBB opening with excessive exposure levels may cause tissue/cell damage and short-term behavioral changes. Here, we investigated whether the EA-mediated BBB opening cause serious adverse events in the normal rat brain tissue and motor behavior, such as brain tissue damage, histopathologic alteration, or aggravated behavioral changes. EA was performed on the accupoint GV26 and GV20. Evans Blue Assay (EBA) and fluorescein isothiocyanate (FITC)–dextran Assay were performed to assess the BBB permeability. Immunofluorescence of GFAP-positive cells was marked and Hematoxylin-eosin (HE) Staining was carried out for assessing brain cortex damage. Tunel Assay was conducted to assess cell apoptosis. ELISA was used to measure the contents of TNF-α and IL-1β in brain tissue to explore the inflammatory response. The open field test, balance beam test and Y-Maze test were carried out to assess and neural behaviour. EA can induce an increase in BBB permeability on rats, allowing the macromolecular tracer Evans blue (EB) and Fluorescein Isothiocyanate (FITC)–Dextran to enter the brain. Histological analysis indicated that there was no obvious red blood cell leakage and a cellular apoptotic response in the brain tissue of rats with EA treatment. In addition, EA stimulation at specific parameters did not impair the rats’ motor ability, balance and coordination, and short-term spatial learning and memory. Our results suggest that EA can safely and effectively open the BBB in rats without causing brain damage and behavioral memory impairment.

In this narrative review, a theoretical framework on the crosstalk between physical exercise and blood-brain barrier (BBB) permeability is presented. We discuss the influence of physical activity on the factors affecting BBB permeability such as systemic inflammation, the brain renin-angiotensin and noradrenergic systems, central autonomic function and the kynurenine pathway. The positive role of exercise in multiple sclerosis and Alzheimer’s disease is described. Finally, the potential role of conditioning as well as the effect of exercise on BBB tight junctions is outlined. There is a body of evidence that regular physical exercise diminishes BBB permeability as it reinforces antioxidative capacity, reduces oxidative stress and has anti-inflammatory effects. It improves endothelial function and might increase the density of brain capillaries. Thus, physical training can be emphasised as a component of prevention programs developed for patients to minimise the risk of the onset of neuroinflammatory diseases as well as an augmentation of existing treatment. Unfortunately, despite a sound theoretical background, it remains unclear as to whether exercise training is effective in modulating BBB permeability in several specific diseases. Further research is needed as the impact of exercise is yet to be fully elucidated.

1. Unlike some interfaces between the blood and the nervous system (e.g., nerve perineurium), the brain endothelium forming the blood-brain barrier can be modulated by a range of inflammatory mediators. The mechanisms underlying this modulation are reviewed, and the implications for therapy of the brain discussed. 2. Methods for measuring blood-brain barrier permeability in situ include the use of radiolabeled tracers in parenchymal vessels and measurements of transendothelial resistance and rate of loss of fluorescent dye in single pial microvessels. In vitro studies on culture models provide details of the signal transduction mechanisms involved. 3. Routes for penetration of polar solutes across the brain endothelium include the paracellular tight junctional pathway (usually very tight) and vesicular mechanisms. Inflammatory mediators have been reported to influence both pathways, but the clearest evidence is for modulation of tight junctions. 4. In addition to the brain endothelium, cell types involved in inflammatory reactions include several closely associated cells including pericytes, astrocytes, smooth muscle, microglia, mast cells, and neurons. In situ it is often difficult to identify the site of action of a vasoactive agent. In vitro models of brain endothelium are experimentally simpler but may also lack important features generated in situ by cell:cell interaction (e.g. induction, signaling). 5. Many inflammatory agents increase both endothelial permeability and vessel diameter, together contributing to significant leak across the blood-brain barrier and cerebral edema. This review concentrates on changes in endothelial permeability by focusing on studies in which changes in vessel diameter are minimized. 6. Bradykinin (Bk) increases blood-brain barrier permeability by acting on B2 receptors. The downstream events reported include elevation of [Ca2+]i, activation of phospholipase A2, release of arachidonic acid, and production of free radicals, with evidence that IL-1 beta potentiates the actions of Bk in ischemia. 7. Serotonin (5HT) has been reported to increase blood-brain barrier permeability in some but not all studies. Where barrier opening was seen, there was evidence for activation of 5-HT2 receptors and a calcium-dependent permeability increase. 8. Histamine is one of the few central nervous system neurotransmitters found to cause consistent blood-brain barrier opening. The earlier literature was unclear, but studies of pial vessels and cultured endothelium reveal increased permeability mediated by H2 receptors and elevation of [Ca2+]i and an H1 receptor-mediated reduction in permeability coupled to an elevation of cAMP. 9. Brain endothelial cells express nucleotide receptors for ATP, UTP, and ADP, with activation causing increased blood-brain barrier permeability. The effects are mediated predominantly via a P2U (P2Y2) G-protein-coupled receptor causing an elevation of [Ca2+]i; a P2Y1 receptor acting via inhibition of adenyl cyclase has been reported in some in vitro preparations. 10. Arachidonic acid is elevated in some neural pathologies and causes gross opening of the blood-brain barrier to large molecules including proteins. There is evidence that arachidonic acid acts via generation of free radicals in the course of its metabolism by cyclooxygenase and lipoxygenase pathways. 11. The mechanisms described reveal a range of interrelated pathways by which influences from the brain side or the blood side can modulate blood-brain barrier permeability. Knowledge of the mechanisms is already being exploited for deliberate opening of the blood-brain barrier for drug delivery to the brain, and the pathways capable of reducing permeability hold promise for therapeutic treatment of inflammation and cerebral edema.

Malignancies in the central nervous system (CNS) are among the most prevalent and lethal tumors. Tumor treating fields (TTFields), a physical therapeutic strategy, show significant potential in treating CNS tumors by inducing cell apoptosis, cell-cycle arrest, immune activation, and enhancing anti-PD-1 therapy efficacy. Additionally, TTFields can increase blood-brain barrier (BBB) permeability, further supporting their application in CNS malignancies. This review aims to summarize the advances and mechanisms of TTFields in CNS tumor treatment while addressing its current limitations and challenges. We reviewed existing literature on TTFields, focusing on their effects on glioma and brain metastasis (BM)-related primary tumors. The mechanisms investigated included mitosis and cell cycle interference, inhibition of cell migration and invasion, promotion of apoptosis and protective autophagy, activation of immunogenic cell death (ICD) and immune responses, and modulation of BBB permeability. TTFields demonstrate inhibitory effects on CNS malignancies, particularly in glioma. They also suppress brain metastasis from primary tumors such as lung cancer, breast cancer, melanoma, and colorectal cancer. Mechanistically, TTFields act through multiple pathways, including disrupting mitosis, impeding cell migration and invasion, enhancing apoptosis and autophagy, activating immune responses, and increasing BBB permeability. TTFields exhibit therapeutic potential in CNS malignancies, especially glioblastoma (GBM), through diverse biological mechanisms. Their ability to enhance BBB permeability and target metastatic tumors suggests promise for broader clinical applications, including brain metastasis treatment.

To investigate the molecular mechanisms underlying the beneficial effect of electroacupuncture (EA) in experimental models of Alzheimer's disease (AD) in vivo. Senescence-accelerated mouse prone 8 (SAMP8) mice were used as AD models and received EA at Yingxiang (LI 20, bilateral) and Yintang (GV 29) points for 20 days. For certain experiments, SAMP8 mice were injected intravenously with human fibrin (2 mg). The Morris water maze test was used to assess cognitive and memory abilities. The changes of tight junctions of blood-brain barrier (BBB) in mice were observed by transmission electron microscope. The expressions of fibrin, amyloid- β (Aβ), and ionized calcium-binding adapter molecule 1 (IBa-1) in mouse hippocampus (CA1/CA3) were detected by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), Western blot or immunohistochemical staining. The expression of fibrin in mouse plasma was detected by enzyme-linked immunosorbent assay. The expressions of tight junction proteins zonula occludens-1 and claudin-5 in hippocampus were detected by qRT-PCR and immunofluorescence staining. Apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Fibrin was time-dependently deposited in the hippocampus of SAMP8 mice and this was inhibited by EA treatment (P<0.05 or P<0.01). Furthermore, EA treatment suppressed the accumulation of Aβ in the hippocampus of SAMP8 mice (P<0.01), which was reversed by fibrin injection (P<0.05 or P<0.01). EA improved SAMP8 mice cognitive impairment and BBB permeability (P<0.05 or P<0.01). Moreover, EA decreased reactive oxygen species levels and neuroinflammation in the hippocampus of SAMP8 mice, which was reversed by fibrin injection (P<0.05 or P<0.01). Mechanistically, EA inhibited the promoting effect of fibrin on the high mobility group box protein 1 (HMGB1)/toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nicotinamide adenine dinucleotide phosphate (NADPH) signaling pathways (P<0.01). EA may potentially improve cognitive impairment in AD via inhibition of fibrin/A β deposition and deactivation of the HMGB1/TLR4 and RAGE/NADPH signaling pathways.

The blood-brain barrier (BBB) plays a significant pathophysiological role in multiple sclerosis (MS). Vasopressin (AVP) is released after brain injury and contributes to the inflammatory response. We propose that AVP may be modulating BBB permeability and hence affecting EAE clinical signs. Female Lewis rats were immunized s.c. with guinea-pig brain extract suspended in complete Freund’s adjuvant. Prior to that, animals were subjected to Neurointermediate pituitary lobectomy (NIL) or treated with AVP receptor antagonist (conivaptan). BBB permeability assays were performed. Western blot for claudin-5 and histological analysis were performed in conivaptan treated EAE rats. EAE increase in BBB permeability to Evans blue was reverted by the NIL surgery. AVP receptor blockade reverted the EAE BBB hyperpermeability to Evans blue and 10-kDa FITC-dextran in almost all brain regions. Both, AVP low levels and AVP receptor blockade attenuated EAE clinical signs. Conivaptan reduced perivascular cuffs in EAE rats. A decrease in claudin-5 expression was observed in EAE rats and conivaptan treatment partially restored normal levels. Our data indicate that V1a and V2 AVP receptors can modulate BBB permeability and consequently are involved in the CNS inflammatory process during EAE. Future research is required to characterize the utility of vasopressin antagonist in MS treatment.

Alzheimer’s disease (AD) is an irreversible and progressive neurodegenerative disease accompanied by aging, followed by memory impairment and cognitive decline. Although numerous attempts have been made to develop treatments for AD, most clinical trials have failed to delay or stop the progression of AD. Electroacupuncture (EA) is a complementary alternative medicine technique widely used to treat pain, inflammation, and neurodegenerative diseases. Additionally, blood-brain barrier (BBB) disruption is a known pathophysiology of neurodegenerative diseases, including AD. Moreover, amyloid beta deposition increases BBB permeability and produces inflammatory cytokines induced by glial activation. However, our previous study revealed that EA treatment at the Taegye acupoints (KI3) improves memory impairment through anti-neuroinflammation and increases glucose metabolism in 5XFAD mice. Therefore, we evaluated whether EA treatment at KI3 regulates BBB dysfunction in the prefrontal cortex of 5XFAD mice. For this study, 6.5-month-old 5XFAD mice were treated with EA stimulation at KI3 three times a week for two weeks. Western blotting, immunohistochemistry, and flow cytometry were used to evaluate the effects of EA treatment on BBB dysfunction. We found that EA stimulation attenuates BBB integrity by protecting BBB tight junction proteins (CD31, aquaporin 4, occludin, and claudin 5) in the prefrontal cortex of 5XFAD mice. In addition, EA treatment regulated inflammatory cytokines (IL-1α, IL-1β, IL-17, IL-23, IFN-ɣ, monocyte chemoattractant protein 1 (MCP-1), granulocyte-macrophage colony stimulating factors [GM-CSF], and IL-10) in the peripheral circulation of 5XFAD mice. Therefore, our data suggest that EA treatment could be a therapeutic agent for enhancing BBB dysfunction in AD.

Electroacupuncture prevents depression via PINK1/Parkin-driven suppression of NLRP3 activation.