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

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 AD^9,10. In recent years, traditional Chinese medicine (TCM) has made significant progress, and it is expected to offer new possibilities for preventing and treating AD^11,12. 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 obesity^13,14, 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 research^20,21 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

1. 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.
2. 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.
3. 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.
4. Continue intraperitoneal administration of D-galactose for 7-weeks to induce Alzheimer's disease-related pathological manifestations in rats²².

2. Preparation for TEAT

1. Sterilize surgical instruments such as ophthalmic scissors, trays, and tweezers and disinfect. Clean the surfaces in the operating room environment thoroughly and disinfect.
2. 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.
3. 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.
4. Wash hands with soap and water for 20 s, and then dry them. Afterward, wear sterile gloves.
5. 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)

1. 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).
2. Shave the head area around the acupoint of the rat using a trimmer (usually an electric men's beard trimmer).
3. 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.
4. 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.
5. Pinch the area around Baihui (GV 20) with thumb and index finger on one hand, while holding the needle with the other.
6. 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).
7. 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).
8. 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)

1. 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 research^20,21, this acupoint pairing has been established for its efficacy.
2. 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).
3. 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.
4. 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.
5. 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.
6. Quickly insert the needle perpendicularly into the point 0.25 cm below the Shenshu (BL 23). See Figure 4A.
7. 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.
8. 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).
9. 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

1. 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.
2. 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

1. Following the intervention, at week 8, perform the Morris Water Maze (MWM) experiment^2,24 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.
2. 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.
3. After the behavioral assessment, anesthetize the rats as above and euthanize with intraperitoneal injections of 150 mg/kg body weight of pentobarbital sodium.
4. 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.
5. Remove the rat brain and place it in a specimen bottle containing 4% paraformaldehyde for 24-48 h at 4 °C^25,26. 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.

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 environmen...

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, transmissi...

Materials

Name	Company	Catalog Number	Comments
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