Academic Editor: Rafael Franco
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 DeadEndTM 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.
Ischemic stroke is a disease of the brain that can lead to disability and death in adults. In 85% of stroke cases, survivors suffer from long-term disability, such as movement and sensory disorders and impairment of learning and memory ability due to a variety of factors, such as blood-brain barrier (BBB) disruption, excitotoxic cell death, and edema formation [1, 2]. Ischemic stroke mainly involves occlusion of the middle cerebral artery (MCA), which provides sensory and motor areas with oxygen and nutrients. The therapeutic window for treating acute ischemic stroke is within 6 h [3], so our study focused on ischemic stroke during convalescence. Researchers have attempted to use a middle cerebral artery occlusion (MCAO) model to investigate the timing of BBB repair under pathological conditions, but an agreement on whether two or three weeks are needed has yet to be reached [4, 5]. Regardless of the duration of BBB repair, obvious cerebral ischemia/reperfusion injury can be observed after 24 to 72 h [6].
The BBB is a complex structure that is mainly composed of endothelial cells, pericytes, astrocytic foot processes, basement membrane and tight junctions (TJs) [7]. An intact BBB under physiological conditions and a repaired BBB under pathological conditions prevent the entry of peripheral inflammatory substances and regulate the exchange of toxins and nutrients between the brain and blood [8]. However, the BBB is an obstacle to the transport of large molecules into the brain, including those used for the treatment of CNS diseases such as stroke and Alzheimer’s disease (AD) [9]. Many experimental research studies have aimed to disrupt or bypass the BBB to deliver therapeutic drugs using, for example, focused ultrasound, mannitol, intranasal delivery and intraventricular injection [10, 11, 12], but these methods are difficult to apply in the clinic. Therefore, the exploration of practical methods that safely, effectively, noninvasively, and repeatedly promote the permeability of the BBB to facilitate drug delivery to the brain is both crucial and of significant clinical interest [13, 14].
The stimulation parameters of electronic acupuncture (EA), a combination of traditional Chinese medicine theory and modern electronic technology, can be used to treat strokes [15, 16]. Moreover, our previous study demonstrated that EA stimulation can open the BBB of rats with cerebral ischemia reperfusion induced by contusion while not inducing obvious brain edema [17]. In a recent systematic review, the therapeutic effect of Baihui (GV20)-based scalp acupuncture was confirmed in animal models of focal cerebral ischemia [18]. Another study indicated that multiple applications of EA at GV20 and Shuigou (GV26) could significantly alleviate cerebral infarction and enhance the sensorimotor ability of ischemic rats [19]. According to traditional Chinese medicine (TCM), because GV20 and GV26 are both on the “Du meridian”, they govern blood vessels. Recent studies have attempted to clarify the ability of EA stimulation at GV20 and GV26 to increase cerebral blood flow and improve cerebral vascular circulation [20, 21]. The acupuncture points, approach and parameters of EA can be adjusted to exert different effects on the CNS. Therefore, EA may be a reliable and effective method for treating CNS diseases such as stroke in the clinic [22].
Nerve growth factor (NGF), the first neurotrophic drug to be discovered, plays an active role in regulating neuronal development, differentiation, plasticity, cell death and survival [23]. However, the molecular weight of NGF is approximately 13.4 kDa, making it difficult to cross the BBB under normal conditions and reach its effective drug concentration to exert its therapeutic effect [24]. Thus, we believe that EA at GV20 and GV26 can be used to open the BBB and facilitate the entry of drugs such as NGF into the brain to treat CNS diseases. Our previous study showed that the dilatational waves delivered through EA at GV20 and GV26 can promote the passage of Evans blue (EB) through the BBB, indicating that similar passage of NGF through the BBB may be possible [25]. Subsequently, we conducted a preliminary study on the ability of EA to open the BBB with fluorescein isothiocyanate-dextran (FITC-dextran) as a tracer. Later, we found that 20 kDa FITC-dextran, which has a molecular similar to that of NGF, can also be induced to pass through the BBB via EA (data not shown). Inspired by these findings, we labeled NGF with FITC to allow visual observation of its entry into the brain.
Therefore, in this study, the effect of EA on facilitating the passage of NGF through the BBB and its influence on learning and memory in MCAO/R rats were assessed. These experiments were conducted in three parts. First, we determined the duration of BBB repair after MCAO. Second, based on the first experiment, EA was used with specific parameters to preliminarily explore its ability to induce NGF passage through the BBB and to clearly determine whether NGF is taken up by neurons. Finally, we assessed the therapeutic effects of NGF on learning and memory in MCAO/R rats under EA stimulation to provide a theoretical basis for the use of EA to induce the entry of CNS-targeting drugs through the BBB for the treatment of ischemic stroke.
Adult male Sprague-Dawley (SD) rats (3 months old) weighing 250–270 g were obtained from Zhejiang Chinese Medical University. All rats were kept on a 12-h light/dark cycle and provided ad libitum access to food and water. All procedures in this study were performed in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. All efforts were made to alleviate animal suffering, minimize the number of animals used, and utilize alternatives to in vivo techniques when possible.
The rats were subjected to MCAO using an intraluminal thread according to a
previously described method with some modifications. Briefly, the rats were
allowed free access to water and then anesthetized with sodium pentobarbital (50
mg/kg) intraperitoneally (i.p.). The right common carotid artery (CCA), external
carotid artery (ECA) and internal carotid artery (ICA) were carefully exposed and
isolated from the vagus nerve under sterile conditions. The MCA was occluded by
inserting a thread into the ICA through the CCA stump and advancing it until it
blocked the origin of the MCA. Body temperature was monitored and maintained at
36.5–37.5
Neurological deficit scores were determined by an examiner who was blinded to the group assignments 24 h after MCAO. A modified scoring system was used for this evaluation as follows: 0, no apparent neurological deficits (normal); 1, inability to extend the left forepaw when lifting the tail (mild deficit); 2, mild circling to the contralateral side when walking (moderate deficit); 3, slumping toward the contralateral (paralyzed) side (severe deficit); and 4, inability to walk autonomously without loss of consciousness (very severe deficit). MCAO was considered to be successfully induced in rats with a neurological deficit score of 1 to 3. In this study, 38 rats that died from pulmonary insufficiency or subarachnoid hemorrhage and asphyxia were eliminated.
Male SD rats were randomly divided into the following five groups (n = 13 rats each): the control group; the MCAO-24 hour group; the MCAO-72 hour group; the MCAO-2 week group; and the MCAO-3 week group. The rats in the MCAO groups underwent the MCAO-induction operation, while the control group did not. Each group was further randomly divided into three subgroups (A, B, and C), and the cerebral infarction volume, ultrastructural changes in the BBB and permeability of the BBB were assessed. The cerebral infarction volume was measured in the rats in the subgroups (n = 5 rats each). Ultrastructural changes in the BBB were observed using transmission electron microscopy in the rats in the B subgroups (n = 3 rats each). The permeability of the BBB was examined in the rats in the C subgroups (n = 5 rats each) after injection with 2% EB in saline via the caudal vein using an indwelling needle (4 mL/kg). The rats in each group were decapitated and brains were taken at corresponding time points. For instance, rats in MCAO-3 week group were sacrificed 3 weeks after MCAO surgery, and rats in the control group were sacrificed at the same time as MCAO-3 week group.
2,3,5-triphenyltetrazolium chloride (TTC, T8170, Solarbio, Beijing) staining was
exploited to assess the infarct volume. Briefly, rats were deeply anesthetized
with sodium pentobarbital intraperitoneally and sacrificed by
decapitation at the appropriate time point. The brains were rapidly dissected and
sectioned into five coronal slices with an approximate thickness of 2 mm; these
slices were then stained for 20 min at 37
The rats were anesthetized with sodium pentobarbital intraperitoneally.
After perfusion, the tissues of the ischemic penumbra (size:
1.0 mm
The brains of the rats in the C subgroups were sectioned into 30
The brains of rats in the C subgroups were placed in a small animal physiological signal telemetry device (IVIS Lumina LT, PerkinElmer, Waltham, MA, USA). The Cy5.5 channel was used, and ovals of the same size were drawn. Then, the mean fluorescence intensity with the ovals was measured to reflect the deposition of EB in the brain tissue.
The group in which the BBB was basically repaired based on experiment 1 was
further used as the MCAO/R model group (3 weeks after MCAO operation). These
MCAO/R rats were randomly divided into the MCAO model group and the MCAO model-EA
group. Additionally, a control group and control-EA group were included (n = 10
rats each). Acupuncture needles (length 25 mm, diameter 0.30 mm; Hwato, Suzhou
Medical Supplies Factory Co, Ltd, China) were inserted at GV20 (Baihui, the
center of the parietal bone) and GV26 (Shuigou, 1 mm below the tip of the nose)
in the rats in the EA groups. The needles were then connected to an acupuncture
point nerve stimulator (HANS-200, Nanjing Jinsheng, Ltd, China), and
stimulation with a frequency of 2/100 Hz and an intensity of 2
mA was administered for 40 min (a homemade relay cycled power to the electrode
for 6 sec on and 6 sec off). Each group was further randomly divided into two
subgroups (n = 5 rats each) based on the drug (NGF solution or FITC-NGF solution)
injected into the caudal vein. The rats in the A subgroups were injected with NGF
in PBS (10
NGF solution (XF8415011, R&D Systems, 10
A 0.15 mol/L NaCl solution was prepared and mixed with a molar amount of
NaHCO
Immunofluorescence analyses were performed according to standard protocols.
Briefly, the brains were isolated and postfixed in 4% PFA overnight at 4
FITC-NGF signal was observed in frozen 30
Based on the results of experiment 2, the frequency for delivering electronic
acupuncture was determined. The MCAO/R rats (3 weeks after MCAO operation) were
randomly divided into the MCAO model group, the MCAO model-NGF group, the MCAO
model-EA group and the MCAO model-EA-NGF group; a control group was also included
(n = 5 rats each). The rats in the MCAO model-NGF group and MCAO model-EA-NGF
group were injected with NGF (2018070423005, Hiteck, China) solution (10
Male SD rats were trained on the Morris water maze to assess their spatial
learning and memory. The water maze consisted of a circular pool measuring 180 cm
in diameter and was surrounded by 70 cm-high walls. The depth of the pool was 50
cm, and the water temperature was 23
Before the trial, the ability of the rats to swim to and climb onto the platform was evaluated. Rats that could not swim or only swam to but could not climb onto the platform were excluded. Next, the rats were randomly divided into groups.
In the learning phase of the experiment (days 1–5), each rat was placed individually on the platform for 30 sec. Then, for each of four trials they performed per day, the rats were placed in the tank at one of four entry points, i.e., north, south, east, or west, in turn. The entry point was different from the one used in the prior trial, but in any trial, the entry point could be the same for all rats. In each trial, the rats had 90 sec to reach the platform, on which they were allowed to stay for 30 sec. If a rat could not find the platform within the allowed time, it was guided to and placed onto the platform, and the time was recorded as 90 sec. After each trial, the rats were placed on the platform for 30 sec. On day 5, the results for each trial were recorded as a measure of spatial learning.
On day 6, a probe trial in which the platform was removed was conducted to assess spatial memory. During the probe trial, the rats were allowed to search for the platform for 90 sec. The SMART 3.0 system (Panlab, Barcelona, Spain) was used to record the number of times the rats crossed the former location of the platform and the swimming path.
On day 6, the rats were deeply anesthetized and perfused with PBS until the
brains exhibited no irritating odor. The brains were then cut into 4
Data are plotted in graphs as the means
After MCAO, extensive lesions developed in the lateral cortices of the rats.
Fig. 1A,B show that in the MCAO groups, cerebral infarctions of varying degrees
occurred in the region of the brain to which the middle cerebral artery supplies
blood and its vicinity. Normal tissue stained deep red, while the infarct area
appeared white. Previous our studies have found that sham surgery does not cause
brain infarction. Supplementary Fig. 1 demonstrates the cerebral infarction
volume is related to the insertion of thread during MCAO surgery, not other
surgical procedures. Fig. 1B shows that in the MCAO groups, the volume of the
cerebral infarctions was higher than that in the control group (p
BBB repaired time after MCAO operation. (A) Representative
images of brain sections stained with TTC. L, Left cerebral hemisphere. (B)
Summary of cerebral infarct size in brain. The infarct volume was expressed as
the percentage of the whole brain area. Data were expressed as mean
We also examined changes in the BBB ultrastructure and EB permeability. As shown
in Fig. 1C, transmission electron microscopy revealed that in rats in the control
group, the vascular lumen was normal in size and shape, and the vascular
endothelial cells were smooth, not swollen, and closely arranged. Additionally,
the basement membrane (BM) was intact and continuous. The nuclei were normal in
size and shape, and the structure of the mitochondria and endoplasmic reticulum
was intact. In the MCAO-24 hour group, MCAO-72 group and MCAO-2 week group, edema
and “vacuolization” could be seen around the vascular endothelial cells, and
the BM was incomplete or discontinuous. The nuclei were hyperchromatic, and some
mitochondria were swollen. However, in the MCAO-3 week group, the shape of the
vascular lumen was basically restored, and the thickness of the cell membrane was
essentially normal. Compared with that of the other groups, these edema around
the cells was significantly reduced in the MCAO-3 week group. Additionally, the
BM and the nuclear structure were basically intact, the perinuclear space was
uniform, and mitochondrial damage was minimal. To determine the permeability of
the BBB, we use EB as a tracer. As shown in Fig. 2A, under laser confocal
microscopy, fluorescence imaging revealed that in the control group, EB was
barely visible through the blood vessels in either cortical hemisphere. In the
rats in the MCAO groups, EB was not observed to penetrate the blood vessels on
the left side (nonischemic side), similar to the control group. On the right side
(ischemia-penumbra), however, transport of EB across the BBB, as evidenced by a
“lantern”-shaped pattern, could be seen in the MCAO-24 hour group, MCAO-72 hour
group, and MCAO-2 week group but not the control group. However, in the rats in
the MCAO-3 week group, this change in vascular permeability of
the right ischemic penumbra was not obvious. To calculate the fluorescence
intensity of EB in the rat cerebral cortex, a small animal physiological signal
telemetry device was used. The heat map revealed that the fluorescence intensity
in the control group was lower than that in the MCAO-24 hour group and MCAO-72
hour group (p
BBB repaired time after MCAO operation. (A) The permeability of
the BBB in the control, MCAO-24 hour, MCAO-72 hour, MCAO-2 week and MCAO-3 week
groups were observed under a fluorescence microscope, yellow arrows point to
vessels. Focal ischemia is in right cerebral hemisphere. (B) In bioluminescent
imaging of brains derived from rats injected with EB in vein tail under a small
animal physiologicalsignal telemetry device. Representative images are shown. (C)
The mean fluorescence intensity was taken to reflect the deposition of EB in the
brain tissue; ** p
Specific frequency EA stimulation can induce NGF and FITC-NGF
through BBB. (A) NGF content in the prefrontal cortex in the control,
control-EA, model, model-EA group;
We determined the specific frequency at which EA induced the most robust
enhancement of BBB permeability by measuring the level of NGF and FITC-NGF
penetration into the rat prefrontal cortex after EA stimulation. Fig. 3A shows
that NGF penetration in the control-EA group was higher than that in the control
group, but the difference was not significant (p
After identifying the specific frequency at which EA had the most robust effect
on BBB permeability, we used EA at this frequency to assess the passage of
FITC-NGF through the BBB. As shown in the fluorescence images of brain tissue
sections in Fig. 4, FITC-NGF showed lantern-like infiltration of the blood
vessels in the control-EA group and model-EA group but not in the control group
or model group. After evaluating FITC-NGF penetration through the blood vessels,
we speculated that it could be taken up by neurons. Therefore, we obtained
fluorescence images to assess FITC-NGF uptake. As shown in Fig. 5A,B, more
neuronal FITC-NGF uptake was observed in the control-EA group than in the control
group in the prefrontal cortex (p
Specific frequency EA stimulation can open the BBB and facilitate FITC-NGF trough vessel. Fluorescence images showing that FITC-NGF diffused around the blood vessels in the control-EA group, and model-EA group in the prefrontal cortex, but control and model group had no such phenomenon.
EA stimulation induces FITC-NGF uptake by prefrontal neurons
through blood vessels. (A) Fluorescence images showing that FITC-NGF uptake by
prefrontal neurons in the control-EA group, and model-EA group in the prefrontal
cortex, but control and model group had no obvious phenomenon. Positive cells
were shown in the yellow dotted box. (B) FITC-NGF uptake by prefrontal neurons in
control, control-EA, model and model-EA group. The mean positive cell rate was
used to assess the uptake of FITC-NGF by neurons. * p
EA stimulation can enhance BBB permeability, facilitating the uptake of NGF by
neurons by allowing it to pass through the BBB. Therefore, we further
investigated the effects of NGF entry into brain tissue. Fig. 6 shows changes in
the learning and memory ability in the rats. The control group traveled a shorter
distance and showed a shorter latency in finding the platform on day 5 than the
model group and the model-NGF group (p
Induction of NGF entry into the brain by EA can
promote learning and memory ability. (A) Trajectories diagram of finding target
platform in control, model, model-NGF, model-EA and model-EA-NGF group. (B) Time
on latency of finding target platform in each group; * p
Apoptosis of neurons in the hippocampus. (A) Fluorescence
images showing TUNEL staining in control, model, model-NGF, model-EA and
model-EA-NGF group. (B) Fluorescence analysis of apoptosis levels; ** p
Ischemic stroke is a common clinical disease associated with high mortality and disability rates. Therefore, it is vital to establish an animal model of the disease and identify potential treatments to improve the quality of life of patients. The MCA is involved in ischemic stroke. The pathological process initiated by occlusion of the MCA is similar to that of clinical stroke, and therefore this procedure is widely regarded as a standard method for inducing an animal model of focal cerebral ischemia. In this experiment, an MCAO/R model was established by using the Zea Longa method. Blood flow through the right MCA of rats was blocked and restored by inserting and removing an intraluminal thread, producing the MCAO/R model [26]. In this model, blood flow can be restored in a timely manner under waking conditions, affecting the rats little. Thus, it can be used for research on the recovery process that occurs after brain injury. However, there are some limitation in simulating stroke in 3-month-old rats, because stroke is common in middle-aged and elderly people. Other literatures demonstrate that BBB function can also be influenced by age [27]. Consistently, 24-month-old mice have significantly lower occludin levels and ZO-1 expression than those in young adult mice [28]. Therefore, the opening effect and mechanism of BBB in aged rats may be different from that in young rats.
In the acute stage after cerebral ischemia, i.e., from day 1 to day 3, BBB dysfunction first triggers cytotoxic edema within minutes followed by the onset of vasogenic edema associated with BBB breakdown [29]. The results of our study showed that the volume of cerebral infarction at 24 hours and 72 hours was significantly increased and tended to be lower at 3 weeks. This is consistent with the results from another research group, who observed severe brain damage and high permeability 24 hours and 72 hours after cerebral ischemia. Although some research groups indicate that 2 weeks of recovery after an MCAO operation is sufficient, others support the hypothesis that the BBB is essentially repaired only after 3 weeks [30, 31]. Our findings can be explained by the fact that AQP4-dependent transcellular water flux is critical to the movement of edema fluid across the astrocyte cell membrane in the glia limitans into the CSF [32]. In addition to inducing edema, cerebral ischemia also results in cellular reactions such as angiogenesis and the reconstruction of functional microvasculature to facilitate stroke recovery. VEGF and angiopoietins are crucial for angiogenesis and protection against ischemic injury [33], and the upregulation of VEGF expression not only promotes angiogenesis but also enhances microvascular permeability [34]. Interestingly, we found that EB was barely visible in the control group and on the nonischemic side in the MCAO group because the BBB was still intact, allowing it to limit entry of the EB-albumin complex. Similarly, the 3-week group did not demonstrate EB penetration of blood vessels, indicating that the barrier function of the BBB may have been restored. The heat map of the fluorescence intensity obtained in our study also supports this hypothesis. We used transmission electron microscopy to visually assess BBB structure and function after inducing MCAO, observing signs of endothelial cell swelling, mitochondrial membrane structural damage and surrounding cavitation necrosis, which is in line with studies demonstrating that connexin channels are targets for manipulating endothelial calcium dynamics in the brain and BBB permeability [35]. Furthermore, we observed fewer swollen mitochondria and relatively intact endothelial cell structures in the 3-week group. The pathological process here potentially involves salvaging tissue in the ischemic penumbra [36]. A recent review of the literature revealed that following cerebral ischemia, vascular permeability is changed and the BBB is damaged, leading to brain edema, inflammation, neuronal necrosis and apoptosis [37]. These outcomes are related to alterations in BBB ultrastructure and function [38]. However, the data obtained in this study showed recovery of the BBB ultrastructure, limited passage of EB through the BBB and a decreased brain edema volume in the 3-week group, suggesting that the BBB was basically repaired three weeks after MCAO.
The BBB, a physical and metabolic barrier essential for maintaining CNS homeostasis and preventing potentially harmful circulating substances from entering the brain, mainly comprises brain endothelial cells, pericytes, astrocytes, microglia, neurons, and extracellular matrix components. However, due to its low permeability, the BBB prevents drugs with molecular weights greater than 500 Da from entering the brain under physiological conditions [39]. Hence, a safe and effective method for increasing the permeability of the BBB is urgently needed. Previous studies have demonstrated the effectiveness of EA in increasing the permeability of the BBB in rats recovering from MCAO [40]. Current methods for opening the BBB include techniques based on ultrasound, hypertonic saline solutions and drugs. However, these opening methods have disadvantages, such as the generation of an inflammatory response, a short or uncontrollable opening time, and dose dependence [41, 42]. In contrast, EA stimulation increases the permeability of the BBB and can be regulated bidirectionally. Under physiological conditions, high-frequency EA can open the BBB to a certain extent while protecting the damaged BBB in pathological conditions [43]. Therefore, we selected safe and controllable EA as a means of opening the BBB. Additionally, ELISA revealed that with specific parameters, EA stimulation increased the amount of exogenous NGF in the brain under physiological and pathological conditions but did not affect the physical structure of the BBB, which is important for preserving cellular integrity and the health of the brain and the CNS [44]. To visually observe the passage of NGF across the BBB, we labeled the primary amine group of NGF with FITC, establishing a stable urea bond covalently to form FITC-NGF [45]. In general, FITC-NGF emits yellow-green fluorescence that permits tracing of the bonded NGF. The results revealed that EA at a specific frequency can open the BBB and induce the entry of FITC-NGF into the brain. We also observed very low levels of NGF and FITC-NGF in the control group, indicating the BBB has a strong barrier effect. Under the intervention of EA, NGF has an increasing trend in the control-EA group, and FITC-NGF can be detected directly by fluorescence method, showing that EA can also be used in physiological BBB. Moreover, because NGF binds to two types of membrane receptors, tropomyosin receptor kinase A (TrkA) and pan-neurotrophin receptor p75 (p75NTR) [46], we explored whether entry of exogenous NGF into the brain may be taken up by neurons. The findings agreed with our hypothesis; as anticipated, EA stimulation enhanced BBB permeability and induce NGF uptake by prefrontal neurons, suggesting a way for exogenous NGF to exert its neurotrophic effects in the CNS.
Increasing evidence has demonstrated that although it interacts with both
receptors, NGF has a high affinity for TrkA and a low affinity for p75 [47]. TrkA
has a positive effect, maintaining neuronal survival and growth [48], whereas p75
often has a negative effect, such as inducing apoptosis [49]; however, both are
single transmembrane proteins on cells [50]. More recent evidence confirmed that
I
This study was an extension of our earlier study in which 2/100 Hz EA was applied to the GV20 and GV26 acupoints for 40 min, effectively increasing BBB permeability in rats. Initially, our research team was inspired by the basic theory of traditional Chinese medicine (TCM), in which the GV20 (Baihui) and GV26 (Shuigou) acupoints are situated on the head and face and refresh and calm the mind following stimulation. A recent study suggested that certain nerves and blood vessels, such as the greater occipital nerve and branch of the frontal nerve, are superficially and richly distributed beneath GV20, which plays a role in regulating blood circulation due to its effect on the CNS. Meanwhile, the second branch of the trigeminal nerve and the buccal branch of the facial nerve are distributed under the GV26, and stimulation of this acupoint can stimulate the trigeminal nerve and facial nerve, acting on the brainstem to promote respiratory rhythm, hypertension and other brain functions [57]. Accordingly, we selected GV20 and GV26 as EA stimulation points and observed the effects on BBB permeability. According to previous experimental results, we preliminarily determined the parameters of EA stimulation required for stable opening of the BBB. It’s an interesting phenomenon that BBB opening in prefrontal cortex was obvious under EA at a specific frequency. Moreover, BBB permeability was most notable following the 8 min EA stimulation. The opening of BBB by EA is time-dependent, while the BBB was immediately closed after EA stimulation was removed. We also found that enhancement of BBB permeability may be related to disruption of interendothelial TJs, causing the formation of gaps between endothelial cells resulting from the activation of neurons that release SP and a decline in ZO-1 and occludin expression [58]. Therefore, we aimed to apply EA at currently used parameters to a pathological model such as the MCAO model to facilitate the entry of macromolecule drugs such as NGF into the brain and provide a new method for the treatment of CNS diseases. However, in the present study, the precise timing of the opening and closing of the BBB under EA stimulation was not determined. Additionally, the cumulative effect of specific parameters of EA on opening the BBB, the difference in brain region opening and the relationship between brain regions, such as the frontal cortex and hippocampus, will be the focus of our next research. Moreover, the specific frequency at which EA facilitates drug entry into the brain and whether the opening mechanism involves astrocytes, pericytes, enzymes, and diverse transport systems, such as P-glycoprotein (P-gp), require further research.
In conclusion, our results demonstrated that the BBB was basically repaired in rats 3 weeks after MCAO, which simulates the sequelae of cerebral ischemia in the clinic. For an intact BBB under physiological conditions and a repaired BBB under pathological conditions, methods to increase the permeability are vital to facilitate the entry of beneficial substances into the CNS. This paper showed that under EA stimulation at a specific frequency, NGF reached the rat brain through the BBB and was taken up by prefrontal cortex neurons, allowing it to exert its pharmacological effects. More critically, we showed that entry of NGF into the brain by EA stimulation at a specific frequency improved the learning and memory ability of rats and inhibited the apoptosis of neurons in the hippocampus, providing a new method for treating central nervous system diseases with macromolecule drugs.
YZ and XL conceived and designed the experiments. PG, SZ and XM performed the experiments. HW and LG analyzed the data. YZ drafted the manuscript. XL revised the manuscript. All authors contributed to the article and approved the submitted version.
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University (Approval No. 10635).
Not applicable.
This study was supported by the National Natural Science Foundation of China (no. 82174502).
The authors declare no conflict of interest.