Electroacupuncture Enhances Cognition by Promoting Brain Glucose Metabolism and Inhibiting Inflammation in the APP/PS1 Mouse Model of Alzheimer’s Disease: A Pilot Study

Abstract

Background:

Alzheimer’s disease (AD) is a neurodegenerative disease, yet there is no effective treatment. Electroacupuncture (EA) is a complementary alternative medicine approach. In clinical and animal studies, EA promotes cognition in AD and vascular dementia. It has been previously reported that cognitive decline in AD might be closely related to reduced glucose intake in the brain. It is worth mentioning that the regions of glucose hypometabolism are usually found to be associated with neuroinflammation.

Objective:

This study is to explore whether the protective mechanism of EA on cognition is related to the regulation of glucose metabolism and neuroinflammation.

Methods:

APP/PS1 mice were randomly divided into AD group and the treatment (AD + EA) group. In the AD + EA group, EA was applied on Baihui (GV20) and Yintang (GV29) for 20 min and then pricked at Shuigou (GV26), once every alternate day for 4 weeks. Morris water maze (MWM) tests were performed to evaluate the effects of EA treatment on cognitive functions. ¹⁸F-FDG PET, immunofluorescence, and western blot were used to examine the mechanisms underlying EA effects.

Results:

From MWM tests, EA treatment significantly improved cognition of APP/PS1 mice. From the ¹⁸F-FDG PET, the levels of uptake rate of glucose in frontal lobe were higher than the AD group after EA. From immunofluorescence and western blot, amyloid-β (Aβ) and neuroinflammation were reduced after EA.

Conclusion:

These results suggest that EA may prevent cognitive decline in AD mouse models by enhancing glucose metabolism and inhibiting inflammation-mediated Aβ deposition in the frontal lobe.

Keywords

Alzheimer’s disease cognition electroacupuncture frontal lobe glucose metabolism neuroinflammation

INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disease that is associated with old age and has become a major burden on health care systems worldwide [1, 2]. It is still one of the urgent problems in the medical field to explore the pathogenesis of AD and find effective ways to prevent it. Neuroimaging has significant advantages in the diagnostic accuracy of AD, as well as the evaluation of therapeutic effects [3]. ¹⁸F-FDG PET is a well-established imaging technique for assessing cognitive decline in AD that can quantify neuropathology in the brain by using glucose analogues. Studies have suggested that cognitive impairment to a certain extent results from decreased cerebral glucose metabolism in AD patients [4]. It is especially interesting that the regions of glucose hypometabolism are usually found to be associated with neuroinflammation [5], and that is a key part of the etiopathogenesis of AD [6]. Microglia, the resident phagocytes of the innate immune system in the central nervous system, can be activated by the pathological process of AD, such as Aβ. Activated microglial cells play a role in the recognition and phagocytosis of Aβ [7]. While the initial inflammatory response is conducive to ameliorating neuronal injury, prolonged abnormal microglial activation potentially exacerbates neurodegeneration and has a series of harmful effects.

Despite new advances in understanding the neurobiology and pathophysiology of AD, only a handful of drugs are currently available for patients. Based on this consideration, many non-drug therapies have been proposed for prevention. Acupuncture is a therapeutic approach that has curative effects and few side effects. EA is widely used due to its adjustable strength and easy quantification in China compared to manual acupuncture [8]. In recent years, a functional MRI study confirmed that acupuncture can activate certain cognitive-related regions in AD patients, and acupuncture improved learning-memory ability in AD mice [9, 10]. In addition, we have previously found that the expressions of Aβ in the hippocampus of APP/PS1 mice were reduced after EA therapy, as well as cognitive impairment was improved, suggesting that EA intervention is helpful to the cognitive decline of AD [11, 12, 11, 12].

The aim of the current study was to investigate the mechanism of EA in enhancing cognition. We hypothesized that EA at Governor Vessel (GV) acupoints would alter cerebral glucose metabolism in a manner associated with neuroinflammation to enhance cognition. In this study, 6-month-old APP/PS1 mice were used, which was a well-established transgenic AD mouse model. First, we assessed the learning and memory abilities of the AD animals after EA treatment using the MWM test. Next, we evaluated changes in glucose metabolism in the frontal lobe using ¹⁸F-FDG PET. Finally, the changes of Aβ and inflammatory response in the frontal lobe were tested using immunofluorescence and western blot. The schematic diagram of this study is detailed in Fig. 1.

Fig.1

Schematic diagram of this study to detect the effect of EA on AD.

MATERIALS AND METHODS

Experimental animals

6-month-old male APP/PS1 mice and age- and gender-matched C57BL/6N mice were purchased from Beijing HFK Bioscience Company, Ltd. (experimental animal license number: SCXK (Jing) 2014-0004). The mice weighed 28.0±2.0 g and were housed separately in standard mouse cages at the Experimental Animal Center of Beijing University of Chinese Medicine. The room temperature was maintained at 24±2°C, and the humidity was 40–60%. The mice were maintained on a standard 12 h light-dark cycle (dark cycle 8 : 00 PM–8 : 00 AM). All mice had ad libitum access to food and water. The bedding material was replaced daily to keep the cages clean and dry. The experiment began 8 days after the animals entered the room so that the mice could acclimate to the housing conditions. Every effort was made to minimize the suffering of the mice during the experimental procedure.

Apparatuses

The primary apparatuses are listed in Table 1.

Table 1

The primary apparatus used in this study

Apparatus	Source	Details
Disposable sterile acupuncture needle	Beijing Zhongyan Taihe Medical Instrument, Co., Ltd	Model: ZYTH2013030504; specification: 0.25×13 mm
Electroacupuncture (EA) apparatus	Peking University Institute of Science Nerve and Beijing Hua Wei Industrial Development Company	Model: HANS-LH202
Morris water maze	Shanghai Xinruan Information Technology, Co., Ltd	Model: XR-XM101
Software of image acquisition and analyzing system	China Daheng Group, Inc.	China Daheng Group, Inc., Beijing Image Vision Technology Branch
Micro-PET scanner	the Chinese Medicine Research Institute PET Room	–
PET imaging system	Siemens INVEON PET/CT imaging system	–

Animal grouping and intervention

A total of twenty APP/PS1 mice were randomly and equally divided into AD model (AD) and EA treatment (AD + EA) groups (n = 10 per group). Ten C57BL/6N mice composed the control group (C).

In this experiment, GV20, GV29, and GV26 were selected based on traditional Chinese medicine (TCM) theory and our previous studies. First of all, acupoints have a near-therapeutic effect. The three acupoints are all located at the head, so they have the effect of regulating mental activities. In addition, according to the theory of TCM, GV and brain are closely related. GV20, GV29, and GV26 are the important points in the GV, so stimulation at the three acupoints can improve brain function. The acupuncture points GV20, GV29, and GV26 on the mice were located based on the Acupoint Standard for Experimental Animals.

In the AD + EA group, EA was applied once every alternate day for 4 weeks. Mice in the C and AD groups were immobilized for 20 min when EA was performed. To immobilize the mice, we made special bags based on the size of the mice. After the mice crawled inside the bags, two clips were put in place to close the open backs of the bags, which absolutely controlled and immobilized the mice well. As the front third of each bag was made of mesh, the experimental mice could breathe unimpeded. The acupoint locations and operations of EA in this study are shown in Table 2 and Fig. 2 [12].

Table 2

Acupoints location and operation of EA in this study

Acupoint	Location	Operation of EA
GV20	On the top of the head, at the midpoint of the line connecting the ears.	First, insert needle obliquely in an upward direction to a depth of 5 mm. Then, connect the needle to the EA apparatus set to a frequency of 2 Hz and a current of 1 mA. Continue the intervention for 20 min.
GV26	On the face, in the upper one-third of the philtrum.	Deliver a fast prick after the end of EA treatment.
GV29	On the forehead, in the depression between the two eyebrows medial end.	Insert needle obliquely in a downward direction to a depth of 5 mm. The other operations are the same as for GV20.

Fig.2

The location of GV acupoints applied in this study. Red points indicate the locations of GV20, GV26, and GV29 on the head of mice (left: top view; right: side view).

MWM test

The MWM test system consists of a water maze device, water maze image automatic acquisition, and software analysis system. The water maze device is the main site of the experiment, which consists of a circular pool (diameter: 90 cm; height: 50 cm; divided into four quadrants: I, II, III, and IV) and a removable circular platform. The camera is placed 2 m above the center of the pool to automatically collect the swimming images of the animal. These signals are directly input into the computer for automatic analysis and processing by the automatic image acquisition and analysis system (Daheng Group, China). The experimental surroundings are required to maintain sound insulation, and the objects in the room were unchanged for the duration of the test. During the formal experiment, the pool was filled with water up to 30 cm depth, and the addition of milk powder rendered the water opaque. A thermometer was used to monitor the water temperature that was maintained at 20±1°C, keeping the indoor light intensity consistent during the experiment.

In training trials, the platform was fixed in the center of quadrant III (the target quadrant), located 2 cm below the water surface. The animals were first placed on the platform for 10 s in each training trial. Then, the mice were gently placed into the water from quadrants I, II, and IV. The entry point is the equal distance to the center of the pool in quadrants I, II, and IV. The time of finding the platform was recorded. When the mouse stayed on the platform for 5 s, the collection stopped automatically, which was deemed successful, and time was recorded. If the animal failed to climb onto the platform within 60 s, the escape latency was recorded as 60 s. The training trial lasted 4 days.

A probe trials were performed 1 day after the last training trial. In the probe trials, the platform was removed, and the animals were gently placed into the water from the entry points of quadrants I, II, and IV. The swimming trajectory and the duration spent in quadrant III were recorded. This spatial probe trial was used to evaluate the spatial memory ability of the mice.

Micro-PET imaging

Four mice from each group were randomly selected for micro-PET imaging. Before the micro-PET scan, we first monitored the blood glucose of the mice. When the levels of blood glucose were in the normal range (7.0–10.0 mmol/L), we could start experiments. Before the micro-PET scan, the mice were deprived of water for 6 h. After the mice were completely anesthetized with isoflurane inhalation (2% in 100% oxygen, 1 L/min). ¹⁸F-FDG tracer at a dose of 14.8–16.5 MBq was injected via the tail vein. After the injection for 60 min, the mouse was placed on the scan bed in the prone position, the mouse and scanner long axis were parallel, and the head of the mouse was located within the scanner field of view. Then, the micro-PET began to collect the image. The scan lasted 10 min, and mice wore breath mask with isoflurane continuously carried to ensure entirely unconscious during this progress.

Micro-PET captured a single frame image, and then filtered back projection and CT photon attenuation correction were used for image reconstruction (pixel size 0.2×0.2×0.8 mm, 30 s/frame). Next, the three-dimensional region of interest selection (ROIs) of PET/CT images in the frontal lobe were selected manually by the experimenter, which were in transverse, coronal, and sagittal planes. Finally, we calculated the uptake rate per gram with ROIs.

Immunofluorescence

Twenty-four hours after micro-PET scanning, four mice in each group were randomly anesthetized. After heart perfusion, brains were fixed in 4% phosphate-buffered paraformaldehyde for 24 h, followed by ethanol dehydration, xylene treatment for transparency, paraffin embedding, and coronal sectioning. Slices were incubated at 4°C with anti-Iba-1 (1 : 2000, ab178847, Abcam, England), anti-IL-1β (1 : 100, ab9722, Abcam, England), and anti-IL-10 (1 : 100, ab9969, Abcam, England) antibodies. After permeabilization and blocking overnight, appropriate secondary antibodies (GB21303, Servicebio, China) were used at a dilution of 1 : 300. After the sections were washed 3 times with PBS, they were incubated with DAPI (G1012, Servicebio, China) for 10 min, followed by live imaging. We captured and scanned the frontal lobe images with a confocal laser microscope (FV1000, Olympus, Japan) at 200× and 400× magnification.

Western blot

Twenty-four hours after micro-PET scanning, six mice in each group were randomly anesthetized, and the frontal lobe was collected. Tissues were lysed with RIPA lysis buffer (Sainobo, China). The protein concentration was determined using a bicinchoninic acid assay kit (Sainobo, China) according to the manufacturer’s instructions. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto PVDF membranes at 4°C at 300 mA for 2 h. The levels of Aβ₄₀, Aβ₄₂, Iba-1, IL-1β, and IL-10 were measured by incubating with the primary antibodies against Aβ₄₀ (1 : 1000, ab17295, Abcam, England), Aβ₄₂ (1 : 2000, ab201060, Abcam, England), Iba-1 (1 : 2000, ab178847, Abcam, England), IL-1β (1 : 2000, ab9722, Abcam, England), and IL-10 (1 : 2000, ab9969, Abcam, England) at 4°C overnight. GAPDH (1 : 20000, YM3029, Immunoway, China) was used as an internal control. Subsequently, the membranes were washed with TBST and incubated with the corresponding secondary antibody for 1 h at room temperature. The immunoreactive bands were detected using a chemiluminescence gel imaging system (C600, Azure Biosystems, USA). Quantitative results are expressed as a ratio of target proteins to GAPDH and then compared to all groups to measure relative changes.

Statistical analysis

All data are expressed as the means±SD. Statistical analysis was performed using IBM SPSS Statistics 20 software. Comparisons between groups were analyzed using repeated measures two-way ANOVA in the training trial. One-way ANOVA followed by the least significant difference (LSD) multiple-range test was used to analyze group differences in the space probe trial and the uptake rate of ¹⁸F-FDG and WB. Statistical significance was set at p < 0.05, and high statistical significance was set to p < 0.01.

RESULTS

EA treatment alleviated cognitive impairments in APP/PS1 mice

To assess the spatial learning ability of the mice, a training trial was conducted from day 1 to day 4 (Fig. 3A, B). We recorded the time spent by the mice in searching to climb onto the platform to escape the water (escape latency). The mice in the AD group showed a longer escape latency than the C group. The results suggested that the ability of spatial learning in the AD group had obvious deficits. However, the escape latency of the AD + EA group gradually shortened with the training time. Compared to day 1, the mice from the AD + EA group showed significantly lower escape latency on days 3 and 4 (Fig. 3C).

Fig.3

The MWM device and the results of the MWM test. A) The MWM device. B) The mouse was tested with a MWM apparatus. C) Training trial results. D) Space probe trial results. E) Representative swimming trajectories of the three groups (the black squares indicate the water entry points of mice.) n = 10 per group, the results are presented as the means±SD. ^★★Compared with Day 1, p < 0.01; ^▴▴Compared with C group, p < 0.01; ^♦♦Compared with AD group, p < 0.01.

After assessing the spatial learning ability, the maintenance of memory was tested by a probe trial on day 5. The longer duration that the mice stayed in quadrant III (platform quadrant), the better their memory retention [13]. Compared with the C group, the AD group showed a significantly lower platform crossing number and duration percentage in quadrant III. After EA treatment, the AD + EA group showed a significantly longer duration in quadrant III compared with the AD group (Fig. 3D). Representative probe traces of each group intuitively reflected the search strategy of mice (Fig. 3E). The above results demonstrated that 6-month-old APP/PS1 mice had significant cognitive impairment, and EA treatment ameliorated cognitive decline by enhancing spatial learning and memory.

EA treatment enhanced glucose metabolic activity in the frontal lobe of APP/PS1 mice

After observing that EA alleviated cognitive impairments, we investigated the mechanism responsible for enhancing cognition. The dysregulation of energy metabolism in the brain is a significant causative factor in the development of AD. A reduction in glucose metabolism could result in neuronal dysfunction. ¹⁸F-FDG PET displays the rate of cerebral glucose metabolism, and the ROIs for PET data analysis are a well-established evaluation of the metabolic rate of glucose [14]. From the image, the same color standard and color code from top high to bottom low were used to display the metabolic rate of the glucose. ROIs of PET images in the frontal lobe of APP/PS1 mice were shown in Fig. 4A. Obviously, the metabolic rates of glucose from micro-PET scanning in the C and AD + EA groups were higher than that in the AD group (Fig. 4B). The ROI data confirmed that the uptake rates of ¹⁸F-FDG in the frontal lobe of the AD group were significantly lower than that in the C group, which recovered after EA stimulation in the AD + EA group (Fig. 4C).

Fig.4

The frontal lobe of APP/PS1 mice in each group using ¹⁸F-FDG PET scanning images. A) ROIs of the frontal lobe of mice are shown in red. B) The frontal lobe of ¹⁸F-FDG PET scanning image of each group. Color code: min = 0, max = 6.5. C) Comparison of the uptake rate of ¹⁸F-FDG per gram in the frontal lobe of each group. n = 4 per group, the results are presented as the means±SD. ^▴▴Compared with C group, p < 0.01; ^♦Compared with AD group, p < 0.05.

EA treatment attenuated Aβ deposition in the frontal lobe of APP/PS1 mice

As Aβ deposition contributed to AD pathogenesis, we hypothesized that the improvement of glucose metabolism could be attribute to the reduction of Aβ load. To test this hypothesis, we measured expressions of Aβ₄₀ and Aβ₄₂ by western blot. The results showed that the levels of both Aβ₄₀ and Aβ₄₂ in the frontal lobe of AD + EA group were significantly markedly reduced following EA treatment as compared with levels in the AD group (Fig. 5).

Fig.5

Effects of EA on Aβ deposition in the frontal lobe of APP/PS1 mice in each group. A) The expression levels of Aβ₄₀ were determined by western blotting analysis. B) The expression levels of Aβ₄₂ were determined by western blotting analysis. GAPDH was used as a loading control. n = 6 per group, the results are presented as the means±SD. ^▴▴Compared with C group, p < 0.01; ^♦Compared with AD group, p < 0.05.

EA treatment inhibited inflammatory response in the frontal lobe of APP/PS1 mice

We hypothesized that the accumulation of Aβ could be due to an inflammatory response. The abnormal activation of microglia in the brain appeared crucial to the pathogenesis of Aβ [6, 15]. As expected, we clearly observed the localization of microglia, Iba-1 immunopositive areas in the frontal lobe were detected in the mice of the AD group (Fig. 6A). Western blot results showed that Iba-1 of the frontal lobe in the AD group were obviously higher than that in the C group, which decreased after EA stimulation in the AD+EA group (Fig. 6B). These results suggested that microglial activation were present in the brains of 6-month-old APP/PS1 mice. We further confirmed the inflammatory response-inhibiting effect of EA by measuring inflammatory cytokines. Activated microglia was determined by repertoire of pro-inflammatory cytokines in brain environment. Therefore, we next examined the effect of EA treatment on the key cytokines, such as IL-1β. The results showed that compared with AD group, the level of pro-inflammatory cytokine IL-1β in AD + EA group was decreased when treated with EA (Fig. 7), and reversely, the level of anti-inflammatory cytokine IL-10 in AD + EA group was significantly increase after EA intervention (Fig. 8). Taken together, these findings showed that EA treatment inhibited inflammation in the frontal lobe of APP/PS1 mice.

Fig.6

Effects of EA on microglial activation in the frontal lobe of APP/PS1 mice in each group. A) Microglia (red) were detected in the frontal lobe region using immunofluorescence. n = 4 per group. Scale bar = 100μm, magnification×200. B) The expression levels of Iba-1 were determined by western blotting analysis. GAPDH was used as a loading control. n = 6 per group, the results are presented as the means±SD. ^▴▴Compared with C group, p < 0.01; ^♦Compared with AD group, p < 0.05.

Fig.7

Effects of EA on expression of pro-inflammatory cytokine IL-1β in the frontal lobe of APP/PS1 mice in each group. A) IL-1β (red) were detected in the frontal lobe region using immunofluorescence. n = 4 per group. Scale bar = 50μm, magnification×400. B) The expression levels of IL-1β were determined by western blotting analysis. GAPDH was used as a loading control. n = 6 per group, the results are presented as the means±SD. ^▴▴Compared with C group, p < 0.01; ^♦Compared with AD group, p < 0.05.

Fig.8

Effects of EA on expression of anti-inflammatory cytokine IL-10 in the frontal lobe of APP/PS1 mice in each group. A) IL-10 (red) were detected in the frontal lobe region using immunofluorescence. n = 4 per group. Scale bar = 50μm, magnification×400. B) The expression levels of IL-10 were determined by western blotting analysis. GAPDH was used as a loading control. n = 6 per group, the results are presented as the means±SD. ^▴▴Compared with C group, p < 0.01; ^♦Compared with AD group, p < 0.05.

DISCUSSION

In the current work, our findings supported the notion that EA therapy exerted beneficial effects on cognition in AD mice by stimulating GV acupoints, at least partially through the mechanism that involved glucose metabolism elevation and neuroinflammation inhibition in the frontal lobe.

Despite growing acknowledgement of AD, effective and safe therapeutic interventions remain to be found. Many people focus now on the prevention of cognitive impairments that may lead to AD. EA is one of the complementary alternative medicine techniques, which shows clinically neuroprotective effects in patients with mild cognitive impairment [16]. According to the theory of meridians and collaterals, brain function is closely related to the GV [17]. Recent studies have shown that regulating GV can promote the compensation and recombination of brain function [18, 19]. Most studies chose GV20 as the main acupoint, which might be related to the specific neural response pattern induced by EA at acupoints [20]. Herein, we use GV20, GV26, and GV29, which are attached to the GV. The function of these acupoints are restoring consciousness, regulating mentality, and benefiting intelligence, and in clinical practice, they are often used to treat diseases of the central nervous system [21 –23]. The combination of these acupoints, which we call the method of “dredging the governor vessel to wake up the mind” in accordance with the theory of TCM, is an effective therapy for cognitive decline.

Cognitive dysfunction is the core symptom of AD patients. The cognition process in the brains of animals could not be observed directly. The changes could be predicted only according to the observed stimulus response. Cognitive abilities can be analyzed by measuring the performance or reaction time of the model at a specific interval after learning or performing a certain task [24]. In this experiment, the mice were trained to learn to find the hidden platform in the fixed position underwater for stable spatial position cognition [25, 26]. AD mice performed worse than controls and showed a random search strategy for the target (platform) without the obvious inclination. This phenomenon indicated that the learning and memory abilities of APP/PS1 mice at the age of 6 months were markedly degraded, enthusiasm to avoid water was decreased, and physical fitness and activity were reduced. After EA treatment, they performed better, thereby suggesting that EA promoted the cognition of mice.

However, it should be emphasized that the cognitive function is a very complex process that is the result of whole brain activity. Therefore, the complex cognitive process should not be simply located in a specific brain region [27, 28]. Previous studies on AD have focused on the hippocampus [29]. Recent studies have found that the transcriptional levels of some factors closely related to brain aging change with age (in months) only in the frontal lobe [30]. It reported that early cognitive deficits in AD mice were related to the frontal cortex before hippocampus-dependent impairments [31]. This suggests that there may be some important changes in the frontal lobe different from the hippocampus and basal forebrain that are not yet known. The frontal lobe, which makes up roughly one-third of the cerebral lobe, is the most recently developed and highly evolved part of the brain and is responsible for the integration of all senses and perceptions. The prefrontal lobe is closely related to higher cognitive function [32]. The frontal lobe contains many neurotransmitters and receptors, such as 5-HT [33], adenosine receptors [34]. Also, prefrontal lobe function is regulated by dopamine neurons [35], which can improve the excitability of frontal cortex pyramidal neurons and plays an important role in synaptic plasticity [36, 37]. The receptor of dopamine in the frontal lobe is mainly D1, which can reduce the release of glutamate in L5 pyramidal cells of the prefrontal cortex when stimulated [38]. They are related to attention, prediction, short-term memory tasks, reasoning, decision-making, emotion and other high-level psychological activities, and play a very important role in the long-term memory maintenance of nontasks. In previous work, we conducted extensive studies of the hippocampus. Therefore, our focus in this study is on the frontal lobe.

Glucose is a key source of energy for brain tissue in mammals and maintains the normal function of neurons, including cognition. However, neurons cannot synthesize or store glucose, so they depend only on glucose input. Cognitive deficits in AD are usually associated with changes in glucose metabolism in the brain [14]. PET provides a noninvasive way to quantify brain glucose uptake, and ¹⁸F-FDG is the most commonly used radioactive tracer of brain glucose [39]. In this study, we used ¹⁸F-FDG PET as an approach to assess the effects of EA on the frontal lobe. In the AD group, ¹⁸F-FDG metabolism in the frontal lobe was lower than that in the C group but increased after EA treatment. Reduced glucose utilization in specific brain regions in AD mice may be due to Aβ deposition and excessive neuronal loss; the former is not conducive to the use of glucose, and the latter leads to a direct decrease in glucose metabolism [40]. In turn, abnormal glucose metabolism can lead to an increase in the production of Aβ, which is a vicious cycle. In our previous study, EA therapy was demonstrated to reduce Aβ in the hippocampus [11]. Insulin resistance exists in the AD brain, so that the brain tissue’s ability to absorb glucose is significantly reduced. The above results indicated that EA had a protective effect on AD directly or indirectly by promoting glucose metabolism in the frontal lobe, which might be related to the regulation of the PI3K/AKT signaling pathway [41].

Growing evidence suggested that inflammatory processes driven by microglia contribute to the pathogenesis of AD. We discovered that EA treatment inhibited inflammatory response. It is known that microglia plays a detrimental role in AD. Inflammatory response mediated by activated microglia is a major contributor to neurodegeneration [42]. Additionally, microglial activation can secret pro-inflammatory cytokine, such as IL-1β [43], which reduces LTP by inhibition of glutamate release in aged rats [44]. It is worth noting that Aβ is the main component of senile plaques, the characteristic pathological product of AD [45], and its excess production or aggregation is the key for the pathogenesis of AD [46]. Studies found that Aβ constantly stimulated the activation of microglia and released a large number of cytokines, which led to continuous inflammation in the brain and eventually led to neurodegeneration [47, 48]. Additionally, activated microglia can release toxic substances such as peroxides, NO, and reactive oxygen radicals, thus damaging cellular mitochondria, triggering oxidative stress injury, and inducing cell apoptosis [49], which will aggravate the disorder of glucose metabolism in the brain [50]. Actually, Aβ may be the key mediator between neuroinflammation and glucose metabolism in the brain [5]. Chronic inflammation is known to exacerbate insulin resistance associated with systemic disease-states. Neuroinflammation have an important role in the brain insulin and insulin-like growth factor-1 resistances that occur in AD and PD [51]. Insulin can block Aβ binding to synapses, thereby preventing the ensuing neurotoxicity and Aβ-induced loss of insulin receptors from neuronal dendrites [52]. Thus, the insulin-related signaling pathway may be a key pathway for EA to improve cognitive ability.

In the last few years, the number of publications about the ketogenic diet (KD) for neurological diseases have been progressively increasing. KD has been of interest as a treatment for potential neurodegenerative diseases such as AD [53]. It has been shown in AD, ketone bodies become the alternative energy source to glucose for the brain due to their ability to cross the blood-brain barrier carried by specific transporters that are not downregulated during AD [54]. Besides, KD was shown to decrease the production of amyloid-β protein precursor and Aβ [55]. Moreover, KD was related to the activation of peroxisome proliferator-activated receptor gamma and then to the decrease of inflammation [56].

In conclusion, the results of the current study demonstrate that EA stimulation has a useful degree of efficacy in AD mice. The effects of EA treatment on cognition are at least partially dependent on enhancing glucose metabolism and inhibiting inflammatory response in the frontal lobe of APP/PS1 mice, which may be closely related to the regulation of GV. Results from this study imply that EA may represent a potential therapeutic strategy for the prevention of cognitive decline in AD. Indeed, there are many other valuable acupoints to improve cognition. More work in future studies is needed to explore the positive role of EA treatment in age-related neurodegenerative diseases, such as AD. In addition, clinical trials should be considered for future studies.

Footnotes

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (No.81503654, No.81603678).

Authors’ disclosures available online ().

References

Guillozet

, Weintraub

, Mash

, Mesulam

(2003) Neurofibrillary tangles, amyloid, and memory in aging and mild cognitive impairment. Arch Neurol 60, 729–736.

Lozano

, Naghavi

, Foreman

, Lim

, Shibuya

, Aboyans

, Abraham

, Adair

, Aggarwal

, Ahn

, Alvarado

, Anderson

, Andrews

, Atkinson

, Baddour

, Barker-Collo

, Bartels

, Bell

, Benjamin

, Bennett

, Bhalla

, Bikbov

, Bin Abdulhak

, Birbeck

, Blyth

, Bolliger

, Boufous

, Bucello

, Burch

, Burney

, Carapetis

, Chen

, Chou

, Chugh

, Coffeng

, Colan

, Colquhoun

, Colson

, Condon

, Connor

, Cooper

, Corriere

, Cortinovis

, de Vaccaro

, Couser

, Cowie

, Criqui

, Cross

, Dabhadkar

, Dahodwala

, De Leo

, Degenhardt

, Delossantos

, Denenberg

, Des Jarlais

, Dharmaratne

, Dorsey

, Driscoll

, Duber

, Ebel

, Erwin

, Espindola

, Ezzati

, Feigin

, Flaxman

, Forouzanfar

, Fowkes

, Franklin

, Fransen

, Freeman

, Gabriel

, Gakidou

, Gaspari

, Gillum

, Gonzalez-Medina

, Halasa

, Haring

, Harrison

, Havmoeller

, Hay

, Hoen

, Hotez

, Hoy

, Jacobsen

, James

, Jasrasaria

, Jayaraman

, Johns

, Karthikeyan

, Kassebaum

, Keren

, Khoo

, Knowlton

, Kobusingye

, Koranteng

, Krishnamurthi

, Lipnick

, Lipshultz

, Ohno

, Mabweijano

, MacIntyre

, Mallinger

, March

, Marks

, Matsumori

, Matzopoulos

, Mayosi

, McAnulty

, McDermott

, McGrath

, Mensah

, Merriman

, Michaud

, Miller

, Mock

, Mocumbi

, Mokdad

, Moran

, Mulholland

, Nair

, Naldi

, Narayan

, Nasseri

, Norman

, O’Donnell

, Omer

, Ortblad

, Osborne

, Ozgediz

, Pahari

, Pandian

, Rivero

, Padilla

, Perez-Ruiz

, Perico

, Phillips

, Pierce

, Pope

3rd , Porrini

, Pourmalek

, Raju

, Ranganathan

, Rehm

, Rein

, Remuzzi

, Rivara

, Roberts

, De Leon

, Rosenfeld

, Rushton

, Sacco

, Salomon

, Sampson

, Sanman

, Schwebel

, Segui-Gomez

, Shepard

, Singh

, Singleton

, Sliwa

, Smith

, Steer

, Taylor

, Thomas

, Tleyjeh

, Towbin

, Truelsen

, Undurraga

, Venketasubramanian

, Vijayakumar

, Vos

, Wagner

, Wang

, Watt

, Weinstock

, Weintraub

, Wilkinson

, Woolf

, Wulf

, Yeh

, Yip

, Zabetian

, Zheng

, Lopez

, Murray

, AlMazroa

, Memish

(2012) Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: A systematic analysis for the Global Burden of Disease Study 2010. Lancet 380, 2095–2128.

McKhann

, Knopman

, Chertkow

, Hyman

, Jack

Jr. , Kawas

, Klunk

, Koroshetz

, Manly

, Mayeux

, Mohs

, Morris

, Rossor

, Scheltens

, Carrillo

, Thies

, Weintraub

, Phelps

(2011) The diagnosis of dementia due to Alzheimer’s disease: Recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 7, 263–269.

Albert

, DeKosky

, Dickson

, Dubois

, Feldman

, Fox

, Gamst

, Holtzman

, Jagust

, Petersen

, Snyder

, Carrillo

, Thies

, Phelps

(2011) The diagnosis of mild cognitive impairment due to Alzheimer’s disease: Recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 7, 270–279.

Brendel

, Probst

, Jaworska

, Overhoff

, Korzhova

, Albert

, Beck

, Lindner

, Gildehaus

, Baumann

, Bartenstein

, Kleinberger

, Haass

, Herms

, Rominger

(2016) Glial activation and glucose metabolism in a transgenic amyloid mouse model: A triple-tracer PET study. J Nucl Med 57, 954–960.

Passamonti

, Tsvetanov

, Jones

, Bevan-Jones

, Arnold

, Borchert

, Mak

, Su

, O’Brien

, Rowe

(2019) Neuroinflammation and functional connectivity in Alzheimer’s disease: Interactive influences on cognitive performance. J Neurosci 39, 7218–7226.

Yokokura

, Mori

, Yagi

, Yoshikawa

, Kikuchi

, Yoshihara

, Wakuda

, Sugihara

, Takebayashi

, Suda

, Iwata

, Ueki

, Tsuchiya

, Suzuki

, Nakamura

, Ouchi

(2011) In vivo changes in microglial activation and amyloid deposits in brain regions with hypometabolism in Alzheimer’s disease. Eur J Nucl Med Mol Imaging 38, 343–351.

Wei

, Yang

, Yin

, Wang

, Zheng

(2016) The quality of reporting of randomized controlled trials of electroacupuncture for stroke. BMC Complement Altern Med 16, 512.

Jiang

, Gao

, Zhou

, Xu

, Shi

, Liu

, Li

(2015) Electroacupuncture treatment improves learning-memory ability and brain glucose metabolism in a mouse model of Alzheimer’s disease: Using morris water maze and micro-PET. Evid Based Complement Alternat Med 2015, 142129.

10.

Wang

, Nie

, Li

, Zhao

, Han

, Song

, Xu

, Shan

, Lu

, Li

(2012) Effect of acupuncture in mild cognitive impairment and Alzheimer disease: A functional MRI study. PLoS One 7, e42730.

11.

Wang

, Miao

, Abulizi

, Li

, Mo

, Xue

, Li

(2016) Improvement of electroacupuncture on APP/PS1 transgenic mice in spatial learning and memory probably due to expression of Abeta and LRP1 in hippocampus. Evid Based Complement Alternat Med 2016, 7603975.

12.

Tang

, Shao

, Guo

, Zhou

, Cao

, Xu

, Wu

, Li

, Xiang

(2019) Electroacupuncture mitigates hippocampal cognitive impairments by reducing BACE1 deposition and activating PKA in APP/PS1 double transgenic mice. Neural Plast 2019, 2823679.

13.

Vorhees

, Williams

(2006) Morris water maze: Procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1, 848–858.

14.

Bouter

, Bouter

(2019) (18)F-FDG-PET in mouse models of Alzheimer’s disease. Front Med (Lausanne) 6, 71.

15.

Frautschy

, Yang

, Irrizarry

, Hyman

, Saido

, Hsiao

, Cole

(1998) Microglial response to amyloid plaques in APPsw transgenic mice. Am J Pathol 152, 307–317.

16.

Tan

, Wang

, Huang

, Zhou

, Yuan

, Liang

, Yin

, Xie

, Jia

, Shi

, Wang

, Yang

, Chen

(2017) Modulatory effects of acupuncture on brain networks in mild cognitive impairment patients. Neural Regen Res 12, 250–258.

17.

Liu

, Qi

, Pan

, Ma

, Qian

, Fu

(2013) Clinical observation on treatment of clearing the governor vessel and refreshing the mind needling in neural development and remediation of children with cerebral palsy. Chin J Integr Med 19, 505–509.

18.

Cao

, Tang

, Li

, Gao

, Shi

, Li

(2017) Behavioral changes and hippocampus glucose metabolism in APP/PS1 transgenic mice via electro-acupuncture at governor vessel acupoints. Front Aging Neurosci 9.

19.

, Wu

, Mai

, Fan

, Du

, Qian

, Zhu

(2019) “Governor vessel-unblocking and mind-regulating’ acupuncture therapy ameliorates cognitive dysfunction in a rat model of middle cerebral artery occlusion. Int J Mol Med 43, 221–232.

20.

Deng

, Duan

, Liao

, Liu

, Wang

, Liu

, Tang

, Pang

, Tao

, He

, Yuan

, Liu

(2016) Changes in regional brain homogeneity induced by electro-acupuncture stimulation at the Baihui acupoint in healthy subjects: A functional magnetic resonance imaging study. J Altern Complement Med 22, 794–799.

21.

, Wang

, Kong

, Shen

, Ma

, Du

, Zhou

(2018) Acupoint combinations used for treatment of Alzheimer’s disease: A data mining analysis. J Tradit Chin Med 38, 943–952.

22.

Yang

, Yue

, Zhu

, Han

, Li

, Liu

, Wu

, Yu

(2014) Electroacupuncture promotes proliferation of amplifying neural progenitors and preserves quiescent neural progenitors from apoptosis to alleviate depressive-like and anxiety-like behaviours. Evid Based Complement Alternat Med 2014, 872568.

23.

, Ruan

, Zeng

, Zhou

, Ding

, Zhou

(2015) Improvement in acupoint selection for acupuncture of nerves surrounding the injury site: Electro-acupuncture with Governor vessel with local meridian acupoints. Neural Regen Res 10, 128–135.

24.

Samaey

, Schreurs

, Stroobants

, Balschun

(2019) Early cognitive and behavioral deficits in mouse models for tauopathy and Alzheimer’s disease. Front Aging Neurosci 11, 335.

25.

Reynolds

, Zhong

, Clendinen

, Moffat

, Magnusson

(2019) Age-related differences in brain activations during spatial memory formation in a well-learned virtual Morris water maze (vMWM) task. Neuroimage 202, 116069.

26.

Jacobsen

, Wu

, Redwine

, Comery

, Arias

, Bowlby

, Martone

, Morrison

, Pangalos

, Reinhart

, Bloom

(2006) Early-onset behavioral and synaptic deficits in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A 103, 5161–5166.

27.

Eustache

, Piolino

, Giffard

, Viader

, Sayette

VDL

, Baron

, Desgranges

(2004) ‘In the course of time’: A PET study of the cerebral substrates of autobiographical amnesia in Alzheimer’s disease. Brain 127, 1549–1560.

28.

Meulenbroek

, Rijpkema

, Kessels

RPC

, Rikkert

MGMO

, Fernandez

(2010) Autobiographical memory retrieval in patients with Alzheimer’s disease. Neuroimage 53, 331–340.

29.

Bird

, Chan

, Hartley

, Pijnenburg

, Rossor

, Burgess

(2010) Topographical short-term memory differentiates Alzheimer’s disease from frontotemporal lobar degeneration. Hippocampus 20, 1154–1169.

30.

Bertoux

, Ramanan

, Slachevsky

, Wong

, Henriquez

, Musa

, Delgado

, Flanagan

, Bottlaender

, Sarazin

, Hornberger

, Dubois

(2016) So close yet so far: Executive contribution to memory processing in behavioral variant frontotemporal dementia. J Alzheimers Dis 54, 1005–1014.

31.

Girard

, Baranger

, Gauthier

, Jacquet

, Bernard

, Escoffier

, Marchetti

, Khrestchatisky

, Rivera

, Roman

(2013) Evidence for early cognitive impairment related to frontal cortex in the 5XFAD mouse model of Alzheimer’s disease. J Alzheimers Dis 33, 781–796.

32.

Manenti

, Sandrini

, Gobbi

, Cobelli

, Brambilla

, Binetti

, Cotelli

(2017) Strengthening of existing episodic memories through non-invasive stimulation of prefrontal cortex in older adults with subjective memory complaints. Front Aging Neurosci 9, 401.

33.

Engelborghs

, Sleegers

, Van der Mussele

, Le Bastard

, Brouwers

, Van Broeckhoven

, De Deyn

(2013) Brain-specific tryptophan hydroxylase, TPH2, and 5-HTTLPR are associated with frontal lobe symptoms in Alzheimer’s disease. J Alzheimers Dis 35, 67–73.

34.

Albasanz

, Perez

, Barrachina

, Ferrer

, Martin

(2008) Up-regulation of adenosine receptors in the frontal cortex in Alzheimer’s disease. Brain Pathol 18, 211–219.

35.

Chandler

, Waterhouse

, Gao

(2014) New perspectives on catecholaminergic regulation of executive circuits: Evidence for independent modulation of prefrontal functions by midbrain dopaminergic and noradrenergic neurons. Front Neural Circuits 8, 53.

36.

Surmeier

, Carrillo-Reid

, Bargas

(2011) Dopaminergic modulation of striatal neurons, circuits, and assemblies. Neuroscience 198, 3–18.

37.

Gao

, Goldman-Rakic

(2003) Selective modulation of excitatory and inhibitory microcircuits by dopamine. Proc Natl Acad Sci U S A 100, 2836–2841.

38.

Wang

, Dever

, Lowe

, Storey

, Bhansali

, Eck

, Nitulescu

, Weimer

, Bamford

(2012) Regulation of prefrontal excitatory neurotransmission by dopamine in the nucleus accumbens core. J Physiol 590, 3743–3769.

39.

Roman

, Pascual

(2012) Contribution of neuroimaging to the diagnosis of Alzheimer’s disease and vascular dementia. Arch Med Res 43, 671–676.

40.

Daulatzai

(2012) Quintessential risk factors: Their role in promoting cognitive dysfunction and Alzheimer’s disease. Neurochem Res 37, 2627–2658.

41.

Talbot

, Wang

H-Y

, Kazi

, Han

L-Y

, Bakshi

, Stucky

, Fuino

, Kawaguchi

, Samoyedny

, Wilson

, Arvanitakis

, Schneider

, Wolf

, Bennett

, Trojanowski

, Arnold

(2012) Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J Clin Invest 122, 1316–1338.

42.

, Tian

, Guo

, Fang

, Zhang

, Yu

, Xie

, Zhang

, Lu

, Wang

(2012) Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury. J Neuroinflammation 9, 178.

43.

Sato

, Ohtaki

, Tsumuraya

, Song

, Ohara

, Asano

, Iwakura

, Atsumi

, Shioda

(2012) Interleukin-1 participates in the classical and alternative activation of microglia/macrophages after spinal cord injury. J Neuroinflammation 9, 65.

44.

Vereker

, O’Donnell

, Lynch

(2000) The inhibitory effect of interleukin-1beta on long-term potentiation is coupled with increased activity of stress-activated protein kinases. J Neurosci 20, 6811–6819.

45.

Gouras

, Tsai

, Naslund

, Vincent

, Edgar

, Checler

, Greenfield

, Haroutunian

, Buxbaum

, Xu

, Greengard

, Relkin

(2000) Intraneuronal Abeta42 accumulation in human brain. Am J Pathol 156, 15–20.

46.

Martikainen

, Kemppainen

, Johansson

, Teuho

, Helin

, Liu

, Helisalmi

, Soininen

, Parkkola

, Ngandu

, Kivipelto

, Rinne

(2019) Brain beta-amyloid and atrophy in individuals at increased risk of cognitive decline. Am J Neuroradiol 40, 80–85.

47.

Blume

, Focke

, Peters

, Deussing

, Albert

, Lindner

, Gildehaus

F-J

, von Ungern-Sternberg

, Ozmen

, Baumann

, Bartenstein

, Rominger

, Herms

, Brendel

(2018) Microglial response to increasing amyloid load saturates with aging: A longitudinal dual tracer in vivo PET-study. J Neuroinflammation 15, 307.

48.

Hickman

, Allison

, El Khoury

(2008) Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer’s disease mice. J Neurosci 28, 8354–8360.

49.

Chong

, Li

, Maiese

(2005) Oxidative stress in the brain: Novel cellular targets that govern survival during neurodegenerative disease. Prog Neurobiol 75, 207–246.

50.

Gotzl

, Brendel

, Werner

, Parhizkar

, Monasor

, Kleinberger

, Colombo

, Deussing

, Wagner

, Winkelmann

, Diehl-Schmid

, Levin

, Fellerer

, Reifschneider

, Bultmann

, Bartenstein

, Rominger

, Tahirovic

, Smith

, Madore

, Butovsky

, Capell

, Haass

(2019) Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism. EMBO Mol Med 11, e9711.

51.

de la Monte

(2017) Insulin resistance and neurodegeneration: Progress towards the development of new therapeutics for Alzheimer’s disease. Drugs 77, 47–65.

52.

De Felice

, Vieira

, Bomfim

, Decker

, Velasco

, Lambert

, Viola

, Zhao

, Ferreira

, Klein

(2009) Protection of synapses against Alzheimer’s-linked toxins: Insulin signaling prevents the pathogenic binding of Abeta oligomers. Proc Natl Acad Sci U S A 106, 1971–1976.

53.

Yang

, Shan

, Zhu

, Wu

, Wang

(2019) Ketone bodies in neurological diseases: Focus on neuroprotection and underlying mechanisms. Front Neurol 10, 11.

54.

Hasselbalch

, Knudsen

, Jakobsen

, Hageman

, Holm

, Paulson

(1995) Blood-brain barrier permeability of glucose and ketone bodies during short-term starvation in humans. Am J Physiol 268, E1161–1166.

55.

Versele

, Corsi

, Fuso

, Sevin

, Businaro

, Gosselet

, Fenart

, Candela

(2020) Ketone bodies promote amyloid-beta(1-40) clearance in a human in vitro blood-brain barrier model. Int J Mol Sci 21, 934.

56.

Jeong

, Jeon

, Shin

, Kim

, Lee

, Kim

, Kang

, Cho

, Choi

, Roh

(2011) Ketogenic diet-induced peroxisome proliferator-activated receptor-gamma activation decreases neuroinflammation in the mouse hippocampus after kainic acid-induced seizures. Exp Neurol 232, 195–202.