This work was supported by S&#38;T Program of Hebei (NO.E2020100001, and NO.22372502D), High-level S &#38; T Innovation and Entrepreneurship Talent Project of Shijiazhuang (No. 07202203).

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 &#176;C and relative humidity of 40%-70%.
1. Establishment of a chronic hypoxia mouse model (Figure 1)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Evaluate the chronic hypoxia-induced cognitive dysfunction model using histology examination and behavioral tests: the open field test15 and the water maze test16.</li>
</ol>
2. Anesthesia (Figure 2)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Once their excitability decreased, pinch the mouse&#39;s toe to check its reflex. Then, transfer the mouse onto the constant temperature heating pad (37 &#176;C).</li>
	<li>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.</li>
</ol>
3. Electro-acupuncture treatment
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Locate the GV24 acupoint 1.3 mm directly above the midpoint of the mouse&#39;s eyes on the midline of the forehead17. The depth of acupuncture needle insertion must be 2 mm.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Prepare disposable acupuncture needles (see Table of Materials) and an electro-acupuncture device (see Table of Materials) for the procedure (Figure 4).</li>
	<li>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.</li>
	<li>Perform acupuncture at GV20 and GV24 acupoints transversely for 2 mm depth, lifting the skin on the mouse&#39;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&#39;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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Administer the daily treatment for 30 min each day for 6 consecutive days, with a single day of rest between each treatment cycle.</li>
</ol>
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.
<ol>
	<li>To conduct the test, prepare a white-walled cube measuring 50 cm &#215; 50 cm &#215; 30 cm, with the bottom divided into 25 equal squares measuring 10 cm &#215; 10 cm.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Use a video tracking system (see Table of Materials) to record the mouse&#39;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.</li>
		<li>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.</li>
		<li>After each test, clean the open field box with 75% ethanol to prevent any false results caused by odor interference when using a mouse.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Place curtains around the circular water tank to prevent the mouse from seeing the researchers during the test.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Throughout the experiment, introduce water into the tank while maintaining a temperature range of 22-24 &#176;C.
	<ol>
		<li>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&#39;s recording of the mouse&#39;s movements and relevant parameters.</li>
	</ol>
	</li>
	<li>Conduct a 5-day continuous spatial exploration test by sequentially placing each mouse in quadrants I, II, III, and IV.
	<ol>
		<li>Position the mouse facing the wall. Move away from the maze to prevent the mouse from using the experimenter&#39;s position as a reference point. Record the time the mouse takes to find the platform.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Dry the mouse with a towel and return it to its cage.</li>
		<li>Rotate the placement of each mouse in each quadrant every 20 min. Record each mouse&#39;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>On Day 6, remove the platform from the target quadrant and conduct a spatial exploration test.
	<ol>
		<li>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. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Place the brain samples in an embedding box. Subsequently, wash the processed brain samples with running water for 6 h.</li>
	<li>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.</li>
	<li>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 &#176;C for 3 h. Finally, embed them in an automatic processor.</li>
	<li>Utilize a rotary slicer to obtain 4 &#181;m sections. Subsequently, subject the sections to hematoxylin staining for a duration ranging from 3-8 min, followed by eosin staining for 1-3 min.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

Cognitive Improvement in Chronic Hypoxia Mice with Acupuncture

The authors have nothing to disclose.

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&#39;s vital energy, or &#34;qi,&#34; 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 pai...

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-&#946; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% paraformaldehyde solution</td>
			<td>Bioroyee (Beijing) Biotechnology Co., Ltd</td>
			<td>RL3234</td>
			<td></td>
		</tr>
		<tr>
			<td>ANY-maze</td>
			<td>Science</td>
			<td>&#160;SA201</td>
			<td>Video tracking system</td>
		</tr>
		<tr>
			<td>C75BL/6J mice</td>
			<td>BEIJING HFK BIOSCIENCE CO.,LTD</td>
			<td>No.110322220103041767</td>
			<td>Gender: Male,&#160; Weight: 18&#8211;22 g</td>
		</tr>
		<tr>
			<td>Electroacupuncture device</td>
			<td>Great Wall</td>
			<td>KWD-808 I</td>
			<td></td>
		</tr>
		<tr>
			<td>Hwato acupuncture&#160; needle</td>
			<td>Suzhou Medical Appliance Factory</td>
			<td>2655519&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-22</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoZoomer Digital Pathology</td>
			<td>Hamamatsu Photonics K. K</td>
			<td>C9600-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal anesthesia machine</td>
			<td>RWD</td>
			<td>YL-LE-A106</td>
			<td></td>
		</tr>
	</tbody>
</table>

acupuncture treatment in a mouse model of chronic hypoxia-induced cognitive dysfunction

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.

Author Spotlight: Improving Anesthesia Protocols for Enhanced Mouse Acupuncture Research

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

Selecting the right anesthesia protocol is crucial for conducting mouse acupuncture. Due to potential cognitive impairment associated with prolonged anesthesia, it represents an essential initial challenge, demanding careful consideration to minimize cognitive function risks in mice during the procedure. In this study, we propose an improved protocol that helps to minimize functional and behavioral abnormalities caused by excessive anesthesia dosing, and enhances the accuracy of the experiments.In the future, our laboratory aims to study the underlying mechanisms of acupuncture despite its observable therapeutic effects on behavior and pathology. Our research will focus on unraveling acupuncture to gain a deeper understanding of its mechanisms.

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 withou...

Watch this Scientific Journal Video about Acupuncture Treatment in a Mouse Model of Chronic Hypoxia-Induced Cognitive Dysfunction at JoVE.com

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.

The treatment of central nervous disorders has consistently posed significant challenges to the medical field. Acupuncture, a non-pharmacological practice ...

acupuncture-treatment-mouse-model-chronic-hypoxia-induced-cognitive

Research

JoVE Journal

Behavior

Electroacupuncture in a Chronic Hypoxia-Induced Cognitive Dysfunction Mouse Model

To begin, place an anesthetized mouse with chronic hypoxia-induced cognitive dysfunction on a heating pad set at 37 degrees Celsius. Then, connect the anesthesia machine to the mouth and nose of the mouse. For electroacupuncture, locate the GV20 acupoint on the midline of the forehead between the line connecting the ear tips.Next, locate the GV24 acupoint 1.3 millimeters directly above the midpoint of the eyes. Then find the ST36 acupoint on the outside of the knee joint about two millimeters below the fibula head. Prepare disposable acupuncture needles and an electroacupuncture device for the procedure.Now place the mouse in the prone position. With the right thumb, index finger and middle finger, hold a stainless steel needle. Puncture the points GV20 and GV24 transversely at a depth of two millimeters.Then, puncture the ST36 acupoint vertically to a depth of three to four millimeters. Connect the proximal end of the needles at GV20 and the left ST36 to one electrode set. Connect the proximal end of the needles at GV24 and the right ST36 to another electrode set.Select the Continuous Wave mode on the electroacupuncture device and set the electric current intensity to two milliamperes and the frequency to two hertz. Confirm the ideal treatment by observing local mild tremors at the acupoints. Use adhesive tape to secure the horizontally inserted needle and the connection line.Administer the treatment for 30 minutes each day for six consecutive days. Electroacupuncture appear to improve the exploratory activity of hypoxia-induced mice. The treated mice also displayed a more organized behavior.

738 Views.  Heilongjiang University of Chinese Medicine.  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. 

Cognitive Improvement in Chronic Hypoxia Mice with Acupuncture

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