Operation Procedure and Precautions of Thread-Embedding Acupuncture Therapy in Alzheimer

Min You; Jing-yan Miao; Meng-ying Yang; Qing Liu; Yue-ang Liu; Yan-jun Du

doi:10.3791/65895

JoVE Journal > Neuroscience

Neuroscience

Operation Procedure and Precautions of Thread-Embedding Acupuncture Therapy in Alzheimer

Published: May 10, 2024

doi:

10.3791/65895

Min You*¹, Jing-yan Miao*¹, Meng-ying Yang¹, Qing Liu¹, Yue-ang Liu¹, Yan-jun Du^1,2,3,4

¹College of Acupuncture and Orthopedics,Hubei University of Chinese Medicine, ²Hubei Shizhen Laboratory, ³Hubei International Science and Technology Cooperation Base of Preventive Treatment by Acupuncture and Moxibustion, ⁴Hubei Provincial Hospital of Traditional Chinese Medicine

Summary

This study presents a protocol for thread-embedding acupuncture therapy in Alzheimer’s disease-like rats.

Abstract

Thread-embedding therapy (TEAT) is a treatment that prevents and manages diseases by inserting a biodegradable suture into an acupoint, providing long-lasting stimulation. TEAT is a simple approach that avoids the discomfort of regular acupuncture and provides sustained therapeutic effects. This article discusses the potential impact of TEAT on the learning and memory abilities of rats with Alzheimer's disease-like symptoms. Since chemically induced neuronal degeneration and cognitive impairments in rats does not entirely reflect the true pathological changes observed in Alzheimer's disease. Consequently, our research group has designated these manifestations as Alzheimer's disease-like symptoms. A protocol has been established to outline the selection of acupoints, the operation process, and necessary precautions for the head and lower back. The experiment was conducted on three groups: a control group, a model group, and a TEAT group, each containing 6 rats. To induce Alzheimer's disease-like symptoms, rats were intraperitoneally injected with D-galactose for 7 weeks (49 days). The rats in the TEAT group received acupoint catgut embedding treatment. Following the intervention period, a Morris Water Maze (MWM) was conducted to evaluate the rats' learning and memory. Subsequently, the rats were sacrificed, and their brain tissue was examined. A histological examination was performed to understand the effects of TEAT on the pathology of rats exhibiting symptoms of Alzheimer's disease. This study suggests that TEAT may improve learning and memory in rats with Alzheimer's disease-like symptoms, indicating a potentially promising new treatment approach for this neurodegenerative condition.

Introduction

Thread-embedding acupuncture (TEA) involves embedding catgut or absorbable sutures into acupoints using a special needle. This technique has a prolonged effect until the sutures are absorbed and degraded. It provides continuous and sustained stimulation to the acupoints for a week or longer¹, achieving a comparable outcome without the necessity for daily repetitive procedures². This reduces the number of patients seeking medical treatment, thus conserving medical resources to some extent. In recent years, TEAT has been increasingly used in various medical fields in China, including internal medicine, external medicine, dermatology, facial features, gynecology, and pediatrics³. Studies have shown that it is effective in producing long-term and reliable effects for certain chronic and complex diseases, such as epilepsy⁴ and facial nerve paralysis⁵. It has the advantages of minimal trauma and simple operation⁶. Additionally, the treatment costs of TEAT have been reported to be lower than those of electroacupuncture (EA)⁷. Therefore, TEAT is increasingly valued in clinical and scientific research.

Alzheimer’s disease (AD) is a neurodegenerative condition that progresses over time. Its pathophysiological process begins to accumulate approximately 20 years before clinical symptoms appear, but its mechanism is still not fully understood⁸. Given the limited availability of effective drugs for AD, research is increasingly focusing on the prodromal and preclinical stages of the disease⁹. Prevention is a crucial strategy for addressing the development and progression of AD⁹^,¹⁰. In recent years, traditional Chinese medicine (TCM) has made significant progress, and it is expected to offer new possibilities for preventing and treating AD¹¹^,¹². As a subtype of acupuncture, TEAT is preferred over other therapies due to its wide treatment range, minimal trauma, simple operation, low treatment frequency, and long-lasting efficacy⁶. Emerging literature suggests that TEAT may benefit individuals with obesity¹³^,¹⁴, diabetes¹⁵, insomnia¹⁶, postmenopausal osteoporosis¹⁷, and depression¹⁸, all of which are risk factors for AD. It also improves senescence in aging rats by regulating mitophagy², improves spatial learning and memory impairment, alleviates pathological damage of the hippocampus, and inhibits inflammation response in vascular dementia (VD) rats¹⁹. It appeared attractive to figure out whether TEAT could also act on pathological processes during AD.

The current research lacks uniformity and standardization in several critical elements of TEAT operation, including the embedding method, embedding tool, thread, and embedding interval period. This lack of standardization affects the evaluation of the therapy’s effect and the exploration of related mechanisms and restricts external promotion and communication. Prior research²⁰^,²¹ has demonstrated that intraperitoneal administration of D-galactose (D-gal) can induce Alzheimer’s disease-like pathological changes in rats, including neuronal degeneration and cognitive impairments. Furthermore, electroacupuncture at the Baihui(GV20) and Shenshu(BL23) has been shown to effectively ameliorate cognitive decline, mitigate neuroinflammation, and reduce neuronal damage. This study takes Alzheimer’s disease-like model rats as an example to introduce the selection of acupoints and the operation process and precautions of TEAT under this model. It discusses the critical operational elements of TEAT in experimental research to provide a reference for future studies.

Protocol

All experiments were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Hubei Provincial Center for Disease Control and Prevention Laboratory Animal Management and Use Committee. A total of 18 3-month-old female Sprague-Dawley (SD) rats weighing 220-250 g were used for the test. All rats were maintained on a 12 h light/dark cycle at 23 ± 1 °C and 40%-50% humidity, with free access to food and water. Following a one-week acclimatization period, during which the rats were allowed to habituate to the laboratory conditions, the experiments were initiated.

1. Model development

The experimental design is shown in Figure 1. Randomly divide 18 Sprague-Dawley 3-month-old female rats weighing 220-250 g into control, model, and TEAT groups. Each group consists of 6 rats.
Inject the model group and TEAT group daily with D-galactose (D-gal; 120 mg/kg/day), while the control group receives saline injections (120 mg/kg/day), for 7 weeks (49 days) in the lower ventral area²². The injection should be administered daily between 8:00 and 9:00 a.m.
Weigh the rats on the 7th day of each week between 9:00 and 10:00 a.m. The experiment lasted for 49 days. Bury the threads every 14 days on the 14^th, 28^th, and 42^nd days. Fix the time for each thread embedding between 9:00-12:00 am.
Continue intraperitoneal administration of D-galactose for 7-weeks to induce Alzheimer's disease-related pathological manifestations in rats²².

2. Preparation for TEAT

Sterilize surgical instruments such as ophthalmic scissors, trays, and tweezers and disinfect. Clean the surfaces in the operating room environment thoroughly and disinfect.
Prepare the surgical platform by laying a small animal heating pad flat on the surface, followed by draping with a sterile towel. Subsequently, place the cleaned and disinfected operating board smoothly on top, with the rough side facing upwards. Then, switch on the heating pad and set the temperature to 32 °C.
For the procedure, select a No. 6 disposable thread-embedding needle with a diameter of 0.6 mm and a 5-0 absorbable surgical suture (collagen suture) with a length of 2 cm. Cut the 2 cm 5-0 absorbable surgical suture into 0.5 cm and 0.2 cm pieces using ophthalmic scissors. Soak the cut thread in 0.9% saline and set aside.
Wash hands with soap and water for 20 s, and then dry them. Afterward, wear sterile gloves.
Anesthetize the rats with an IP injection of chloral hydrate (300 mg/kg) in the lower ventral area, ensuring that anesthesia is confirmed when animals are unresponsive in a surgical plane, based on the lack of response from a toe pinch test, and maintaining a stable physiological state. Additionally, to prevent corneal dryness and potential damage while the rats are under anesthesia, apply a generous amount of veterinary ointment to the eyes, ensuring they remain lubricated throughout the procedure.
NOTE: It is recommended to anesthetize the rat, as it is painful for them to experience the piercing.

3. Thread-embedding operation for Baihui (GV 20)

Locate the acupoint Baihui (GV 20) in rats at the right midpoint of the parietal bone (Figure 2A). Position the rats to fully expose the head, with the ears pulled forward to approximate the method used in humans for connecting the ear apex lines. Identify the Baihui (GV 20) point by palpating the central longitudinal ridge of the skull and pinpointing the location roughly at its midpoint. Confirm this acupoint further at the level where the ears would make contact with the head if laid flat, aligning with the intersection of the sagittal suture and the parietal bone. Use a marker to mark Baihui (GV 20).
Shave the head area around the acupoint of the rat using a trimmer (usually an electric men's beard trimmer).
Prior to surgery, disinfect the acupoints and surrounding skin by applying a 0.5% iodine solution in circular motions, starting from the center, and moving outwards. Subsequently, perform a triple disinfection process: use the iodophor 3x, each followed by an application of alcohol to ensure proper decontamination and prepare the site for aseptic technique.
Use forceps to clamp out the 0.2 cm absorbable surgical suture soaked in saline and place it in the front end of the needle tube. Then, connect it to the needle core.
Pinch the area around Baihui (GV 20) with thumb and index finger on one hand, while holding the needle with the other.
Insert the needle horizontally and quickly at a 15° angle to the skin surface, 0.1 cm below the Baihui (GV 20) point, while pinching the skin (Figure 3A).
After the needle tip reaches 0.1 cm above the Baihui (GV 20) point, push the needle core while retracting the needle tube to fully implant the 0.2 cm absorbable surgical suture into the subcutaneous tissue of the acupoint (Figure 3B).
Slowly remove the needle, making sure no suture is exposed. Press the needle hole with sterile dry cotton for 10 s to prevent bleeding. If the thread is outside the body after embedding the line at acupoints, extract it and re-operate.

4. Thread-embedding operation for Shenshu (BL 23)

The acupoint Shenshu (BL 23) in rats is located 5 mm lateral to the 2^nd lumbar vertebra (Figure 2B). First, locate the 6^th lumbar vertebra based on the hip tubercle of the rat. Then locate the 2^nd lumbar vertebra by counting upward four vertebral bodies. Shenshu (BL 23) will be 5 mm lateral to it²³.
NOTE: The combination of the Baihui (GV 20) and Shenshu (BL 23) acupoints is a frequently utilized set in Traditional Chinese Medicine (TCM) theory for the prevention and treatment of cerebral disorders. As confirmed by previous research²⁰^,²¹, this acupoint pairing has been established for its efficacy.
Use a marker to mark Shenshu (BL 23). Shave the back and waist area around the acupoint of the rat using a trimmer (usually an electric beard trimmer).
Prior to surgery, disinfect the acupoints and surrounding skin by applying 0.5% iodine solution in a circular motion, starting from the center and moving outwards. Subsequently, perform a triple disinfection process: use the iodophor three times, each followed by an application of alcohol to ensure proper decontamination and preparation of the site for aseptic technique.
Use forceps to clamp out the 0.5 cm absorbable surgical suture soaked in saline and place it in the front end of the needle tube. Then, connect it to the needle core.
Stretch the skin with the index and middle fingers of the pressing hand or the thumb and index fingers. Hold the needle with the puncturing hand.
Quickly insert the needle perpendicularly into the point 0.25 cm below the Shenshu (BL 23). See Figure 4A.
After the needle tip reaches 0.5 cm under the skin, adjust the needle tip, making the direction of insertion centripetal, forming a 45° angle with the skin surface and obliquely insert 0.5 cm (Figure 4B).
NOTE: It is important to exercise caution when needling the Shenshu (BL 23), which is located near the kidney. Deep insertion or repeated lifting and thrusting of the needle can cause damage. Therefore, we recommend an insertion depth of around 0.3-0.6 cm.
Push the needle core while retracting the tube and fully implant the 0.5 cm absorbable surgical suture into the acupoint's muscle tissue of the acupoint (Figure 4C).
Slowly pull out the needle, ensuring no suture is exposed. Then, press the needle hole with sterile dry cotton for 10 s to prevent bleeding. If the thread is outside the body after embedding the line at acupoints, extract it and re-operate.

5. Post-operative care

Postoperatively, place the rat on a small animal heating pad set at a temperature of 32°C to facilitate recovery from anesthesia.Ensure the animal is fully conscious and its condition is satisfactory before returning it to its housing enclosure.
After surgery, observe signs of sterile inflammatory responses in the affected area within a week. These signs may include redness, swelling, or a pus-like discharge. Typically, these symptoms do not require any treatment. However, if there is an excessive amount of exudate, drain and disinfect the affected area. In such cases, apply erythromycin ointment until the wound fully recovers.

6. Behavioral testing and histological examination

Following the intervention, at week 8, perform the Morris Water Maze (MWM) experiment²^,²⁴ to evaluate their learning and memory capabilities. Perform the learning trials over 5 days, with four trials per day. Then administer a probe trial after 24 h from the last acquisition day.
Perform statistical comparisons using statistical analysis software. Analyze the behavioral data of learning ability by one-way analysis of variance (ANOVA) and post-hoc Tukey's test. Assess the significance of escape latency data by two-way repeated measure analysis of variance (ANOVA) followed by the post-hoc Bonferroni test. A value of p <0.05 is considered significant in all tests.
After the behavioral assessment, anesthetize the rats as above and euthanize with intraperitoneal injections of 150 mg/kg body weight of pentobarbital sodium.
Fix the rat's brain through perfusion using 4% paraformaldehyde perfused via the vascular system²⁵.
1. Insertion and fixation of the gavage needle
  1. Prepare a 12G needle and ensure it is straight and suitable for passing through the ventricle of the rat. Locate the ventricle of the rat, which is typically the left ventricle, and insert the needle through it into the ascending aorta. This step requires precision and a good understanding of rat anatomy.
  2. Once the needle is in place, use a hemostat to clamp the gavage needle near the point where it enters the aorta. This will help to secure the needle and prevent it from moving.
  3. Ensure that the clamp is tight enough to hold the needle securely but not so tight as to damage the aorta or impede blood flow. The gavage needle should remain in the ascending aorta throughout the procedure to maintain proper perfusion.
2. Perfusion with normal saline
  1. Prepare a 50cc syringe without a needle for the perfusion. Attach the other end of the gavage needle, which is outside the rat's body, to the syringe filled with normal saline.
  2. Make a small incision to cut the right atrial appendage to allow blood to flow out of the heart. This step ensures that the blood is displaced by the saline during perfusion.
  3. Begin injecting the normal saline into the ascending aorta at a steady and constant rate. This will flush out the blood and prepare the tissues for fixation.
  4. Monitor the fluid coming out of the right atrial appendage and be prepared to change the syringe as needed to maintain a continuous perfusion with fresh saline.
    placed, prepare to perfuse with the fixative.
  5. Replace the syringe containing saline with one containing 4% paraformaldehyde, a commonly used fixative in histology. Proceed with the perfusion using the paraformaldehyde to fix the tissues in place for subsequent processing and analysis.
    NOTE: Throughout this procedure, it is crucial to maintain aseptic techniques to prevent infection and to handle the rat gently to minimize injury. Additionally, all personnel involved should be trained in the technique to ensure consistency and effectiveness.
Remove the rat brain and place it in a specimen bottle containing 4% paraformaldehyde for 24-48 h at 4 °C²⁵^,²⁶. Perform ethanol dehydration and xylene treatment step by step, followed by conventional continuous coronal sections (4-5 µm) for measurement. Evaluate the morphology and loss of hippocampal neurons in each group using Hematoxylin and Eosin (H&E) staining (200x).
NOTE: A necropsy examination to assess for gastrointestinal (GI) lesions was not conducted on the rodents based on the absence of clinical signs indicative of GI disturbances throughout the experimental period. The animals exhibited no overt adverse reactions that would suggest the presence of such pathologies.

Representative Results

This article introduces the specific operation method of TEAT in Alzheimer's disease-like rats. It discusses the critical operational elements of TEAT in experimental research to provide a reference for future studies.

TEAT improves learning in Alzheimer's disease-like rats
As illustrated in the flowchart (Figure 5), during the adaptive training period, it was observed that rats in each group were observing the surrounding environment while swimming. The analysis of swimming speed (Figure 6) showed no significant difference among the groups, indicating consistent baseline levels of movement speed and visual perception. Any observed differences in experimental results were not due to variations in movement ability or visual perception.

The rats underwent 5 days of platform search training. In Figure 7A, it can be observed that the movement trajectories of each group of rats differed in the early and late stages of training. On day 2, most of the escape-driven rat groups swam randomly. However, on day 5, the rats in the control group swam with direction, while the model group rats swam randomly along the pool wall. The TEAT group rats' movement became denser in the target quadrant. The experiment measured escape latency, with shorter latency indicating stronger abilities. Figure 7B shows that the model group exhibited reduced learning and memory abilities compared to the control group. However, the TEAT group showed a significant improvement in spatial reference memory impairment in rats with AD-like pathology.

TEAT improves spatial memory in Alzheimer's disease-like rats
In the probe trial, the location where the animal spends time indicates spatial bias²⁷. Rats use four primary methods to search for platforms: edge-based, random-based, trend-based, and linear-based²⁸. The results indicate that each group of rats employed different search strategies (Figure 8A). The control group primarily used linear and directional search strategies, suggesting a strong memory and ability to recall the platform's location. In contrast, the rats in the model group exhibited an edge-like main search strategy, indicating a significant decline in their memory ability. They did not learn to find the platform or remember its location. The rats in the TEAT group showed a trend-based search strategy. The trajectory density was highest in the quadrant where the original platform was located, as well as in the adjacent quadrants. This indicates a memory improvement compared to the model group.

Figure 8B and Figure 8C show the results of the duration of stay in the quadrant of the original platform and the number of times the rats crossed the platform. The model group rats exhibited a significant reduction in their stay time in the original platform quadrant and the number of times they crossed the platform compared to the control group (p <0.01). Compared to the model group, the TEAT group rats exhibited a significant increase in their stay time in the original platform quadrant and the number of times they crossed the platform (p <0.01). These results suggest that TEAT can improve spatial learning and memory impairment in rats with AD-like pathology.

TEAT ameliorates hippocampal aging in rats (Figure 9). Compared to the control group, rats in the model group exhibited severe pathological changes, including a significant reduction in the number of pyramidal cells and their irregular arrangement. In addition, nuclear condensation, neuronal degeneration, and other abnormalities were observed. However, following TEAT treatment, the number of hippocampal pyramidal cells in rats increased and were more organized. Although some nuclei exhibited condensation, the overall cell morphology appeared more normal. Cells were more densely distributed and ordered, cytoplasmic staining was more consistent, and the edema surrounding the cells was significantly reduced. Notably, normal pyramidal cells were observed.

Figure 1: Flow diagram of the experiment. After 1 week of adaption (week 0), the study allocated 18 rats into three groups: Control, Model, and TEAT. During weeks 1 to 7, the control group received saline injections, while the model and TEAT groups were administered D-galactose. The TEAT group additionally underwent thread-embedding acupuncture therapy on weeks 2, 4, and 6. All rats were subjected to the Morris water maze test in week 8, followed by euthanasia. Abbreviations: Adapt = adaptation, Con = Control group, Mod = Model group, TEAT = Thread-embedding Acupuncture Therapy group, D-gal = D-galactose, MWM = the Morris water maze test. Please click here to view a larger version of this figure.

Figure 2: The location of the acupoint. (A) The acupoint Baihui (GV 20) in rats. It is located at the right midpoint of the parietal bone. (B) The acupoint Shenshu (BL 23) in rats. It is located 5 mm lateral to the second lumbar vertebra. Abbreviations: GV = Governor Vessel, a concept in Traditional Chinese Medicine (TCM) that refers to the main meridian running along the midline of the body's posterior aspect, BL = Bladder, stands for the Bladder Meridian, which in TCM corresponds to the Foot-Taiyang meridian, one of the six yang meridians, running along the back of the body and legs. Please click here to view a larger version of this figure.

Figure 3: Detailed thread-embedding operation of Baihui (GV 20). (A) The needle is inserted horizontally at a 15° angle to the skin surface, 0.1 cm below the Baihui (GV 20) acupoint, with the skin pinched to facilitate the procedure. (B) Once the needle tip reaches a position 0.1 cm above the Baihui (GV 20) acupoint, the needle core is pushed while retracting the needle tube to fully implant the 0.2 cm absorbable surgical suture into the subcutaneous tissue at the acupoint. Abbreviations: GV = Governor Vessel, a concept in Traditional Chinese Medicine (TCM) that refers to the main meridian running along the midline of the body's posterior aspect. Please click here to view a larger version of this figure.

Figure 4: Detailed thread-embedding operation of Shenshu (BL 23). (A) The needle is rapidly inserted perpendicularly into the acupoint to a depth of 0.25 cm below the Shenshu (BL 23). (B) Once the needle tip has reached a depth of 0.5 cm beneath the skin surface, the needle is redirected towards the center of the acupoint, creating a 45°angle with the skin, and then obliquely inserted to a depth of 0.5 cm. (C) While the needle shaft is advanced, the tube is retracted, ensuring the complete implantation of a 0.5 cm segment of absorbable surgical suture into the muscle tissue at the Shenshu (BL 23) acupoint. Abbreviations: BL = Bladder, stands for the Bladder Meridian, which in TCM corresponds to the Foot-Taiyang meridian, one of the six yang meridians, running along the back of the body and legs. Please click here to view a larger version of this figure.

Figure 5: The flow chart of the WMW test. The adaption training prior to the experiment is denoted as Day 0, during which the rats' motor capabilities are observed and the swimming velocity is statistically analyzed. This is followed by a five-day place navigation phase (Days 1 to 5), where the latency to escape (escape latency) is compared and analyzed among the different groups. On Day 6, a spatial probe trial is conducted to assess the time spent in the target quadrant (target quadrant dwell time) and the number of platform crossings. Please click here to view a larger version of this figure.

Figure 6: Comparison of swimming speed (m/s) among groups. This figure delineates the mean swimming velocities (in meters per second) of rats across various experimental groups, each comprising six animals (n=6). The error bars show standard deviation. No inter-group differences were obtained, as indicated by ns (p > 0.05). The statistical analysis was executed using SPSS software, with all data adhering to a normal distribution. Please click here to view a larger version of this figure.

Figure 7: The results of WMW's place navigation. (A) This panel presents the representative motion trajectory diagrams, illustrating the navigational paths of rats within each experimental group during the place navigation trials. (B) The escape latency, measured in seconds (s), is compared across the groups, with each group consisting of six rats (n=6). Statistical analysis was performed using two-way repeated measures ANOVA to assess the within-subjects' effects over the course of the navigation trials and the between-subjects differences among the groups. Data are compliant with normal distribution assumptions and are depicted as the group mean ± standard deviation. Significant differences are indicated by asterisks (*p < 0.05, **p < 0.01) compared to the control group and by hashtags (#p < 0.05, ##p < 0.01) compared to the model group. Please click here to view a larger version of this figure.

Figure 8: The results of WMW's probe trail. (A) The panel displays representative motion trajectory diagrams that capture the spatial exploration behaviors of rats in the probe trial within each experimental group. (B) This graph compares the dwell time (in seconds, s) within the target quadrant among the groups, reflecting the rats' memory retention of the platform location. (C) The graph illustrates the frequency (freq) with which rats crossed the platform area, indicating their spatial memory performance. Each group comprised six rats (n=6), and the data were statistically analyzed using one-way ANOVA to assess differences across the groups. The data adhere to a normal distribution and are presented as the mean ± standard deviation. Significant differences are indicated by asterisks (**p < 0.01) compared to the control group and by hashtags (#p < 0.05, ##p < 0.01) compared to the model group. **p < 0.01 vs. control group, #p < 0.05 vs. model group, ##p < 0.01 vs. model group. Please click here to view a larger version of this figure.

Figure 9: Hematoxylin and Eosin Staining Assessment of Hippocampal CA1 Region in Rat Groups at 200x magnification. (A) Control group exhibits typical neuronal structure with well-preserved pyramidal cells. (B) Model group displays pronounced pathological alterations, characterized by a marked decrease in the density of pyramidal cells, darkly stained degenerated neurons, and disordered arrangement. (C) TEAT group demonstrates enhanced hippocampal architecture, with an increased number of pyramidal cells that are more orderly and possess a more normative morphology. Despite occasional nuclear condensation, the cells are more densely packed, exhibit uniform cytoplasmic staining, and show a significant reduction in pericellular edema. The presence of normative pyramidal cells is notably observed. Scale bar = 50 µm. Please click here to view a larger version of this figure.

Discussion

Intervention time
The etiology of AD is complex, and its pathogenesis remains uncertain. Currently, there is no effective treatment for the disease, and its course is irreversible. Therefore, the academic community has reached a consensus on the importance of focusing on the prevention and treatment of AD. The prevention and treatment strategies for AD emphasize early intervention, following the TCM concept of treating conditions before they occur, emphasizing treatment before its onset, transmission, and recurrence. The accumulated research in humans²⁹^,³⁰ and rodents³¹^,³² has shown that early acupuncture and moxibustion intervention can achieve significant results without apparent side effects, making it a promising potential method for preventing and treating AD. Therefore, this study administered treatment during the modeling process. TEAT was implemented at the beginning of the D-gal intraperitoneal injection.

Acupoint selection
AD is a severe neurodegenerative disease that primarily affects the brain regions responsible for memory, language, reasoning, and social behavior. The brain (Nao) is considered the sea of marrow (Sui) in TCM. The marrow (Sui) is made of essence (Jing), a substance unique in TCM that is believed to be the body's essential constitutional strength, vitality, and resistance. Essence (Jing) is mainly stored in the kidneys (Shen). If the kidney essence (Shen Jing) is weak, the brain (Nao) may lack nourishment, and cognitive impairment may occur. Therefore, TCM suggests tonifying the kidneys and benefiting the essence (Jing) can help improve brain function. The brain can be directly influenced by certain meridians, such as the Governor vessel (GV) and the bladder meridian of foot Taiyang (BL), as they enter the brain. The combination of the Governor vessel (GV) and the bladder meridian of foot Taiyang (BL) used for preventing and treating AD is highly utilized³³^,³⁴. Baihui (GV 20) is an acupoint located at the vertex of the head and is part of the Governor vessel. According to literature, in studies using acupuncture and moxibustion for the prevention and treatment of AD, Baihui (GV 20) has the highest frequency of occurrence and is considered a critical point in the formula³⁵. The Shenshu (BL 23) is an acupoint on the bladder meridian of foot Taiyang. It is located 1.5 cun lateral to the lower border of the spinous process of the second lumbar vertebra. As the kidney back transporting-Shu point, it can strengthen all aspects of the kidneys, including kidney yin, yang, essence (Jing), and qi. Acupuncture and moxibustion at Baihui (GV 20) and Shenshu (BL 23) may improve cognitive function and alleviate AD symptoms in various ways³⁶^,³⁷.

Interval time
The operating standards of TEAT³⁸ dictate that the treatment interval and course of treatment should be determined based on the disease's condition and the selected operating site, with an interval of 1 week to 1 month. The course of treatment can range from 1 to 5 times. This suggestion can also be followed in experimental animal research. Based on the reviewed literature³⁹, treatment is generally administered once a week or every 2 weeks, with most cases involving 4-8 consecutive treatments. The experiment lasted 49 days, during which the threads were buried on the 14^th, 28^th, and 42^nd days.

Tool and methods
Various thread-embedding tools are used in current experimental animal research. These include No. 7⁴⁰ or No. 9⁴¹ disposable thread-embedding needles, self-reinvented needles, and No. 12 lumbar puncture needle⁴². The self-reinvented needles use No. 5-7 syringes as the tube and an appropriate filiform needle for the needle core. The needle tip has been pre-smoothed in advance. For example, in a study using SD rats weighing 250 ± 20 g (month age not specified in the text), choose the No.7 syringe and the 0.30 x 0.40 mm filiform needle⁴³^,⁴⁴. However, the self-reinvented needles must be strictly disinfected, making them unable to be used widely. If the needle tip is too large or sharp, it can damage blood vessels and nerves, increasing the likelihood of bruising⁴⁵. Lumbar puncture needles, which have a large diameter and sharp tips, are more likely to damage blood vessels and nerves. The disposable embedding needle is the most used in clinical practice³⁹. It is more convenient to use and safer to operate compared to self-reinvented needles and lumbar puncture needles, making it ideal for TEAT. Additionally, the goal of the experiment is to improve human health. Therefore, animal studies should reflect the clinical situation. Hence, it is advisable to use disposable thread-embedding needles as thread-embedding tools.

Catgut was the primary thread material used in TEAT, but due to tissue rejection and animal-derived ingredients, it has been replaced by biosynthetic absorbable surgical sutures. Sutures are available in various sizes and should be matched to the needle, such as No.7 for 4-0 or 3-0 sutures. In animal research, 3-0⁴¹, 4-0⁴⁶, and 5-0⁴⁷ are mainly used. Most sutures are 1-2 cm long. However, rodents have much smaller bodies than humans, so there is a lack of appropriate size for their body size. Therefore, researchers must cut sutures into 0.2-0.5 cm segments to make them suitable for rodents. Therefore, it is recommended that sutures be rinsed with clean and sterile distilled water or saline after cutting and before soaking for later use or immediate application to reduce the risk of infection⁴⁶.

Some studies have proposed detailed requirements for the embedding method, such as rapidly inserting needles into the acupoint from about 0.5 cm below the acupoint towards the acupoint⁴⁸. This is important because rodents are small. In addition to having abundant muscle tissue in their lower limbs, their subcutaneous tissue and muscle layers in the head, back, and abdomen is relatively weak. Therefore, the issue of TEAT's insertion points and insertion angle in animal research needs to be emphasized here. When performing TEAT on the lower limbs of rodents, direct needling at the acupoints is feasible. As for the body, it is recommended to insert the needle below the acupoints (the distance can be flexibly adjusted according to the length of the thread body) using oblique needling (<45°) or flat needling (<15°) to avoid damage to the internal organs.

Potential of TEAT for the prevention and treatment of Alzheimer's disease
Recently, research on TEAT is emerging, although its effects and mechanisms remain elusive. As mentioned above, TEAT can improve AD-related risk factors such as obesity¹³^,¹⁴, diabetes¹⁵, insomnia¹⁶, postmenopausal osteoporosis¹⁷, and depression¹⁸. It has a beneficial effect on learning and memory impairment in VD rats¹⁹. TEAT regulates lipid metabolism through the PPAR pathway to ameliorate tissue injury in exercise-induced fatigued rats⁴⁹ and exerts anti-inflammatory and analgesic effects in intermittent cold stress-induced mice through the TRPV1 pathway⁵⁰. In the present study, we demonstrated that thread-embedding treatment on AD-like rats could alleviate learning and memory impairments by ameliorating spatial memory loss and reducing degenerated cells in hippocampal CA1 regions. However, due to the current research's lack of uniformity and standardization in several critical elements of TEAT operation, including the embedding method, embedding tool, thread, and embedding interval period, this article mainly discusses the critical operational elements of TEAT in experimental research. This research was only a preliminary exploration of the effect of TEAT on AD, which required much more complement and improvement in clarifying the exact molecular and interaction between them. Nevertheless, it offered a new trial for the inevitable AD process, which may somehow relieve the heavy burden on the public health care system.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported with funds from the National Natural Science Foundation of China (NSFC) Nos. 82374564 (to YJD) and Nos. 82074566 (to YJD) and from Sun Guojie Inheritance Base for TCM Acupuncture-Moxibustion of World Federation of Acupuncture-Moxibustion Societies in Wuhan, China (World Federation of Acupuncture-Moxibustion Societies [2019] No. 26) (to YJD) and the National Famous TCM Inheritance Studio construction project of the National Administration of Traditional Chinese Medicine, China (the National Administration of Traditional Chinese Medicine [2022] No. 5) (to YJD).

Materials

0.9% saline, 4% paraformaldehyde, Hematoxylin, Eosin Y (water-soluble), absolute ethanol, 0.01M PBS powder, xylene, hydrochloric acid, embedded paraffin, neutral gum	sinopharm (www.sinopharm.com)
5-0 absorbable surgical suture	bodamedical (www.bodamedical.com)
70% alcohol, iodophor, cotton ball			Used for disinfection.
chloral hydrate	sigma	C8383
coverslips, glass slides	Guangzhou Wuqiang Experimental Equipment Co., Ltd
Dehydrator	Wuhan Junjie JT-12J Computerized Biological Tissue Dehydrator
DELUXE THERMOSTAT HEAT MAT	Zhenhua Instruments		For maintaining the body temperature of rats.
D-galactose	sigma	G0750
electric men's beard trimmer			Used for shaving rats.
Electronic scale for small animals (ZK-DST)	Henan Zhike Information Technology Co., Ltd
erythromycin ointment	frontpharm
glass dish			Used to place cut absorbable sutures.
High-Pressure Steam Sterilization Pot (YM100Z)	Shanghai Sanshen medical equipment factory		For sterilization of surgical instruments.
Ice Maker (CMB40)	Shanghai Sheyan Instrument Co., Ltd.
Microscope (BX-53)	OLYMPUS
milk powder			Used for the MWM test.
Morris Water Maze Video Tracking System & Data Analysis Software (Version: Watermaze 2.0)	Chengdu Taimeng Technology Co., Ltd
No.6 thread-embedding needle	bodamedical (www.bodamedical.com)
ophthalmic scissors, trays, tweezers
Pathology Microtome (RM 2016)	LEICA, Germany
pentobarbital	Germany	P11011
permanent marker			Used to mark the acupoint.
Slicing knives (R35)	Feathers, Japanese
soap			Used to wash hands.
sterile gloves

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You, M., Miao, J., Yang, M., Liu, Q., Liu, Y., Du, Y. Operation Procedure and Precautions of Thread-Embedding Acupuncture Therapy in Alzheimer. J. Vis. Exp. (207), e65895, doi:10.3791/65895 (2024).

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