Evaluation of the Electroacupuncture Effect on Cerebral Blood Flow in APP/PS1 Mice by Using the Laser Speckle Technique

Summary

Here, we present a protocol centered on laser speckle contrast imaging to quantify cerebral blood flow, combined with the Morris Water Maze, for evaluating electroacupuncture's effects in APP/PS1 (AD model) mice, valuable for AD cerebrovascular research.

Abstract

Alzheimer's disease (AD) is characterized by progressive cognitive decline and cerebrovascular dysfunction, including reduced cerebral blood flow (CBF) and neurovascular unit impairment. Electroacupuncture (EA) has shown potential in improving neurological functions, its mechanism may be related to regulating CBF. Laser speckle imaging is a wide-field method for real-time assessment of CBF and can precisely evaluate changes in cerebral blood perfusion. In this experiment, we observed the effects of electroacupuncture on CBF and cognitive ability in AD model mice (APP/PS1 transgenic mice). The blood perfusion of the middle cerebral artery and its branch vessels in mice was observed by laser speckle imaging. The spatial cognitive function of mice was evaluated using the Morris water maze. The results showed that EA could improve the cognitive function of APP/PS1 mice, and the escape latency was significantly decreased in the Morris water maze test (P<0.05). Furthermore, Laser speckle contrast imaging (LSCI) confirmed significant cerebral hypoperfusion in AD model mice compared to wild-type controls (P < 0.05), and importantly, EA treatment induced a statistically significant increase in CBF (P < 0.05), reversing the cerebral hypoperfusion in AD mice. Laser speckle imaging is a visualization technique for evaluating EA-induced cerebrovascular improvements. These findings support EA as a potential adjunct therapy for AD by targeting cerebrovascular dysfunction.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the progressive cognitive impairment and memory decline. It processes the pathological features of the deposition of extracellular beta-amyloid (Aβ) plaques, intracellular neurofibrillary tangle (NFTs), and neuronal loss¹. While these classic features have been the primary focus of therapeutic research, growing evidence underscores the critical role of decreased cerebral blood flow (CBF) in the pathogenesis and progression of AD². CBF reduction occurs in the early stage of AD onset and is associated with the severity of cognitive dysfunction³. Therefore, the treatment strategy for improving cerebrovascular damage is an effective way to alleviate AD symptoms and improve the course of the disease.

Electroacupuncture (EA), a modern technique that integrates traditional acupuncture with electrical stimulation, has shown potential in improving cognitive function in AD. Studies suggest that EA may exert its beneficial effects through multiple mechanisms, such as reducing Aβ deposition, attenuating neuroinflammation, and promoting synaptic plasticity⁴. Notably, there is increasing speculation that the neuroprotective effects of EA may be closely linked to its ability to modulate CBF⁵. However, direct, real-time evidence demonstrating the impact of EA on cerebral hemodynamics in AD models remains limited. The lack of such evidence hinders a comprehensive understanding of how EA improves brain function.

Technological advancements in optical imaging have provided powerful tools for investigating cerebral hemodynamics. As a fast, wide-field optical technique, LSCI delivers high spatiotemporal resolution for visualizing volume-integrated tissue blood flow maps⁶, enabling real-time monitoring of electroacupuncture-induced perfusion dynamics. While skull exposure is required for optimal optical access, LSCI avoids invasive tissue penetration. LSCI was introduced in the 1990s and has been widely applied in neuroscience in the past few decades⁷. Like all imaging techniques, LSCI has inherent limitations: its optical penetration depth is constrained, favoring superficial cortical vasculature while limiting access to deep brain regions (e.g., hippocampus, basal ganglia), and it quantifies relative blood flow (perfusion units) rather than absolute CBF values, with accuracy subtly influenced by vascular morphology and tissue optical scattering^8,9. However, these limitations do not undermine LSCI's utility but define its specialized application in real-time assessment of superficial cortical blood flow. For our study focusing on EA-induced cortical perfusion changes, LSCI provides reliable, actionable data. Specifically, we established a standardized LSCI protocol with targeted ROI delineation and optimized parameters to quantify CBF in APP/PS1 mice, complemented by Morris Water Maze tests to link cerebrovascular improvements with cognitive outcomes.

Protocol

This protocol was approved by the Animal Ethics Committee of Beijing University of Chinese Medicine (BUCM20250708-005), and it was in accordance with all guidelines for the Care and Use of Laboratory Animals of China.

1. Preparation

1. Twelve 6-month-old male APP/PS1 mice and six 6-month-old male C57bl/6 mice were used in this experiment.
2. All mice were housed in the Animal Experiment Center of Beijing University of Chinese Medicine, with three mice per cage. They had free access to sterile drinking water and standard pellet diet.
3. The housing environment was maintained on a 12 h light/dark cycle, with the ambient temperature controlled at 23 ± 2 °C.

2. Animal grouping and interventions

1. Randomly allocate twelve 6-month-old male APP/PS1 mice (weight 27.88 g ± 0.48 g) to two experimental groups (n = 6/ group): the Alzheimer's disease (AD) model group and the electroacupuncture (EA) treatment group.
2. Use six 6-month-old male C57BL/6 mice (weight 29.90 g ± 0.81 g) as the wild-type (WT) group.
3. For the WT control and AD model groups, gently restrain the mice in the well-ventilated mouse restrainers and administer no experimental interventions.
4. For the EA treatment group, gently restrain the mice in the same mouse restrainer as used for the WT control and AD model groups, which limits excessive movement while ensuring unobstructed breathing. Under this standardized restraint condition, insert the disposable sterile acupuncture needles (0.25 mm × 13 mm) into the Baihui (GV20) and Yintang (GV29) acupoints (Figure 1A) on the head, directed toward the nasal region, with a consistent depth of 2-3 mm. Connect the positive and negative terminals of the EA device to these two acupoints, respectively, and deliver stimulation for 15 min (Figure 1B). Configure the stimulation parameters as a disperse-dense wave pattern, with an intensity of 0.1 mA and a frequency of 2 Hz.
5. Administer the interventions to each group once every 48 h, with each session lasting 15 min, for a total duration of 30 days.
   NOTE: Perform acupoint selection, localization, and needling procedures in accordance with T/CAAM 0002-2020: Names and Locations of Commonly Used Acupuncture Points for Laboratory Animals Part 3: Mice¹⁰, a standard issued by the China Association for Acupuncture and Moxibustion on May 15, 2020.

3. MWM test

NOTE: 24 h following the 30-day intervention period, subject all mice across the three experimental groups to the Morris Water Maze (MWM) test, which included both the hidden platform (place navigation) trial and probe trial^11,12. Implement a 1-day acclimation period prior to formal testing to minimize stress, allowing mice to habituate to the experimental room, MWM apparatus, and handling procedures.

1. Preparatory procedures
   1. Position the MWM apparatus and its associated signal acquisition/processing system in a sound-attenuated experimental room. Maintain the ambient temperature of the testing environment at approximately 25 °C, and standardize lighting conditions as follows: mount four 60-W LED tube lights above the four quadrants of the maze at a height sufficient to avoid capture by the ceiling camera. Opaque drapes surrounded the entire testing area to block external environmental interference and prevent unintended spatial cues from affecting the mice's navigation.
   2. Place a circular white tank (90 cm diameter × 50 cm height) at the center of the MWM setup. Connect a ceiling-mounted video camera to an automated tracking system and recorder for continuous data capture throughout testing.
   3. Divide the tank into four equal regions, marked north (N), south (S), east (E), and west (W), and further segment into four congruent quadrants, including northeast (NE), northwest (NW), southwest (SW), and southeast (SE).
   4. Affix distinctly shaped visual shapes (square, triangle, and circle) to the outer wall of each quadrant, positioned within the mice's line of sight to serve as spatial reference points.
   5. Fill the tank with 30 cm of water maintained at 24 ± 1 °C with an electric heater; add white edible pigment to render the water opaque.
2. Hidden platform trial
   1. Fix a submerged platform in the SE quadrant for the duration of this trial.
   2. Randomly release each mouse into the pool from three alternate quadrants (NE, NW, SW), with initial orientation facing the tank wall for consistency.
   3. Allow each mouse a maximum of 60 s to locate the hidden platform independently.
   4. Record escape latency (the time from release to platform ascent) for each trial to assess spatial learning.
      ​NOTE: Each mouse completed three trials per day over five consecutive days. If a mouse failed to find the platform within 60 s, it was gently guided to the platform and allowed to remain there for 10 s to reinforce spatial memory before concluding the trial.
3. Probe Trial
   1. Remove the platform.
   2. Release each mouse once into the pool facing the tank wall and permit 60 s of unconstrained swimming.
   3. Record key outcome measures: average swimming speed and the number of crossings over the former platform location.
4. Post-test care
   1. After each MWM session, dry the mice thoroughly with soft towels and gently warm them using an electric heater to prevent hypothermia.

4. Laser speckle contrast imaging

1. Randomly select three mice from each group and monitor their CBF in real time and in full field using the LSCI system.
2. Animal preparation: Perform all animal operations under anesthesia. Anesthetize mice with 1.25% tribromoethanol injection (0.02 mL/g) intraperitoneally to ensure continuous spontaneous breathing and place them on a horizontal operating plate. Place anesthetized mice on a thermostatically controlled heating pad maintained at 37 °C to stabilize core body temperature. Shave the scalp and carefully dissect the underlying muscles/meninges to expose the skull; keep the exposed cranial surface moist with 0.9% sterile physiological saline throughout the procedure to prevent desiccation.
3. Imaging setup: Position the system's scanning head perpendicular to the exposed cranial window at a fixed distance of 20 cm. Enable the auto-focus and auto-gain functions to optimize image clarity and contrast, with gain values automatically maintained within a range of 140-160 across all experiments. Configure additional standardized imaging parameters as follows: exposure time = 20 ms, time constant = 1.0 s, operating mode = Temporal, filter = 250 frames, sample interval = 5000 ms, and image resolution = 1032 × 772.
4. Data acquisition: Acquire continuous speckle images at a 25 Hz sampling rate, and synchronize collection to the preconfigured temporal and filter parameters.
5. ROI analysis: Use the analysis software to define regions of interest (ROIs) over the prefrontal cortex, specifically targeting the anterior and middle cerebral artery territories within this cortical area. Delineate ROIs over these vascular regions, and extract mean flux values (expressed in perfusion units) from each ROI for subsequent statistical comparison.

5. Statistical analysis

1. Perform statistical analyses using SPSS 27.0.
2. Analyze group differences in the hidden platform, probe trial outcomes, and CBF were analyzed using the Student's t-test. Define statistical significance as P < 0.05, and set high statistical significance at P < 0.01.

Results

Effects of EA on spatial learning and memory in AD model mice

The results of the Morris water maze test are shown in Figure 2. Figure 2A shows the escape latency during the hidden platform test over five days. The escape latency of AD group showed a fluctuating trend, while the WT and EA groups decreased steadily. The escape latency of the AD group was significantly longer than that of the WT group from Day 4 to Day 5...

Discussion

The present study demonstrates that long-term EA intervention at Baihui (GV20) and Yintang (GV29) acupoints significantly ameliorates spatial learning deficits in APP/PS1 transgenic mice, as reflected by the shortened escape latency in the Morris Water Maze hidden platform test. This finding is consistent with previous reports that EA improves cognitive function in various AD models via mechanisms such as reduced Aβ deposition and attenuated neuroinflammation¹³. Notably, acupoint selection wa...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
acupuncture needle	(Beijing Zhongyan Taihe Medicine Company, Ltd., China	0.25 mm × 13 mm	acupuncture needle
Laser speckle contrast imaging system	Gene&I Scientific. Ltd.	MoorFLPI-2
mouse restrainer	Hengtai Laboratory Equipment Center	HT-A2
Tribromoethanol	Nanjing Aibei Biotechnology Co.,Ltd.	M2910	anesthesia