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 DeadEnd TM 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.

Background:The blood-brain barrier (BBB) maintains the balance of the internal environment of the brain and strictly controls substance exchange between the brain and blood dynamically but stably. Transient increases in the permeability of the BBB plays an important role in helping macromolecular drugs enter the brain to exert their pharmacological effects. Previous research has revealed that electronic acupuncture (EA) stimulation connecting Baihui (GV20) and Shuigou (GV26) at a specific frequency can enhance the permeability of the BBB at 8 minutes after the intervention and induce the entry of 20 kDa fluorescein isothiocyanate-dextran (FITC-dextran) into the cerebral cortex, but whether it can also allow drugs to pass the BBB remains unknown. We hypothesized that EA at a specific frequency could open the BBB and induce the entry of nerve growth factor (NGF) into the brain to exert its therapeutic effect. Methods: First, the middle cerebral artery occlusion (MCAO) model is adopted and changes in the permeability and structure of the BBB are assessed by measuring both the intensity of Evans blue (EB) staining and the cerebral infarction volume, and by evaluating the ultrastructure of the BBB. Then, a laser spectrometer and immunofluorescence are used to observe entry of NGF into the brain. Finally, the learning and memory ability of rats are assessed and the DeadEnd TM Fluorometric TUNEL System is applied to assess apoptosis in the hippocampus. Results: Our results showed that, in the first, the BBB was essentially repaired three weeks after MCAO operation. Secondly, Electronic Acupuncture (EA) stimulation at a specific frequency can enhance BBB permeability in the prefrontal cortex and induce NGF uptake by prefrontal neurons. Finally, in the presence of EA stimulation, entry of NGF into the brain promoted learning and memory in rats and inhibited the apoptosis of neurons in the hippocampus. Conclusions: In this study, the timing of BBB repair in the MCAO model was determined under pathological conditions and the EA stimulation can induce the entry of NGF into the brain to exert its therapeutic effect. EA could serve as a new strategy for delivering therapeutics to the central nervous system (CNS), given that EA stimulation at a specific frequency was shown to increase the permeability of the BBB. Further study of the mechanism underlying the opening of the BBB and its timing is needed.

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.

The blood-brain barrier (BBB) maintains the balance of the internal environment of the brain and strictly controls substance exchange between the brain and blood dynamically but stably. Transient increases in the permeability of the BBB plays an important role in helping macromolecular drugs enter the brain to exert their pharmacological effects. Previous research has revealed that electronic acupuncture (EA) stimulation at a specific frequency can enhance the permeability of the BBB and induce the entry of 20 kDa fluorescein isothiocyanate-dextran (FITC-dextran) into the cerebral cortex, but whether it can also allow drugs to pass the BBB remains unknown. We hypothesized that EA at a specific frequency could open the BBB and induce the entry of nerve growth factor (NGF) into the brain to exert its therapeutic effect. To simulate the clinical apoplexy sequelae observed in patients and determine the basic timing of BBB repair under pathological conditions, we employed the middle cerebral artery occlusion (MCAO) model and assessed changes in the permeability and structure of the BBB by measuring both the intensity of Evans blue (EB) staining and the cerebral infarction volume and evaluating the ultrastructure of the BBB. Then, we used a laser spectrometer and immunofluorescence to observe entry of NGF into the brain. Finally, we assessed the learning and memory ability of rats and used the DeadEnd TM Fluorometric TUNEL System to assess apoptosis in the hippocampus. Our results showed that the BBB was essentially repaired three weeks after MCAO, indicating that EA stimulation at a specific frequency can enhance BBB permeability and induce NGF uptake by prefrontal neurons. In the presence of EA stimulation, entry of NGF into the brain promoted learning and memory in rats and inhibited the apoptosis of neurons in the hippocampus. In this study, the MCAO model was used to determine the timing of BBB repair under pathological conditions and assess the EA stimulation-induced entry of NGF into the brain to exert its therapeutic effect. EA could serve as a new strategy for delivering therapeutics to the central nervous system (CNS), given that EA stimulation at a specific frequency was shown to increase the permeability of the BBB. Further study of the mechanism underlying the opening of the BBB and its timing 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.