Method Article

Electroacupuncture Therapy for Cognitive Impairment after Spinal Cord Injury

DOI:

10.3791/69346

⸱

November 21st, 2025

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1School of Acupuncture, Moxibustion and Tuina, Beijing University of Chinese Medicine, 2Department of Rehabilitation, The Third Affiliated Hospital of Beijing University of Chinese Medicine, 3Department of Rehabilitation, Jiangsu Changshu Second People's Hospital, 4Traditional Chinese Medicine Department, Shenzhen Third People's Hospital, Southern University of Science and Technology

* These authors contributed equally

In This Article

Summary

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This study established a standardized electroacupuncture procedure aimed at treating cognitive impairments after spinal cord injury. Moreover, the protocol for performing the Y maze and novel object recognition experiments to assess the efficacy of electroacupuncture in ameliorating cognitive deficits was described in detail.

Abstract

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Spinal cord injury (SCI) is a highly disabling and destructive disease of the central nervous system (CNS), which not only leads to limb motor and sensory dysfunction but also disrupts the structure and function of the hippocampus and cortex, leading to cognitive impairment, which seriously affects patients' quality of life. Thus, it is imperative to explore effective treatment approaches and assessment methods. Electroacupuncture (EA) is a novel therapy that integrates traditional acupuncture with contemporary electrotherapy technology, which has the advantages of high safety and few side effects, and has been widely utilized in the management of SCI. According to the theory of traditional Chinese medicine (TCM), the Governor Vessel connects the brain and the spinal cord. Therefore, EA at the acupoints located on the Governor Vessel can effectively restore the functions of the brain and the spinal cord. In this protocol, we employed Allen's method to construct the SCI model. We introduced an EA procedure aimed at treating cognitive impairments in rats with SCI. Furthermore, we described in detail the operation process of the Y maze and novel object recognition (NOR) experiment to assess the efficacy of EA in ameliorating cognitive deficits in rats. This research not only provides a treatment protocol that serves as a reference for cognitive impairment after SCI but also offers preliminary experimental support for the validation of therapeutic strategies for this condition and subsequent research exploration.

Introduction

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Spinal cord injury (SCI) is a common central nervous system (CNS) disorder, typically resulting from damage to the spinal cord or its surrounding structures due to external forces. This condition can lead to sensory, motor, and autonomic nervous dysfunction below the injury level1. More critically, SCI can also induce secondary complications such as anxiety, depression, and cognitive impairment2,3, which are detrimental to patients' rehabilitation and significantly diminish their quality of life. Multiple epidemiological studies have found that the risk of Alzheimer's disease (AD) in SCI patients is considerably higher than that in the general population4,5,6. Another study has confirmed that the incidence of cognitive impairment in SCI patients may reach as high as 60%5,7. SCI not only leads to atrophy of the motor cortex but also reduces neural activity in brain regions associated with cognitive function8. The increased inflammation in the hippocampus, thalamus, and cortex following SCI is regarded as a significant contributor to cognitive impairment9,10,11. Improving complications related to SCI, such as cognitive impairment, is crucial for enhancing patients' rehabilitation and facilitating their reintegration into society.

Acupuncture is a significant component of traditional Chinese medicine (TCM), renowned for its remarkable clinical efficacy. Electroacupuncture (EA) represents an innovative therapy that integrates traditional acupuncture with contemporary electrotherapy techniques12. It stimulates nerves and muscles by applying low-voltage current to specific acupoints, offering therapeutic benefits for functional recovery following chronic pain and nervous system injuries13,14,15. Numerous studies have demonstrated that EA can promote neurological functional recovery after SCI and mitigate complications such as neuropathic pain, neurogenic bladder, and spasticity16,17,18,19. According to the theory of TCM, the Governor Vessel is connected to the brain and spinal cord, influencing their functions20. We have found that EA applied at the Dazhui (GV14) and Mingmen (GV4) acupoints on the Governor Vessel can alleviate neuroinflammation and inhibit the formation of glial scars, thereby facilitating motor function recovery post-SCI21. Furthermore, our investigations have established that EA at the Baihui (GV20) and Yintang (GV29) acupoints on the Governor Vessel can enhance cognitive abilities in AD model mice22,23. Nonetheless, the potential of EA in addressing cognitive impairment following SCI remains inadequately explored.

In this study, we describe the operational methods of EA for treating cognitive impairment in SCI rats, encompassing acupoint selection, acupuncture angle, treatment duration, and other details. Additionally, we employed the Y-maze and NOR tests, along with pathological evaluations, to assess the efficacy of EA in treating cognitive impairment. This study aims to provide a reference for the clinical treatment of this condition.

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Protocol

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All experimental procedures were performed in strict compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Beijing University of Chinese Medicine (Approval ID: BUCM-2024042505-2065).

1. Animal preparation

  1. Select male adult Sprague Dawley (SD) rats weighing 220.0 g ± 20.0 g for the experiment.
  2. House the animals in the Experimental Animal Center of the Beijing University of Chinese Medicine at a controlled temperature (24 ± 2 °C) and under a 12-h dark/light cycle, with sterile drinking water and a standard pellet diet available ad libitum.
  3. Acclimate all the animals to the environment for 7 days before experimentation.
  4. Randomly divide 24 SD rats into three groups (n = 8 per group): the sham-operation (Sham) group, the SCI model (Model) group, and the electroacupuncture (EA) group. The time axis diagram of experimental design is illustrated in Figure 1.

2. Establishment of the SCI model

NOTE: Rats were deprived of water and food for 8 h before the operation, and the surgical instruments were sterilized using high-temperature steam. The SCI model was constructed using a modification of Allen's method24.

  1. Place the rat in the anesthesia induction box and induce them with oxygen containing 4% isoflurane (1 L/min) until the rat is completely anesthetized.
  2. Remove the rat from the induction box and position its nose and mouth at the anesthesia mask. Adjust the isoflurane concentration to 1.5%-2% to maintain anesthesia.
    NOTE: During the rat anesthesia process, it is imperative to closely monitor the rat's condition. If the rat exhibits shallow and rapid breathing or a drop in body temperature, reduce the isoflurane concentration to avoid over-anesthesia.
  3. Shave the hair from the back of the rat to fully expose the surgical area. Sterilize the operating area with iodophor and alcohol.
  4. Locate the tenth thoracic vertebra (T10). Create a 2.5 cm median longitudinal incision centered on T10.
  5. Use surgical blades to separate the back muscles, exposing the spinous process and the muscles adjacent to the thoracic vertebrae. Remove the spinous process and lamina of T10 with hemostatic forceps, exposing approximately a 10 mm segment of the spinal cord.
  6. Impact the exposed dura mater (200 kilodyne) accurately using a modified Allen's device to induce moderate contusion.
    NOTE: The success of the SCI model is marked by congestion and edema of the dura mater at the injury site and the tail-spasm swing. For the Sham group, the rats underwent the same surgical procedure, including the removal of the vertebral lamina, but the spinal cord was not damaged.
  7. Cover the dura mater with a gelatin sponge and suture the wound layer by layer.
  8. Place the rat on a constant temperature heating pad set to 37 °C and monitor it until it regains consciousness.
  9. Administer penicillin sodium injection into the muscle of the rat once daily for three days post-operation to prevent infection. Additionally, perform bladder massage on the rat 2-3 times daily to facilitate urination.

3. EA treatment

  1. Perform EA intervention 24 h after successfully establishing the SCI model.
  2. Prepare disposable sterile acupuncture needles (diameter: 0.30 mm; length: 25 mm).
  3. According to TCM theory and clinical experience, select Baihui (GV20), Dazhui (GV14), and Mingmen (GV4) acupoints for EA treatment.
  4. Prepare black silk stockings. Guide the rat to drill into the black stockings. Bind and fix the rat onto a board using a tie.
    NOTE: The rope should be tightened moderately to prevent asphyxiation of the rat from overly tight binding or escape due to excessively loose binding.
  5. Locate the GV20 acupoint at the center of the parietal bone, at the intersection of the forehead's midline and the midpoint of the line connecting the two ear tips. Locate GV14 between the seventh cervical vertebra (C7) and the first thoracic vertebra (T1), on the median line of the back. Locate GV4 in the depression under the spinous process of the second lumbar vertebra (L2), on the median line of the back (Figure 2).
  6. Disinfect the acupoints with medical iodophor and 75% medical alcohol.
  7. Insert the needles obliquely downward at an angle of 15° to a depth of 2-3 mm at the GV20. Insert the needles into GV14 and GV4 points at an angle of 45°, with a depth of 5-7 mm (Figure 3A).
  8. Connect the needles to the EA therapeutic apparatus at a frequency of 2 Hz and an intensity of 1 mA. Select the waveform as a continuous wave (Figure 3B, C).
    NOTE: This EA intervention was conducted daily for 28 days, with each session lasting 20 min. Rats in the Sham and Model groups received no treatment, but they were immobilized for 20 min each day in the same manner as those in the EA group.

4. Evaluation of motor function (Figure 4)

NOTE: On the 1st, 7th, 14th, 21st, and 28th days following the operation, the motor function of rats in each group was evaluated by the Basso, Beattie, and Bresnahan (BBB) score.

  1. Place the rat in an open and quiet place to allow for free movement.
  2. Observe the hind limb movements of the rat and score them according to the established scale25.
    NOTE: Two researchers, who were blind to the grouping of the rats, independently assigned scores, and the final score for each rat was calculated as the average of their assessments.

5. Performing the Y-maze experiment (Figure 5)

NOTE: On the first day after the EA intervention (the 29th day of the experiment), the Y-maze experiment was carried out, including a spontaneous alternation test and a novel arm exploration test. The Y-maze experimental procedure is shown in Figure 5A.

  1. Place the experimental animal cages in the experimental environment before the test and acclimate for 30 min.
  2. Clean the Y-maze device with alcohol, and randomly set the three arms of the Y-maze as the start arm, the novel arm, and the other arm.
  3. During the spontaneous alternation test stage, cover the new arm with a baffle.
  4. Place the rat gently at the end of the starting arm and allow it to explore freely in both arms for 10 min. Record the times, distance, and residence time of the rat entering the start arm and the other arm.
  5. Remove any excreta in the chamber after each rat completes the test. Clean the interior of the chamber with 75% medical alcohol and dry it with clean gauze to prevent interference from odors left by previous test subjects. Keep the indoor environment quiet throughout the experiment.
  6. Remove the baffle and conduct the novel arm exploration experiment 4 h after the spontaneous alternation experiment.
  7. Place the rat gently at the end of the starting arm and allow it to explore freely in the three arms for 5 min.
  8. Record the sequence, number of entries, distance, and residence time of the rat entering each arm within 5 min.

6. Conducting the new object recognition (NOR) experiment (Figure 6)

NOTE: On the second day after EA intervention (the 30th day of the experiment), the NOR experiment was carried out. The NOR experimental procedure is shown in Figure 6A.

  1. Prepare an experimental setup consisting of a box measuring 70 cm × 60 cm × 30 cm, blue cubic objects, and red cylindrical objects.
  2. Introduce the rat into the experimental box and allow it to explore the surroundings freely for 10 min.
  3. After the above experiment is completed for 1 h, place two identical cubic objects symmetrically in two corners of the experimental box, positioned approximately 10 cm away from the walls.
  4. Introduce the rat into the experimental box with its head oriented away from the object block. Allow the rat to explore its surroundings freely for 10 min to become familiar with the object block.
  5. Following the completion of each rat's trial, wipe the experimental box thoroughly with alcohol to minimize the impact of residual odors on the behavior of subsequent rats. Once the box is dry, place the next rat inside.
  6. After the above experiment is completed for 4 h, replace the right object block with a cylindrical object.
  7. Reintroduce the rat into the experimental box with its head facing away from the wall of the object block. Allow the rat to freely explore for 5 min in the box.
  8. Record the time spent exploring the cylindrical and cubic objects and the total distance and time. Calculate the preference index using the formula: (New Object Exploration Time - Old Object Exploration Time)/(New Object Exploration Time + Old Object Exploration Time) × 100%.

7. Conducting the sucrose preference test

NOTE: Single-cage housing is utilized to prevent social competition for water, which could confound drinking behavior.

  1. Prepare a bottle with 1% sucrose solution and a bottle with tap water.
  2. Place the sugar solution and tap water bottles within the cage, ensuring to exchange their positions every 12 h.
  3. Allow the rat to adapt to the sugar water and tap water for a duration of 48 h. Subsequently, deprive the rat of drinking water for 24 h without fasting.
  4. Introduce a bottle of sugar water and a bottle of tap water back into the cage. Change the positions of the bottles after 2 h, and record the consumption of each liquid over a 4 h period. Calculate the sucrose preference percentage using the formula: (sucrose solution intake/(sucrose solution intake + water intake) × 100%.

8. HE staining of the hippocampus

  1. After the behavioral test, anaesthetize the rat with 3% phenobarbital sodium (2.0 mL/kg). Perform cardiac perfusion using paraformaldehyde. Take the brain tissue and embed it in paraffin. Prepare slices with a thickness of 5 µm.
    CAUTION: Paraformaldehyde is an irritant and must be handled in a well-ventilated environment.
  2. Deparaffinize the slices with xylene (I) and xylene (II) for 10 min successively. Soak the slices in gradient alcohol for dehydration, with each gradient lasting for 3 min. Finally, immerse the slices in distilled water for 2 min.
  3. Stain the slices with hematoxylin solution for 10 min. Rinse the excess solution with water for 1-3 min. Immerse the slides quickly in muriatic acid ethanol for 5-10 s. Rinse the slide with water again.
  4. Transfer the slices to a weak alkaline aqueous solution for blue return. Rinse the slices with water for 1-3 min. Immerse the slices in 85% ethanol for 3-5 min.
  5. Stain the slices with eosin solution for 3-5 min, dehydrate with gradient alcohol after washing. Place the slices in xylene for 5-10 min. Lastly, use neutral resin to seal the slices.
  6. Examine the stained slides under a light microscope at 400× magnification.

9. ELISA analysis

  1. After the behavioral test, euthanize the rat with intraperitoneal injection of pentobarbital sodium (200 mg/kg). Harvest the hippocampus tissues.
  2. Weigh the hippocampus tissue and transfer it into a pre-chilled homogenization tube.
  3. Homogenize the tissues on ice using a tissue homogenizer.
  4. Centrifuge the homogenate at 5000 × g for 10 min at 4 °C. Collect the supernatant and determine the total protein concentration.
  5. Determine the concentration of IL-1β and IL-6 in the hippocampus using ELISA kits.

10. Statistical analysis

  1. Use an appropriate statistical analysis software for statistical analysis. Express the data as mean ± standard deviation.
  2. Perform two-way analysis of variance (ANOVA) with repeated measures and least significant difference (LSD) test for dissecting differences among groups in the BBB score data.
  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, conduct the non-parametric test.
  4. If the data conforms to a normal distribution, perform Pearson correlation analysis. Otherwise, conduct Spearman correlation analysis.
  5. Consider differences statistically significant when P < 0.05.

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Results

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EA improves the motor function of SCI rats
On the 1st day after SCI operation, the BBB scores of the Model group were recorded at 0, indicating the successful establishment of the SCI model. Throughout the 1st, 7th, 14th, 21st, and 28th days post-operation, the BBB scores of the Model group were significantly lower than those of the Sham group (P < 0.01). Notably, on the 7th, 14th, and 28th ...

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Discussion

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SCI is a stressful event that inflicts severe trauma on patients' physiology and psychology, leading not only to motor and sensory dysfunction but also to cognitive impairment3. As reported4, the risk of cognitive decline after SCI is 13 times higher than that in healthy individuals. The middle-aged and elderly patients with SCI exhibit a higher susceptibility to AD compared to their peers who have not experienced SCI6. Following an injury, SCI p...

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Disclosures

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All authors have declared no potential conflicts of interest.

Acknowledgements

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This research was supported by the National Natural Science Foundation of China (No. 82374593, 82305381), Shenzhen High-level Hospital Construction Fund (No. 24275G1001), the Supported Project of Changshu Medical and Health Science and Technology Plan (No. CSWSQ202302), and the Independent Research Project of Postgraduates in Beijing University of Chinese Medicine (No. ZJKT2025016).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.9% salineBeijing suolaibao technology Co., Ltd.IN9000, P1022-10
4% paraformaldehydeBeyotimeP0099
75% medical alcohol, iodophorBeijing oubei biology science and technology Co., LtdOB40126-1, HX0098AUsed for disinfection
Acupuncture needlesZhongyan Taihe Co., LtdZY500
Anesthetic machine for small animalsShanghai Yuyan Instruments Co., LtdABM
Animal behavior analysis systemBeijing zhong Shi di Chuang technology Co., LtdZS-XWT-II
Centrifugal machineEppendorf, Germany5811FR080384
Electroacupuncture deviceShantou Medical Instrument Factory Co., Ltd6805-DUsed for electroacupuncture treatment
ELISA kitsJianglai Biology Science and Technology Co., LtdJL18365, JL20896Used to detect IL-1, IL-6 levels
Gelatin spongeJiangxi Xiang'en Medical Technology Development Co., Ltd24092901
Hematoxylin solution, Eosin solutionSigma-Aldrich03971Used for HE staining
Infinite Horizon ImpactorLexington, KY, United StatesIH-0400Used for constructing a spinal cord injury model
IsofluraneShenzhen Ruiwode Life Science and Technology Co., LtdR510-22-10
MicroscopeOLYMPUS
Penicillin sodiumCoolaberCP8271-5g
Phenobarbital SodiumSigma-AldrichP3761
SD ratsSibeifu (Beijing) Biology Technology Co., LtdSCXK2019-0010
SPSSIBMVersion 26.0Statistical software
SucroseBeijing Solarbio Technology Co., LtdS8271
XyleneShanghai Aladdin Biochemical Technology Co., LtdX112050

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Electroacupuncture TherapySpinal Cord InjuryCognitive ImpairmentCentral Nervous SystemHippocampus FunctionCortex FunctionAllen s MethodY Maze TestNovel Object RecognitionGovernor Vessel

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