Acupuncture Strategies in a Mouse Model of Alzheimer's Disease

Summary

This article introduces a mouse bag fixation device capable of batch-fixing mice to reduce the stress response and improve experimental efficiency. Furthermore, the study describes an acupuncture protocol that can synergistically regulate brain and intestinal functions.

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Numerous studies have demonstrated that alterations in intestinal flora can influence the central nervous system (CNS) through multiple pathways, ultimately contributing to the onset and progression of AD. Recent research suggests the limitations of pharmacological therapies, emphasizing the need for multi-targeted interventions in AD treatment. Traditional Chinese medicine (TCM) highlights the physiological and pathological relationship between the brain and the intestine. Therefore, therapeutic strategies targeting gastrointestinal regulation to enhance brain function are of great importance for delaying the pathological progression of AD. This protocol introduces a brain-intestine coordination acupuncture method. The experimental results showed that this acupuncture protocol could modulate intestinal flora, suppress intestinal inflammation and neuroinflammation in AD model mice, thereby achieving bidirectional therapeutic effects on brain-intestine regulation. Moreover, a mouse bag fixation device for acupuncture treatment was described in this study, which can reduce stress reaction and improve experimental efficiency.

Introduction

Alzheimer's disease (AD) is an age-related neurodegenerative disorder, with memory loss, cognitive impairment, personality changes, and mental behavioral abnormalities as its main clinical manifestations¹. The pathogenesis of AD is complex and still unclear, and inflammation has received much attention as a potential factor in the development of AD. Recently, numerous studies have proved that intestinal flora dysbiosis plays a vital role in the pathogenesis of AD^2,3. The pathological characteristics of intestinal flora dysbiosis have been observed in both AD patients and AD model animals in the early stages of the disease, preceding the onset of neuroinflammation⁴. Studies have shown that intestinal flora imbalance will cause the release of endotoxin, which will induce intestinal inflammatory reactions and destroy the intestinal mucosal barrier⁵.Numerous noxious substances, such as bacterial metabolites, pathogens, and inflammatory factors, translocate into the systemic circulation through the impaired intestinal barrier, subsequently infiltrating the central nervous system (CNS) and thereby inducing neuroinflammation⁶. Therefore, intestinal flora dysbiosis is a critical initiating factor in triggering the pathological cascade reactions of AD. Therapeutic strategies aimed at regulating the gastrointestinal system to improve CNS function are crucial for delaying the pathological development of AD.

Acupuncture is one of the important therapies in traditional Chinese medicine (TCM), which is characterized by safety, minimal side effects, and holistic regulation, and has been widely used worldwide⁷. This study has partially elucidated the mechanisms through which acupuncture can treat AD via anti-neuroinflammation^8,9, metabolic disorder inhibition¹⁰, and cerebral blood flow regulation^11,12. According to the meridian theory of TCM, the brain and intestine are closely related in both physiological and pathological contexts. TCM emphasizes the simultaneous treatment of the brain and intestines in treating AD¹³. Based on these TCM theories, this study employed a brain-intestine coordination acupuncture protocol to treat AD. Our previous research has confirmed the effectiveness of this acupuncture protocol in treating AD^14,15,16,17. The discoveries showed that acupuncture can effectively improve both intestinal barrier function and cognitive abilities in AD model animals.

Although many studies have demonstrated that acupuncture is effective in treating AD, it remains essential to further clarify the standardized process for acupuncture operation. Mice are widely used in acupuncture experiments, but performing acupuncture on them is challenging due to their small size and sensitive activities. While some researchers have utilized anesthesia to calm the mice before acupuncture, anesthetic agents can often damage the cognitive functions of the animals^18,19,20. Moreover, repeated experimental stimulation will also lead to stress responses in the animals, affecting experimental outcomes. Consequently, it is urgent to develop an appropriate method for securing mice to minimize animal stress and facilitate the acupuncture operation. This study selected APPswe/PS1ΔE9 (APP/PS1) mice (male, 6-month-old, 28-32 g) as an AD model, and detailed the operational process of acupuncture treatment for APP/PS1 mice, including acupoints positioning, acupuncture depth, and acupuncture manipulation. Furthermore, this study has introduced a method for immobilizing mice using a self-made conical mouse bag. The method is applicable for acupuncture interventions targeting the acupoints on the head, back, and outside limbs, and it can extend the fixation duration of the mice, ensuring the effectiveness of the acupuncture intervention.

Protocol

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Beijing University of Chinese Medicine (ID: bucm-4-2021102701-4032). The reagents and the equipment used are listed in the Table of Materials.

1. Animal preparation

1. Select APP/PS1 transgenic animals (male, 6-month-old, 28-32 g) with C57BL/6 background and their corresponding wild-type C57BL/6 littermates (male, 6-month-old, 28-32 g) for the experiment.
2. House the mice in the animal facility at a temperature of 24 °C ± 2 °C and a 12 h dark/light cycle.
   NOTE: The animals were housed in the Experimental Animal Center of Beijing University of Chinese Medicine for this study.
3. Provide sterile drinking water and a standard pellet diet for mice. Acclimate all the animals to the environment for 7 days before experimentation.
4. Randomly divide sixteen APP/PS1 mice into two groups (n = 8 per group): the AD model (Model) group, the acupuncture (Acu) group. Serve eight C57BL/6 mice as the normal control (Normal) group. The timeline of experimental design is shown in Figure 1.

2. Preparation of mouse bags for fixing mice

1. Prepare the raw materials for making mouse bags in advance, including 2 mm thick cardboard, breathable soft gauze mesh, a stapler, and clips (Figure 2A) .
2. Cut a rectangle measuring 10 cm in length and 7 cm in width from the cardboard to serve as the base plate for supporting the mice.
3. Trim the top left and right corners of the shorter side of the rectangle at an angle of approximately 30° to create a trapezoidal shape.
4. Cut the gauze mesh to match the dimensions of the cardboard.
   NOTE: The upper edge of the gauze mesh should extend at least 2 cm above the upper edge of the cardboard, while the left and right edges should also extend at least 2 cm beyond the corresponding edges of the cardboard. Additionally, the lower edge of the gauze mesh should exceed the lower edge of the cardboard by at least 5 cm.
5. Secure the gauze mesh to the cardboard using stainless steel staples. Depending on the size of the mouse, form a tapered funnel channel between the cardboard and the gauze mesh, with an opening at the bottom of the channel to facilitate entry.
   NOTE: On the basis of experience, this funnel channel is standardized to accommodate the width of two adult fingers. If the channel is too narrow, it may hinder the mice's entry; conversely, if it is too wide, it may allow the mice to escape. The production process of the mouse bags is illustrated in Figure 2B. The floor plan and the three-dimensional view of the mouse bag are depicted in Figures 2C,D.

3. Fixing mice using mouse bags

1. Introduce the mice into the opening at the bottom of the mouse bag.
   NOTE: Given their natural inclination towards dark and confined spaces, the mice will instinctively burrow into the bag.
2. Once the mice enter the mouse bag, tighten the opening immediately. Use clips to secure the bags, ensuring that the mesh fabric envelops the mouse, thereby restricting its movement (Figure 2E).
   NOTE: The clips are used to regulate the tightness of the mouse bag, and their position must be adjusted at any time according to the state of the mouse. If the mice exhibit open-mouth breathing and struggle violently, the clips should be released immediately. After the mice calm down, fix them again. When securing the clips, avoid contact with the limbs and tail of the mouse to prevent inducing stress.
3. Repeat the above steps for three consecutive days for each group of mice to ensure they fully acclimatize to the mouse bags. Maintain immobilization for at least 5 min every day.
   NOTE: Once the bags retain the scent of the mice, they will be more inclined to enter the bags willingly and remain inside in a calm state.

4. Acupuncture treatment

1. Following a consecutive three-day fixation process, commence acupuncture intervention on the 4^th day.
2. Prepare disposable sterile acupuncture needles (diameter, 0.25 mm; length, 13 mm).
3. According to the TCM theory and clinical experience, select Baihui (GV20), Yintang (GV29), and Zusanli (ST36) acupoints for acupuncture treatment^14,15,16,17.
4. Based on the anatomical structure, locate the GV20 acupoint at the center of the parietal bone, where the midline of the forehead intersects with the midpoint of the connecting line between the ear tips.
   1. Locate GV29 acupoint between eyes, along the anterior midline. Locate ST36 acupoint at the posterolateral side of the knee joint, 2 mm below the fibular capitulum (Figure 3A-C). Locate the GV20 acupoint at the center of the parietal bone, where the midline of the forehead intersects with the midpoint of the connecting line between the ear tips.
5. Disinfect the acupoints with medical iodophor and 75% medical alcohol.
6. Immobilize the mice using the mouse bags.
7. Hold the disposable sterile acupuncture needles in the right hand. Insert the needles obliquely downward at an angle of 15° to a depth of 2-3 mm at the GV20 and GV29. Locate the ST36 acupoint by touching the fibular head, and then insert the needle perpendicularly at a depth of 4 mm (Figure 3D).
8. Twist the needle bidirectionally within 90° for 15 s at intervals of 5 min during the needling process.
   NOTE: This acupuncture intervention was conducted daily for 35 days, with each session lasting 20 min. Mice in the Normal and Model groups received no treatment, but they were immobilized for 20 min every day in the same manner as those in the Acu group. The aforementioned interventions were performed throughout the water maze test period.

5. Morris water maze (MWM) test

NOTE: The MWM test is a classic behavioral experiment used to assess the cognitive function of rodents²¹. On the 30^th day of acupuncture intervention, the MWM test was conducted for 6 days.

1. Prepare a circular water pool with a diameter of 90 cm and a height of 50 cm, and a platform with a diameter of 9.5 cm. Divide the pool into four equal quadrants: Northeast (NE), Northwest (NW), Southwest (SW), and Southeast (SE).
2. Position geometric markers with different shapes in the center of the four quadrants of the cylinder wall as visual references.
3. Fill the circular pool with water to a depth of 30 cm, and control the water temperature at 23 °C ± 1 °C using an electric heater.
4. Cover the periphery and the top of the water pool with an opaque curtain. Place the camera 2 meters above the center of the pool. Maintain the indoor environment quiet and ensure that all references in the room are consistent throughout the test.
5. At first, conduct the hidden platform trial for 5 consecutive days.
   1. Place the platform 1-2 cm below the water surface at the center of the SW quadrant.
   2. Before the experiment, exercise adaptive swimming training for each mouse to avoid experimental errors.
   3. Release each mouse into the water from one of the four start locations facing the pool wall. Each mouse had 60 s to search for the platform. If the mice find the platform within 60 s and stay for 3 s, stop timing and record the time at this moment as the escape latency.
      1. If the mice fail to find the platform within 60 s, record the escape latency as 60 s, and guide them to stay on the platform for 20 s. If the mice find the platform within 60 s and stay for 3 s, stop timing and record the time at
         this moment as the escape latency.
   4. Dry the mice immediately with a towel, and return them to their cage. Place the cage on the heating pad to keep the animals warm.
6. 24 h after the end of the hidden platform trial, conduct the probe trial for one day.
   1. Remove the platform and release the mouse from the NE quadrant facing the pool wall.
   2. Record the swimming time ratio in the SW quadrant within 60 s.

6. Sample collection

1. After the MWM test, collect fresh fecal tissues of each group for 16S rRNA sequencing.
2. Euthanize the mice with intraperitoneal injection of pentobarbital sodium (150 mg/kg) (following institutionally approved protocols). Harvest the hippocampus and ileum tissues for ELISA detection.

7. 16S rRNA sequencing

1. Extract the total genomic DNA of the microbial community from the collected fresh fecal samples.
2. Measure DNA concentration and purity by 1% agarose gel electrophoresis (Supplementary Table 1).
3. Perform PCR amplification of the V3-V4 region with the universal primers (338F-ACTCCTACGGGAGGCAGCAG, 806R-GGACTACHVGGGTWTCTAAT).
   NOTE: The PCR reaction mixture including 4 µL 5 × Fast Pfu buffer, 2 µL 2.5 mM dNTPs, 0.8 µL each primer (5 µM), 0.4 µL Fast Pfu polymerase, 0.2 µL BSA, 10 ng of template DNA, and ddH₂O to a final volume of 20 µL.
4. Use the Rapid DNA-Seq Kit to build the library of the purified PCR products.
5. Sequence all libraries on the sequencing platform following published protocols²².
6. Perform bioinformatics analyses based on the online platform of Majorbio Cloud Platform. Cluster the optimized sequences into operational taxonomic units (OTUs) using UPARSE 7.1 with 97% sequence similarity level²².
7. Based on the OTUs' information, perform a PERMANOVA test to assess the percentage of variation explained by the treatment along with its statistical significance using the Vegan package.

8. ELISA analysis

1. Homogenize the ileum and hippocampus tissues. Centrifuge the homogenate at 5000 × g for 10 min at 4 °C, and collect the supernatant.
2. Determine the concentration of TNF-α in the ileum and hippocampus and Aβ42 in the hippocampus using ELISA kits. The representative standard curves are provided in Supplementary File 1.

9. Statistical analysis

1. Use SPSS software for statistical analysis. Express the data as mean ± standard deviation.
2. Perform two-way analysis of variance (ANOVA) with repeated measures and the Least Significant Difference (LSD)'s test for dissecting differences among groups in the hidden platform trial.
3. Perform one-way ANOVA with the LSD method to compare the variability among groups in other trials when the data were normally distributed or had homogenous variance. Otherwise, the non-parametric test would be used.
4. Consider differences statistically significant when P < 0.05.

Results

Behavioral responses of mice to acupuncture intervention with or without the use of the mouse bag fixation device
To evaluate whether using a rodent restraint bag during acupuncture intervention improves experimental efficiency, a total of 16 male C57BL/6 mice were randomly assigned to a device usage group and a non-usage group (n = 8 per group). The experiment was conducted by an experimenter proficient in acupuncture operations. For the device usage group, mice were secured using the mouse bag fixa...

Discussion

The intestinal flora constitutes a significant component of the intestinal immune system. Intestinal flora disorder often leads to excessive activation and release of intestinal inflammatory pathways and local inflammatory mediators^23,24. Studies have confirmed that the increase in intestinal barrier permeability, induced by inflammatory injury to the intestinal mucosa, further triggers neuroinflammation and exacerbates the pathological progression of AD

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% saline, 0.01M PBS powder	Beijing suolaibao technology Co., Ltd.	IN9000, P1022-10
75% medical alcohol, iodophor	Beijing oubei biology science and technology Co., Ltd.	OB40126-1, HX0098A	Used for disinfection
Acupuncture needles	Zhongyan Taihe Co., Ltd.	ZY500
APP/PS1 mice and male C57BL/6 mice	Cavens Biogle (Suzhou) Model Animal Research Co., Ltd.	SCXK (Su) 2018-0002
Cardboard	Wenzhou Aguo Paper Co., Ltd.	1010	Used for making mouse bags
Centrifugal machine	Eppendorf,Germany	5811FR080384
ELISA kits	Jiangsu kete trading Co., Ltd.	F5171-A	Used to detect TNF-α level
Gauze mesh	VAGBG Co., Ltd.	24A	Used for making mouse bags
Illumina Nextseq 2000 platform	Illumina, San Diego, USA	MS-103-1002	Sequence the library
Morris water maze detection system	Chengdu Taimeng Technology Co., Ltd.	WMT 200A
NEXTFLEX Rapid DNA-Seq Kit	Bioo Scientific, Austin, Texas, USA	NOVA-5188	Build the library of the purified PCR products