Acupuncture Treatment in a Mouse Model of Chronic Hypoxia-Induced Cognitive Dysfunction

Fang Wan; Zhifang Guo; Mingye Wang; Yunlong Hou; Lu Wang; Wenyan Li; Ning Kang; Pengyu Zhu; Mengnan Li

doi:10.3791/65784

JoVE Journal > Behavior

Behavior

Acupuncture Treatment in a Mouse Model of Chronic Hypoxia-Induced Cognitive Dysfunction

Published: December 08, 2023

doi:

10.3791/65784

Fang Wan*¹, Zhifang Guo*^2,3, Mingye Wang⁴, Yunlong Hou^5,6, Lu Wang^3,5, Wenyan Li^4,7, Ning Kang^2,3, Pengyu Zhu⁸, Mengnan Li^5,6

¹Heilongjiang University of Chinese Medicine, ²Hebei Provincial Key Laboratory of Luobing, ³Key Laboratory of State Administration of TCM (Cardio-Cerebral Vessel Collateral Disease), ⁴Hebei University of Chinese Medicine, ⁵Shijiazhuang Compound Traditional Chinese Medicine Technology Innovation Center, ⁶National Key Laboratory of Luobing Research and Innovative Chinese Medicine, ⁷Shijiazhuang Yiling Pharmaceutical Co., Ltd, ⁸The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine

Summary

Here, we describe a protocol for implementing mild anesthesia and acupuncture treatment on a chronic hypoxia mouse model and conducting behavioral tests to assess the cognitive alterations post-treatment.

Abstract

The treatment of central nervous disorders has consistently posed significant challenges to the medical field. Acupuncture, a non-pharmacological practice rooted in traditional Chinese medicine, entails the insertion of fine needles into precise points on the body and is commonly employed for the management of diverse conditions. Recently, acupuncture has emerged as a promising therapeutic intervention for a range of neurological diseases, including anxiety and respiratory disorders. However, the potential of acupuncture in treating cognitive dysfunction induced by chronic hypoxia has not yet been explored. This paper presents a comprehensive protocol for establishing a mouse model of chronic hypoxia-induced cognitive impairment, administering mild anesthesia, performing acupuncture treatment, and assessing behavioral changes and memory abilities using open field tests and water mazes. The step-by-step protocol provides detailed instructions on accurately locating and positioning acupoints and needles for cognitive improvement. By employing this protocol, researchers can conduct systematic studies to thoroughly evaluate the therapeutic potential of acupuncture for cognitive dysfunction.

Introduction

The global population is currently facing a critical aging problem, resulting in a rapid increase in the prevalence of cognitive disorders. The worldwide incidence of cognitive impairment is approximately 53.97 per 1000 person-years¹. Chronic cerebral hypoxia caused by vascular dysfunction or circulatory/respiratory disorders remains one of the major risk factors for age-related dementia². Previous studies have demonstrated that cerebral hypoxia can increase amyloid-β deposition by modifying BACE1 expression³. Additionally, hypoxia has been associated with glial-cell dysregulation and neuroinflammation⁴^,⁵. Despite the growing magnitude of this issue, effective Western medicines for preventing chronic hypoxia-induced cognitive decline are currently lacking. Non-pharmacological traditional Chinese medicine, particularly acupuncture, has been used for thousands of years to treat cognitive disorders and has shown promising results in alleviating neurodegenerative diseases⁶^,⁷. The Baihui, Shenting, and Zusanli acupoints are effective points for treating cognitive dysfunction⁸^,⁹. Clinical studies have demonstrated that electro-acupuncture therapy significantly improves the Montreal Cognitive Assessment (MoCA) and Mini-Mental State Examination (MMSE) scores in patients with vascular cognitive impairment and effectively ameliorates cognitive dysfunction⁸. Although studies have suggested that acupuncture can significantly enhance the memory ability of rats with arterial ligation-an acute cerebral hypoxia models¹⁰, an acute cerebral hypoxia model, there is no report on the effects of acupuncture in any rodent model with chronic hypoxia-induced cognitive disorders. The lack of research into the mechanism has considerably impeded its clinical application.

Previous research has demonstrated that subjecting rats to a hypoxic environment for a period of 8 weeks can significantly elevate levels of oxidative stress and inflammation in the brain, resulting in a decline in memory function¹¹. The present study aims to investigate the impact of acupuncture on rodent models in order to further our understanding. It is worth noting, however, that anesthesia is typically required during acupuncture treatment in rodents due to the potential for agitation during repeated stimulation. Prolonged anesthesia can significantly impact cognitive function in mice, as most anesthetic drugs can suppress neural activity and impede information processing, leading to behavioral deficits¹². Several studies have shown that administering 2.5% sevoflurane for a duration of 6 h can notably impair spatial memory, learning ability, and attention in mice¹³. Furthermore, evidence suggests that high doses of anesthesia may result in neuronal death or nerve damage in mice¹⁴. Therefore, it is imperative to identify a suitable approach to minimize the overall amount of anesthesia used. In this study, we introduce an effective acupuncture method for treating mice with cognitive impairment, along with behavioral tests to assess their memory abilities. Importantly, we present a modified pre-treatment anesthesia technique that can effectively reduce the total dosage of anesthesia administered during the experiment.

Protocol

The animal experiments were conducted with the approval of the Committee on Animal Research and Ethics of the Hebei Yiling Medical Research Institute (approval number: N2022148). Male C57BL/6J mice weighing 18-22 g (see Table of Materials) were housed in the new drug evaluation center of Hebei Yiling Medical Research Institute. They were provided with normal food and clean water and exposed to artificial light for 12 h daily. The rooms maintained a controlled temperature range of 20-26 °C and relative humidity of 40%-70%.

1. Establishment of a chronic hypoxia mouse model (Figure 1)

Before commencing the experiment, prepare animal cages under normal atmospheric pressure and cages with a continuous low-oxygen environment. Establish a continuous low-oxygen environment by utilizing an automated gas control delivery system to flush the chamber with a mixture of pure oxygen and nitrogen.
NOTE: This system is programmed to control the electromagnetic valve switch, thereby ensuring precise delivery of gas in terms of both time and concentration.
Randomly divide mice into three groups: a control group (Con), a model group (CH), and an electro-acupuncture group (EA + CH). Place control and model/electro-acupuncture mice separately in the two cages, with 10 mice per cage. Maintain the light cycle at 12 h/12 h (light/dark).
NOTE: No treatment or hypoxia is induced in the control group (Con). The model group (CH) consists of mice with chronic hypoxia. The electro-acupuncture group (EA + CH) comprises hypoxia-induced mice treated with electro-acupuncture.
For developing chronic hypoxia, establish the parameters of the low-oxygen chamber by utilizing a digital oxygen meter to regulate the gas flow rate and maintain an oxygen concentration of 10%. Place the animals within the low-oxygen chamber at 9:00 am and remove them at 5:00 pm, resulting in a total of 8 h of uninterrupted exposure to low oxygen per day for 3 months.
NOTE: When setting up the delivery of nitrogen gas to reduce the oxygen concentration, it is advised to proceed slowly to prevent an excessive introduction of nitrogen gas at once, as it will lead to animal fatalities.
Evaluate the chronic hypoxia-induced cognitive dysfunction model using histology examination and behavioral tests: the open field test¹⁵ and the water maze test¹⁶.

2. Anesthesia (Figure 2)

Prepare the small animal anesthesia machine (see Table of Materials) and the constant temperature heating pad.
NOTE: During anesthesia, animals are susceptible to hypothermia, underscoring the necessity of employing a constant-temperature heating pad for insulation.
Place the mouse in the anesthesia induction box and quickly induce with 2%-2.5% isoflurane in oxygen (see Table of Materials) for approximately 1 min.
NOTE: This short-term pre-treatment is a crucial step to ensure that mice can thrive under low-concentration dosage for an extended period of time.
Once their excitability decreased, pinch the mouse's toe to check its reflex. Then, transfer the mouse onto the constant temperature heating pad (37 °C).
Adjust the anesthesia flow rate to approximately 0.5% concentration. Connect the anesthesia machine to the mouth and nose of the mouse. Proceed with electro-acupuncture treatment while ensuring the maintenance of anesthesia.
NOTE: The effect of anesthesia was confirmed when the mice stopped blinking. The effect of anesthesia can last for at least 30 min.

3. Electro-acupuncture treatment

To effectively improve cognitive dysfunction, select specific acupoints, such as Baihui (GV20), Shenting (GV24), and bilateral Zusanli (ST36), based on traditional Chinese medicine theory and clinical experience (Figure 3). Administer electro-acupuncture treatment 2 weeks prior to the completion of the modeling process.
1. Locate the GV20 acupoint on the midline of the forehead, at the midpoint of a line connecting the tips of the ears⁷. The depth of acupuncture needle insertion must be 2 mm.
2. Locate the GV24 acupoint 1.3 mm directly above the midpoint of the mouse's eyes on the midline of the forehead¹⁷. The depth of acupuncture needle insertion must be 2 mm.
3. Locate the ST36 acupoint on the outside of the knee joint, about 2 mm below the head of the fibula¹⁸^,¹⁹. The depth of acupuncture needle insertion must be 3-4 mm.
Prepare disposable acupuncture needles (see Table of Materials) and an electro-acupuncture device (see Table of Materials) for the procedure (Figure 4).
Place the mouse in the prone position under mild anesthesia with 0.5% isoflurane, ensuring their heads and limbs are immobilized. Hold a stainless-steel needle (diameter: 0.18 mm; length: 7 mm) with the right hand, using the thumb, index finger, and middle finger.
Perform acupuncture at GV20 and GV24 acupoints transversely for 2 mm depth, lifting the skin on the mouse's head with the left hand. Puncture the ST36 acupoint vertically for 3-4 mm depth by touching the fibular head on the lateral side of the mouse's knee joint and pressing on the skin with the left thumb.
NOTE: For the acupoints located on the head, it is advisable to insert the needles in the sequence of GV24 followed by GV20. This order facilitates operational convenience. Acupuncture points are discrete anatomical locations rather than stationary points. Consequently, slight deviations in the angle of needle insertion have no effect on the therapeutic effectiveness, similarly seen in patients receiving electro-acupuncture treatment in clinical settings.
Connect the electronic acupuncture device to the needles, with GV20 and the left ST36 connected to one electrode set and GV24 and the right ST36 connected to another (Figure 4). Select the continuous wave mode, with an electric current intensity of 2 mA and a frequency of 2 Hz²⁰^,²¹. Confirm the ideal treatment by observing local mild tremors at acupoints and quiet tolerance by the mouse.
1. When connecting the electrical acupuncture instrument, connect the proximal end of the needle. This helps minimize the impact caused by the weight of the connection line and consequently improves the prevention of needle detachment. If required, utilize adhesive tape to secure the horizontally inserted needle and the connection line.
Administer the daily treatment for 30 min each day for 6 consecutive days, with a single day of rest between each treatment cycle.

4. Open field test (Figure 5)

NOTE: The open field test is a conventional method used to assess the autonomous behavior, exploratory behavior, cognitive abilities, and anxiety behavior of experimental animals in novel and unfamiliar environments²². It consists of an open-field reaction box and a recording device.

To conduct the test, prepare a white-walled cube measuring 50 cm × 50 cm × 30 cm, with the bottom divided into 25 equal squares measuring 10 cm × 10 cm.
Place the mouse into the open field reaction box for acclimation. Allow the mouse to explore the testing room and familiarize with the new environment during the acclimation period. Conduct the open field test after acclimating the mouse to the experimental environment for 1 h.
NOTE: This guarantees the minimization of anxiety or stress induced by alterations in the environment, thus enabling more precise outcomes during the subsequent behavioral assessments.
Place the mouse in the center of the box and monitor it for 10 min after allowing the mouse to adapt to the environment for 2 min.
1. Use a video tracking system (see Table of Materials) to record the mouse's movement trajectory, total distance traveled, time spent in the central area, speed of crossing the central area, and number of entries into the central area during the test.
2. Perform the relevant operations as instructed in the product manual of the video tracking system. Each mouse undergoes a single test and begins exploration from the same location within the box.
3. After each test, clean the open field box with 75% ethanol to prevent any false results caused by odor interference when using a mouse.

5. Water maze (Figure 5)

NOTE: The water maze test is frequently employed as a behavioral assessment tool in experiments involving mice to evaluate their spatial learning and memory capabilities²³.

Prepare a circular water tank with a diameter of 120 cm and a depth of 30 cm. Divide the tank into four equal quadrants: I, II, III, and IV. If using black mice in the experiment, use a white water tank; for white mice, use a black water tank.
Place curtains around the circular water tank to prevent the mouse from seeing the researchers during the test.
Position different markers on the top surface of the water tank as visual cues for spatial orientation. Ensure that these markers remain stationary throughout the experiment to maintain consistency.
Situate a circular platform with a diameter of 10 cm in quadrant III of the water tank as the designated target area. Ensure the platform can be easily moved and secured in any desired location.
Throughout the experiment, introduce water into the tank while maintaining a temperature range of 22-24 °C.
1. Ensure the water level remains consistently 1 cm above the target platform. Include a 20% concentration of non-toxic titanium dioxide in the water to achieve a distinct contrast between the black mice and the white background. This contrast facilitates the camera's recording of the mouse's movements and relevant parameters.
Conduct a 5-day continuous spatial exploration test by sequentially placing each mouse in quadrants I, II, III, and IV.
1. Position the mouse facing the wall. Move away from the maze to prevent the mouse from using the experimenter's position as a reference point. Record the time the mouse takes to find the platform.
2. If the mouse fails to locate the underwater platform within 90 s, guide the mouse to the platform and provide a 30-s learning period. Additionally, record the latency period as 90 s.
3. If the mouse locates the underwater platform within 90 s, let it remain on the platform for 10 s for learning before removing it from the water tank.
4. Dry the mouse with a towel and return it to its cage.
5. Rotate the placement of each mouse in each quadrant every 20 min. Record each mouse's swimming distance, speed, and time taken to find the platform (the latency period) using the video tracking system (see Table of Materials), performing the relevant operations as instructed in the product manual.
6. Set the platform 1 cm above the water surface on day 1. Place the platform at a depth of 1 cm below the water surface on days 2-5.
On Day 6, remove the platform from the target quadrant and conduct a spatial exploration test.
1. Place the mouse in Quadrant I to explore freely for 90 s. The computer records the swimming trajectory of the mouse, the time spent in the target quadrant, and the number of times it crosses the platform.
  NOTE: In order to minimize experimental errors caused by human factors, it is important to keep the position of the reference point fixed in the water maze experiment. Additionally, the experimenter should immediately retreat after placing the mouse in the water. After the experiment is completed, the mice should be dried with a towel and placed back in their cages to maintain warmth.

6. Hematoxylin and eosin (HE) staining (Figure 6)

NOTE: Histological examination of the hippocampal region assists in assessing the establishment of the hypoxia model and determining the efficacy of acupuncture treatment.

After the behavioral experiment, anesthetize the mouse with an intraperitoneal injection of 20 mg/kg pentobarbital sodium and perfuse it with 10% paraformaldehyde solution (see Table of Materials) to ensure complete body perfusion. Isolate the brain tissue and immerse it in 10% paraformaldehyde solution at room temperature (RT) for 3 days to achieve fixation.
Place the brain samples in an embedding box. Subsequently, wash the processed brain samples with running water for 6 h.
Employ an automated tissue processor to dehydrate the samples using a series of alcohol solutions with increasing concentrations, namely, 60% ethanol for 1 h, 70% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol for 2 h, and finally, 100% ethanol for 2 h.
Immerse the tissue specimens in xylene for 2 h to achieve transparency. Subsequently, following the completion of the dehydration process, transfer the permeabilized samples to paraffin wax heated to 60 °C for 3 h. Finally, embed them in an automatic processor.
Utilize a rotary slicer to obtain 4 µm sections. Subsequently, subject the sections to hematoxylin staining for a duration ranging from 3-8 min, followed by eosin staining for 1-3 min.
Sequentially transfer the stained sections to separate containers of pure alcohol and xylene. Then, seal and secure the stained sections with neutral gum in preparation for pathological examination under an optical microscope.
Employ a slide scanner (see Table of Materials) to scan the slices. Subsequently, utilize the viewing software to obtain the HE staining results for the hippocampal region. Compare the arrangement of neurons and the condensation of neuronal nuclei.

Representative Results

Characterizing mouse locomotion trajectories in the open field experiment
The trajectory map reveals that mice in the normal group exhibit a profound inclination for exploration in unfamiliar environments. Their activity trajectories are primarily concentrated in the corners while covering the entire open field (left panel). In contrast, the long-term hypoxia model group of mice displays a significantly diminished desire to explore novel surroundings. They predominantly linger in the corners without exhibiting any exploratory behavior toward the center of the open field (middle panel). Following acupuncture treatment, the exploratory activity of hypoxia-induced mice shows improvement, and their behavior of venturing toward the center of the open field is reinstated (right panel) (Figure 5A).

Characterization of spatial learning and memory in mice
In the normal group, mice spent a relatively longer amount of time in the target quadrant and crossed the platform more frequently, as shown in the trajectory map (left panel). The long-term hypoxic model group of mice demonstrated weakened spatial memory capabilities compared to the normal group, as indicated by their inability to locate the target quadrant within the specified time (middle panel). Following acupuncture treatment, the mice showed significant improvement in their hypoxia-induced spatial memory capabilities. They displayed more organized exploratory behavior and spent a noticeably longer time in the target quadrant (right panel) (Figure 5B).

Histological examination of mouse brain
In the control group, the arrangement of neurons in the hippocampal region of mice (upper left panel) demonstrated regularity, whereas it was disrupted in the long-term hypoxic model group (upper right panel). Conversely, the treatment group exhibited an improvement in the arrangement of neurons (lower panel). Furthermore, the model group displayed exacerbated shrinkage of mouse neuronal nuclei compared to the control group, but this effect was partially alleviated in the treatment group. (Figure 6).

Figure 1: Establishment of a mouse model for hypoxia-induced cognitive impairment. The mice were exposed to hypoxia from day 1 to day 90. Electric acupuncture therapy was administered daily from day 75, with each treatment cycle lasting 6 days and a total of 2 treatment cycles. There was a 1-day break between cycles. Behavioral testing was conducted on day 93. Histological examination and behavioral testing can be conducted on day 65 to confirm the establishment of the model in the hippocampal region. Abbreviations: Mon: month. Please click here to view a larger version of this figure.

Figure 2: Anesthesia pre-treatment prior to electro-acupuncture. Before undergoing electro-acupuncture treatment, the mice were anesthetized using an (A) anesthesia device. The mice were then placed into a (B) chamber box with (C) 2% Isoflurane in the chamber. (D) The duration of the modified anesthesia method was shorter compared to the classical anesthesia method. (E) Mice subjected to mild anesthesia retain their reaction to foot stimulation. Please click here to view a larger version of this figure.

Figure 3: Anatomical structure of acupuncture points on the mouse head. This figure depicts the anatomical positions of GV20 (Baihui), GV24 (Shenting), and ST36 (Zusanli) in mice. (A) An anatomical view of the mouse head showing the frontal and parietal bones. (B) An anatomical view of the mouse leg showing the tibia, fibula, and fibular head. (C) Locations of acupuncture points on the mouse head. (D) GV20 is located on the midline of the forehead, at the midpoint between the tips of the ears, and directly on top of the parietal bone. GV24 is located on the midline of the forehead, just anterior to the junction of the frontal and parietal bones. ST36 is located on the outer side of the hind leg, approximately 2mm below the fibular head. Please click here to view a larger version of this figure.

Figure 4: Electro-acupuncture treatment. The mice underwent needle stimulation at specific points on GV20 (Baihui), GV24 (Shenting), and bilateral ST36 (Zusanli) while under anesthesia. Please click here to view a larger version of this figure.

Figure 5: Representative results of the open field test and water maze test after electro-acupuncture treatment. (A) The open field test was conducted to evaluate the behavioral changes in mice subjected to chronic hypoxia (CH) and acupuncture (EA) treatment. Three representative trajectory plots were generated from the test. (B) The water maze test was conducted to evaluate the spatial memory of mice subjected to chronic hypoxia and acupuncture treatment. Three representative trajectory plots were generated from the test. Please click here to view a larger version of this figure.

Figure 6: Histological examination of mouse brain after electro-acupuncture treatment. Histological pictures of the mice in the control group (upper left panel), the hypoxia group (upper right panel), and the treatment group (lower panel). Scale bars: 100 µm. Please click here to view a larger version of this figure.

Discussion

Acupuncture, a non-pharmacological medical practice originating in China over 2,000 years ago, involves the insertion of thin needles into specific points on the body known as acupuncture points. These points are believed to be connected by channels or meridians through which the body's vital energy, or "qi," flows²⁴. By stimulating these points, acupuncture aims to restore balance and harmony to the body. It has been shown to effectively treat various conditions, including chronic pain, anxiety/depression, digestive problems, menstrual cramps, and respiratory disorders²⁵^,²⁶^,²⁷^,²⁸^,²⁹. In recent years, acupuncture has emerged as an effective therapeutic intervention for neuronal diseases, including cognitive dysfunction. Multiple studies have demonstrated its ability to modulate neurotransmitters, increase cerebral blood flow, reduce oxidative stress, and enhance neuroplasticity²⁰^,³⁰^,³¹^,³². Consequently, it is increasingly recognized as a safe and effective treatment option, particularly when used alongside conventional medical care³³. However, despite its long history and widespread use, the mechanism of action of acupuncture remains incompletely understood. One theory proposes that acupuncture stimulates the release of endorphins, the body's natural painkillers, thereby alleviating pain and promoting a sense of well-being³⁴. Another theory suggests that acupuncture may affect the autonomic nervous system, which regulates various involuntary bodily functions³⁵^,³⁶. Although our understanding of acupuncture's mechanisms is still developing, there is growing recognition among scientists that a standardized laboratory methodology for acupuncture, especially using rodent models, is essential to guide research in this area.

The selection of an appropriate anesthesia protocol is the initial crucial step in conducting acupuncture in a mouse model. Traditional protocols often involve continuous high-dose anesthesia, which can have significant effects on the mouse's nervous system and may result in false-negative behavioral test results after acupuncture treatment. In this study, we propose an improved protocol that utilizes a sealed anesthesia box to gas-anesthetize the mice until they lose consciousness. Subsequently, a stable state is maintained using a low-dose anesthetic during the acupuncture treatment. This method helps to minimize functional and behavioral abnormalities caused by excessive anesthesia dosing and enhances the accuracy of the experiments. Additionally, researchers can opt for isoflurane instead of ketamine and xylazine as it offers faster recovery time and reduces the systemic toxicity risks associated with ketamine and xylazine³⁷. However, it is important to note that false negative results caused by anesthesia may still occur. Even mild anesthesia continuing for 2 consecutive weeks can have a negative impact on cognition³⁸. In order to more accurately evaluate the effectiveness of the treatment, researchers can incorporate an additional group of anesthetized mice that receive no treatment for comparison purposes. Another critical aspect of acupuncture treatment in mice is determining the combination of acupoints. Commonly used acupoints for central nervous system diseases in humans include Baihui (GV20), Yintang (EX-HN3), Shenting (GV24), and Zusanli (ST36)³⁹^,⁴⁰^,⁴¹. In this study, we focused on the inclusion of Baihui (GV20), Shenting (GV24), and Zusanli (ST36) for the treatment. Despite the challenges posed by the small size of mice in acupoint localization, joint positioning based on anatomical structures proves to be an effective method. Lastly, determining the appropriate stimulation frequency and intensity is another key step in performing acupuncture treatment in mice. In this study, we utilized low-frequency electro-acupuncture at 2 Hz and a moderate intensity of 2 mA. Although the therapeutic outcome of acupuncture is evident, further exploration is required to understand its underlying mechanism.

Despite the broad potential applications of acupuncture in the treatment of neurological disorders, this technique has certain limitations. One limitation is its high dependence on the operator's experience, which can result in suboptimal outcomes or harm to experimental subjects when performed by inexperienced operators. Another limitation is the need for improvement in clinical acupuncture treatment to enhance its effectiveness. Currently, researchers are studying the combination of acupuncture with other therapies, such as pharmacological interventions and cognitive training, in order to improve treatment outcomes⁴². Additionally, technological advancements have led to the development of new techniques, like transcranial magnetic stimulation (TMS), which can be used in conjunction with acupuncture to further enhance cognitive function⁴³. Despite these limitations, acupuncture has shown significant benefits in the treatment of various neurological disorders and holds great potential for future applications, particularly when combined with other therapies. This article provides detailed methods for constructing a mouse model of chronic hypoxia-induced cognitive impairment, the process of acupuncture treatment, and behavioral testing methods. These methods can assist researchers in conducting thorough studies on the application and mechanism of acupuncture, thereby promoting the advancement of traditional Chinese medicine.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by S&T Program of Hebei (NO.E2020100001, and NO.22372502D), High-level S & T Innovation and Entrepreneurship Talent Project of Shijiazhuang (No. 07202203).

Materials

10% paraformaldehyde solution	Bioroyee (Beijing) Biotechnology Co., Ltd	RL3234
ANY-maze	Science	SA201	Video tracking system
C75BL/6J mice	BEIJING HFK BIOSCIENCE CO.,LTD	No.110322220103041767	Gender: Male, Weight: 18–22 g
Electroacupuncture device	Great Wall	KWD-808 I
Hwato acupuncture needle	Suzhou Medical Appliance Factory	2655519
Isoflurane	RWD Life Science Co.,Ltd	R510-22
NanoZoomer Digital Pathology	Hamamatsu Photonics K. K	C9600-01
Small animal anesthesia machine	RWD	YL-LE-A106

References

Pais, R., Ruano, L., Carvalho, O. P., Barros, H. Global cognitive impairment prevalence and incidence in community dwelling older adults- A systemic review. Geriatrics. 5 (4), 84 (2020).
Tian, Z., Ji, X., Liu, J. Neuroinflammation in vascular cognitive impairment and dementia: Current evidence, advances, and prospects. International Journal of Molecular Sciences. 23 (11), 6224 (2022).
Yuan, Y., et al. Activation of ERK-Drp1 signaling promotes hypoxia-induced Aβ accumulation by upregulating mitochondrial fission and BACE1 activity. FEBS open bio. 11 (10), 2740-2755 (2021).
Zhu, X., et al. NLRP3 deficiency protects against hypobaric hypoxia induced neuroinflammation and cognitive dysfunction. Ecotoxicology and Environmental Safety. 255, 114828 (2023).
Li, B., Dasgupta, C., Huang, L., Meng, X., Zhang, L. MiRNA-210 induces microglial activation and regulates microglia-mediated neuroinflammation in neonatal hypoxic-ischemic encephalopathy. Cellular & Molecular Immunology. 17 (9), 976-991 (2020).
Cai, M., Lee, J. H., Yang, E. J. Electro-acupuncture attenuates cognition impairment via anti-neuroinflammation in an Alzheimer’s disease animal model. Journal of Neuroinflammation. 16 (1), 264 (2019).
Xie, L., et al. Electro-acupuncture improves M2 microglia polarization and glia anti-inflammation of hippocampus in Alzheimer’s disease. Frontiers in Neuroscience. 15, 689629 (2021).
Huang, L., et al. Effects of acupuncture on vascular cognitive impairment with no dementia: A randomized controlled trial. Journal of Alzheimer’s Disease: JAD. 81 (4), 1391-1401 (2021).
Xi, L., Fang, F., Yuan, H., Wang, D. Transcutaneous electrical acupoint stimulation for postoperative cognitive dysfunction in geriatric patients with gastrointestinal tumor: a randomized controlled trial. Trials. 22 (1), 563 (2021).
Du, S. Q., et al. Acupuncture inhibits TXNIP-associated oxidative stress and inflammation to attenuate cognitive impairment in vascular dementia rats. CNS Neuroscience & Therapeutics. 24 (1), 39-46 (2018).
Zhang, C. E., et al. Hypoxia-induced tau phosphorylation and memory deficit in rats. Neuro-Degenerative Diseases. 14 (3), 107-116 (2014).
Liang, X., Zhang, R. Effects of minocycline on cognitive impairment, hippocampal inflammatory response, and hippocampal Alzheimer’s related proteins in aged rats after propofol anesthesia. Disease Markers. 2022, 4709019 (2022).
Lee, J. R., et al. Effect of dexmedetomidine on sevoflurane-induced neurodegeneration in neonatal rats. British Journal of Anaesthesia. 126 (5), 1009-1021 (2021).
Matsumoto, Y., Fujino, Y., Furue, H. Anesthesia and surgery induce a functional decrease in excitatory synaptic transmission in prefrontal cortex neurons, and intraoperative administration of dexmedetomidine does not elicit the synaptic dysfunction. Biochemical and Biophysical Research Communications. 572, 27-34 (2021).
Kraeuter, A. K., Guest, P. C., Sarnyai, Z. The open field test for measuring locomotor activity and anxiety-like behavior. Methods in Molecular Biology. 1916, 99-103 (2019).
Bromley-Brits, K., Deng, Y., Song, W. Morris water maze test for learning and memory deficits in Alzheimer’s disease model mice. Journal of Visualized Experiments: JoVE. (53), e2920 (2011).
Lin, W., et al. TNEA therapy promotes the autophagic degradation of NLRP3 inflammasome in a transgenic mouse model of Alzheimer’s disease via TFEB/TFE3 activation. Journal of Neuroinflammation. 20 (1), 21 (2023).
Liu, S., et al. A neuroanatomical basis for electro-acupuncture to drive the vagal-adrenal axis. Nature. 598 (7882), 641-645 (2021).
Jang, J. H., et al. Acupuncture inhibits neuroinflammation and gut microbial dysbiosis in a mouse model of Parkinson’s disease. Brain, Behavior, and Immunity. 89, 641-655 (2020).
Dong, W., et al. Electro-acupuncture improves synaptic function in SAMP8 mice probably via inhibition of the AMPK/eEF2K/eEF2 signaling pathway. Evidence-Based Complementary and Alternative Medicine: eCAM. 2019, 8260815 (2019).
Han, Y. G., et al. Electro-acupuncture prevents cognitive impairment induced by lipopolysaccharide via inhibition of oxidative stress and neuroinflammation. Neuroscience Letters. 683, 190-195 (2018).
Zhang, Q., et al. Electro-acupuncture pre-treatment ameliorates anesthesia and surgery-induced cognitive dysfunction via inhibiting mitochondrial injury and neuroapoptosis in aged rats. Neurochemical Research. 47 (6), 1751-1764 (2022).
Zheng, X., et al. Electro-acupuncture ameliorates beta-amyloid pathology and cognitive impairment in Alzheimer disease via a novel mechanism involving activation of TFEB (transcription factor EB). Autophagy. 17 (11), 3833-3847 (2021).
Zhou, W., Benharash, P. Effects and mechanisms of acupuncture based on the principle of meridians. Journal of Acupuncture and Meridian Studies. 7 (4), 190-193 (2014).
Sun, Y., et al. Efficacy of Acupuncture For Chronic Prostatitis/Chronic Pelvic Pain Syndrome : A Randomized Trial. Annals of Internal Medicine. 174 (10), 1357-1366 (2021).
Jung, J., et al. Lipidomics reveals that acupuncture modulates the lipid metabolism and inflammatory interaction in a mouse model of depression. Brain, Behavior, and Immunity. 94, 424-436 (2021).
Yang, N. N., et al. Electro-acupuncture ameliorates intestinal inflammation by activating α7nAChR-mediated JAK2/STAT3 signaling pathway in postoperative ileus. Theranostics. 11 (9), 4078-4089 (2021).
Shetty, G. B., Shetty, B., Mooventhan, A. Efficacy of acupuncture in the management of primary dysmenorrhea: A randomized controlled trial. Journal of Acupuncture and Meridian Studies. 11 (4), 153-158 (2018).
Nurwati, I., Purwanto, B., Mudigdo, A., Saputra, K., Prasetyo, D. H., Muthmainah, M. Improvement in inflammation and airway remodelling after acupuncture at BL13 and ST36 in a mouse model of chronic asthma. Acupuncture in Medicine. 37 (4), 228-236 (2019).
Li, P., et al. Acupuncture can play an antidepressant role by regulating the intestinal microbes and neurotransmitters in a rat model of depression. Medical Science Monitor. 27, 929027 (2021).
Ding, N., Jiang, J., Xu, A., Tang, Y., Li, Z. Manual acupuncture regulates behavior and cerebral blood flow in the SAMP8 mouse model of Alzheimer’s disease. Frontiers in Neuroscience. 13, 37 (2019).
Yang, J. W., Wang, X. R., Ma, S. M., Yang, N. N., Li, Q. Q., Liu, C. Z. Acupuncture attenuates cognitive impairment, oxidative stress and NF-κB activation in cerebral multi-infarct rats. Acupuncture in Medicine. 37 (5), 283-291 (2019).
Li, X., et al. Traditional Chinese acupoint massage, acupuncture, and moxibustion for people with diabetic gastroparesis: A systematic review and meta-analysis. Medicine. 101 (48), 32058 (2022).
Yang, X. Y., et al. Effect of combined acupuncture-medicine anesthesia in thyroid nodule ablation and its effect on serum β-endorphin. Acupuncture Research. 45 (12), 1006-1009 (2020).
Uchida, C., et al. Effects of Acupuncture Sensations on Transient Heart Rate Reduction and Autonomic Nervous System Function During Acupuncture Stimulation. Medical Acupuncture. 31 (3), 176-184 (2019).
Liang, C., Wang, K. Y., Gong, M. R., Li, Q., Yu, Z., Xu, B. Electro-acupuncture at ST37 and ST25 induce different effects on colonic motility via the enteric nervous system by affecting excitatory and inhibitory neurons. Neurogastroenterology and Motility. 30 (7), 13318 (2018).
Michelson, N. J., Kozai, T. Isoflurane and ketamine differentially influence spontaneous and evoked laminar electrophysiology in mouse V1. Journal of Neurophysiology. 120 (5), 2232-2245 (2018).
Yu, X., Zhang, F., Shi, J. Sevoflurane anesthesia impairs metabotropic glutamate receptor-dependent long-term depression and cognitive functions in senile mice. Geriatrics & Gerontology International. 19 (4), 357-362 (2019).
Jeong, J. H., et al. Investigation of combined treatment of acupuncture and neurofeedback for improving cognitive function in mild neurocognitive disorder: A randomized, assessor-blind, pilot study. Medicine. 100 (37), 27218 (2021).
Lin, Y. K., Liao, H. Y., Watson, K., Yeh, T. P., Chen, I. H. Acupressure improves cognition and quality of life among older adults with cognitive disorders in long-term care settings: A clustered randomized controlled trial. Journal of the American Medical Directors Association. 24 (4), 548-554 (2023).
Wu, W. Z., et al. Effect of Tongdu Tiaoshen acupuncture on serum GABA and CORT levels in patients with chronic insomnia. Chinese Acupuncture & Moxibustion. 41 (7), 721-724 (2021).
Zhuo, P. Y., et al. Efficacy and safety of acupuncture combined with rehabilitation training for poststroke cognitive impairment: A systematic review and meta-analysis. Journal of Stroke and Cerebrovascular Diseases. 32 (9), 107231 (2023).
Li, R. Y., Huang, R. J., Yu, Q. Comparison of different physical therapies combined with acupuncture for poststroke cognitive impairment: A network meta-analysis. Evidence-Based Complementary and Alternative Medicine: eCAM. 2021, 1101101 (2021).

Play Video

PDF

DOI

DOWNLOAD MATERIALS LIST

Cite This Article

Wan, F., Guo, Z., Wang, M., Hou, Y., Wang, L., Li, W., Kang, N., Zhu, P., Li, M. Acupuncture Treatment in a Mouse Model of Chronic Hypoxia-Induced Cognitive Dysfunction. J. Vis. Exp. (202), e65784, doi:10.3791/65784 (2023).

Automatically Generated