Senescence-accelerated mice (SAM) are characterized by a variety of functional disorders with aging and divided into SAM/prone (SAMP) and SAM/resistant (SAMR) mice. Among them, SAMP8 mice show significant age-related deficits of learning and memory. While the physiological index of SAMR1 mice is similar to that in the normal animals, they have been used as homologous controls. The uterus was taken out of 12–16-day-old pregnant SAMR1 mice, and the placenta was removed under sterile conditions. After obtaining the embryo, the hippocampus was separated by opening the cranial cavity. The tissue was cut into pieces of 0.5 mm³ and then digested for 20 min with 0.25% trypsin at 37 °C to obtain a single-cell suspension. After centrifugation for 10 min at 60 g/min, the cells were cultured in DMEM/F12 medium (supplemented with 2% B27, 20 ng/mL EGF, 20 ng/mL bFGF, 100 U/mL penicillin, and 100 μg/mL streptomycin) at 37 °C with 5% CO₂. The positive rate of Nestin staining was evaluated by indirect immu-nofluorescence with a rabbit monoclonal anti-nestin (1:50, Sigma Aldrich, United States), and normal rabbit IgG (1:50, Santa Cruz Biotechnology, United States) was used as a negative control. The proliferation of NSCs was observed by methyl thiazolyl tetrazolium (MTT) assay, and the differentiation into neurons or neurogliocytes was observed by indirect immu-nofluorescence with a rabbit monoclonal anti-neuronal nuclei (NeuN) antibody (1:500, Cell Signaling Technology, United States) or a rabbit monoclonal anti-glial fbrillary acidic protein (GFAP) antibody (1:200, Cell Signaling Technology, United States), respectively, and normal rabbit IgG (1:50, Santa Cruz Biotechnology, United States) was used as a negative control[5]. Adequately proliferating NSCs were labeled with BrdU and injected into the hippocampus at a density of 5 × 10⁵ cells/μL with a small animal stereotaxic device. The coordinates of the NSCs relative to the anterior fontanel were as follows: AP: -2.06, ML: ± 1.75, DV: -1.75. In the sparse area of the dorsal dentate gyrus of the hippocampus, 1 μL of NSCs was injected slowly and evenly into each hippocampus for a sustained injection time of 1 min, and then retained in situ for another 1 min. In the sham transplantation group, 0.9% saline solution was injected in the same position. Acupuncture was administered after 24 h.

The experimental procedures were carried out according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1985). The study protocol was approved by the Ethics Committee of Tianjin University of Traditional Chinese Medicine (approval number: TCM-LAEC2019036). Healthy 8-mo-old male SAMP8 and SAMR1 mice were randomly divided into six groups (n = 9 each): (1) SAMR1 control group (RC): Catching-grasping stimulation; (2) SAMP8 control group (PC): Catching-grasping stimulation; (3) SAMP8 sham operation group (PS): 15 d of catching-grasping stimulation before sham transplantation surgery, and another 15 d of catching-grasping stimulation post-surgery; (4) SAMP8 NSC transplantation group (PT): 15 d of catching-grasping stimulation before NSC transplantation surgery, and another 15 d of catching-grasping stimulation post-surgery; (5) SAMP8 NSC transplantation with acupuncture group (PTA): 15 d of acupuncture intervention before NSC transplantation surgery, and another 15 d of acupuncture intervention post-surgery; and (6) SAMP8 NSC transplantation with non-acupoint acupuncture group (PTN): 15 d of non-acupoint needling intervention before NSC transplantation surgery, and another 15 d of non-acupoint needling intervention post-surgery. The mice were raised under exactly the same conditions and fed freely at 24 ± 2 °C.

Sanjiao acupuncture was administered. The acupoints were Danzhong (CV17), Zhongwan (CV12), Qihai (CV6), bilateral Xuehai (SP10), and bilateral Zusanli (ST36), and the location was identified using the Laboratory Acupuncture and Atlas of Animal Acupoints enacted by Experimental Acupuncture-Moxibustion Research Association of China Academy of Acupuncture and Moxibustion. For mice in the PTA group, locations CV17, CV12, CV6, and bilateral ST36 were needled by twisting reinforcing manipulation method, while bilateral SP10 was needled by twisting reducing manipulation method for 30 s. For mice in the PTN group, two fixed non-acupoints located at the bilateral subcostal area were needled with moderate reinforcing-reducing manipulation method for 210 s. Mice in the other four groups were caught and grasped with the same intensity for the same duration. All intervention protocols were implemented once a day for 15 d and suspended only on day 7.

After 15-d Sanjiao acupuncture treatments, behavioral changes were assessed by the Morris water maze test, which consisted of a circular pool, a platform, and a recording system. All equipment for the test was provided by the Chinese Academy of Medical Sciences (Beijing, China). The pool was 90 cm in diameter, with a height of 50 cm. On the day before the trial, mice were allowed to swim freely for 90 s in the pool once in the morning and once in the afternoon to acclimatize to the maze environment. Then, a cylindrical platform, 9 cm in diameter and 28 cm high, was placed in the center of any quadrant with 2 cm under the water. The pool was divided into four quadrants (northeast, southeast, southwest, and northwest). On the offside of the platform, two points were selected at the same distance from the point of entry. After the animals were placed in the water, the time elapsed from entering the water to finding the platform was recorded as the escape latency. The swimming data of the animals were automatically recorded with Morris 1.0.2 software using an automatic image acquisition and analysis system (Beijing, China). If a mouse could not find the platform within 90 s, the escape latency was recorded as 90 s. Each mouse was tested twice daily from two different water entering points, and the average escape latency was calculated. The hidden platform trial was performed for 5 consecutive days.

Three mice in each experimental group were sacrificed after cardiac perfusion. Coronal paraffin sections of the brain tissue were prepared. Brain sections of mice were embedded in paraffin, cut into 5 µm slices under a stereology microscope (Olympus BX-51TF, Japan), and placed onto hydrophilic adhesive slides. Slides were allowed to air dry overnight and then baked for an hour at 60 °C. After BrdU-positive staining was confirmed under a microscope, adjacent sections were dewaxed and hematoxylin-eosin (HE)-stained. Following deparaffinization and gradient ethanol dehydration, slices were stained with Harris hematoxylin, differentiated with hydrochloric acid ethanol, and washed with warm water to turn blue. After eosin staining, slices were dehydrated, cleared, and sealed. The pathological changes in the brain tissue were observed under a microscope after NSC transplantation. Then the paraffin sections of the hippocampus tissue of the mice were stained by immunofluorescence. The positive rate of BrdU staining was evaluated by indirect immunofluorescence and diaminobenzidine (DAB) staining with a rabbit polyclonal anti-BrdU antibody (1:100, Abcam, United Kingdom), and normal rabbit IgG (1:50, Santa Cruz Biotechnology, United States) was used as a negative control. After nuclei were counterstained with hematoxylin, positive BrdU labeled cells were observed and counted.

Three mice in each group were killed by dislocation, and the whole brain was immediately removed and placed in artificial cerebrospinal fluid (ACSF) (NaCl 124 mmol/L, KCl 3.5 mmol/L, NaH₂PO₄·2H₂O 1.2 mmol/L, MgCl₂·6H₂O 1.3 mmol/L, CaCl₂ 2 mmol/L, NaHCO₃ 25 mmol/L, D-Glucose 10 mmol/L). After cooling, the hippocampus was rapidly dissected and sliced into 400-μm sections parallel to the hippocampal groove fibers. These slices were placed in the upper layer of the Transwell co-culture system. Then, the well-developed BrdU-labeled NSCs (2 × 10⁵/well) were inoculated into the lower layer of the Transwell co-culture system, and NSCs were collected on the 4^th, 7^th, and 10^th d after inoculation. Cells were washed one time with phosphate buffer saline (PBS), digested with 0.02% ethylene diamine tetraacetic acid (EDTA), and resuspended into single-cell suspension. After methanol fixation, penetration, and serum blocking, the rabbit synaptophysin antibody (#5461, Cell Signaling) and PE-conjugated donkey anti-rabbit IgG (DantiR-PE, Saierbio) were added on to the sections. Finally, 2000 cells of each sample were collected, and synaptophysin expression was detected by flow cytometry (BD FACS Calibur).

All data are expressed as the mean ± standard deviation (SD) and were analyzed using SPSS16. Data of multiple groups were compared using one-way ANOVA, and comparison between two groups was conducted by Student-Newman-Keuls (SNK) test. P < 0.05 was considered statistically significant.

The Morris water maze test was utilized to study the learning ability to find a fixed position of the hidden platform and form a stable spatial cognitive and memory ability in brain dysfunction animals, which reflects the acquisition of spatial memory by the animals after multiple training. The results showed that the average escape latency of mice in each group decreased with the passage of training days. In comparison to the RC group, the average escape latency of the PC or PS group was significantly longer (P < 0.05). When compared with the PC group, the escape latency of the PT, PTA, or PTN group was significantly shorter from the 4^th d (P < 0.05). In comparison to the PS group, the escape latency of the PT, PTA, or PTN group was significantly shorter from the 4^th day (P< 0.05). The escape latency of the PTA group was significantly shorter on the 5^th day as compared to that of the PTN group (P < 0.05). The results suggested that the 8-mo-old SAMP8 mice had significant cognitive impairment in learning and memory. Herein, the cognitive impairment ability of mice was significantly improved in all the NSC transplantation groups, among which, the behavioral changes of dementia mice in the PTA group were stronger than those in the other two groups with acupoint-related specificity (Figure 1).

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Figure 1 Morris water maze test for behavior of senescence-accelerated mouse. The time elapsed from entering the water to finding the platform was recorded as escape latency. If the mouse could not find the platform within 90 s, the escape latency was recorded as 90 s. ^aP < 0.05 when compared to senescence-accelerated mouse resistant 1 control group, ^bP < 0.05 when compared to senescence-accelerated mouse prone 8 (SAMP8) control group, ^cP < 0.05 when compared to SAMP8 sham operation group, ^dP < 0.05 when compared to SAMP8 neural stem cells transplantation with non-acupoint group. RC: Senescence-accelerated mouse resistant 1 control group; PC: Senescence-accelerated mouse prone 8 control group; PS: Senescence-accelerated mouse prone 8 sham operation group; PT: Senescence-accelerated mouse prone 8 neural stem cells transplantation group; PTA: Senescence-accelerated mouse prone 8 neural stem cells transplantation with acupuncture group; PTN: Senescence-accelerated mouse prone 8 neural stem cells transplantation with non-acupoint group.

Mice in the RC group showed a clear and complete hippocampal structure, dense and orderly nerve cells, abundant cytoplasm, light staining, nucleus in the middle, and complete and compact arrangement of normal glial cells, while no edema was detected in the pericellular space. In the PC group, hippocampal tissue structure was not clear and the arrangement of the nerve cells was irregular. The cells in the CA1 and CA3 areas were solidified and atrophied and deeply stained, and the intercellular structure was loose. In the PT, PTA, and PTN groups, the hippocampal structure was clear, the cell arrangement was dense and orderly, and the necrosis of cells in the CA1 and CA3 areas was reduced (Figure 2).

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Figure 2 Histopathological changes by hematoxylin-eosin staining. Coronal paraffin sections of the brain tissue of three mice in each experimental group were prepared. After BrdU-positive staining was confirmed under a microscope, adjacent sections were dewaxed and subjected to hematoxylin-eosin staining. A: Senescence-accelerated mouse resistant 1 control group; B: Senescence-accelerated mouse prone 8 control group; C: Senescence-accelerated mouse prone 8 sham operation group; D: Senescence-accelerated mouse prone 8 neural stem cells transplantation group; E: Senescence-accelerated mouse prone 8 neural stem cells transplantation with acupuncture group; F: Senescence-accelerated mouse prone 8 neural stem cells transplantation with non-acupoint group.

BrdU, a cell proliferation marker, is a derivative of thymine. It replaces thymine in the DNA synthesis phase (S phase)[6]. After BrdU is injected in vivo or added to cell culture, the proliferating cells can be shown by anti-BrdU monoclonal antibody and immunofluorescence staining. Consequently, the proliferation of the cells was found in NSC hippocampal transplantation groups, and BrdU-positive cells were more in the PTA group than the PT and PTN groups (P < 0.05) (Figure 3).

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Figure 3 Neural stem cell proliferation by immunofluorescence staining. Paraffin section of hippocampus tissue of mice was evaluated by indirect immunofluorescence and diaminobenzidine staining. After nuclei were counterstained with hematoxylin, positive BrdU-labeled cells were observed and counted. Arrows indicate positive cells marked by BrdU expression. A: Immunofluorescence staining; B: BrdU positive cell counting. ^aP < 0.05 when compared to senescence-accelerated mouse prone 8 (SAMP8) neural stem cells (NSCs) transplantation group, ^bP < 0.05 when compared to SAMP8 NSCs transplantation with non-acupoint group. RC: Senescence-accelerated mouse resistant 1 control group; PC: Senescence-accelerated mouse prone 8 control group; PS: Senescence-accelerated mouse prone 8 sham operation group; PT: Senescence-accelerated mouse prone 8 neural stem cells transplantation group; PTA: Senescence-accelerated mouse prone 8 neural stem cells transplantation with acupuncture group; PTN: Senescence-accelerated mouse prone 8 neural stem cells transplantation with non-acupoint group.

Flow cytometry detection showed that on the 4^th d after co-culture of NSCs with hippocampal slices in vitro, the expression of synaptophysin in the PC, PS, PT, and PTN groups decreased significantly as compared to the RC group (P < 0.05). When compared to the PC or PS group, the expression of synaptophysin in the PT, PTA, and PTN groups increased significantly (P < 0.05); when compared to the PTN group, the expression of synaptophysin in the PTA group increased significantly (P < 0.05). On the 7^th d, the expression of synaptophysin in the PC, PS, PTA, and PTN groups decreased significantly as compared to that in the RC group (P < 0.05); the expression of synaptophysin in the PT, PTA, and PTN groups increased significantly as compared to the PC and PS groups (P < 0.05), and that in the PTA group increased significantly as compared to the PTN group (P < 0.05). On the 10^th d, the expression of synaptophysin decreased significantly in the PC, PS, PT, PTA, and PTN groups as compared to that of the RC group (P < 0.05), while that the PT, PTA, and PTN groups increased significantly as compared to that in the PC or PS group (P < 0.05), and when compared to the PTN group, the expression in the PTA group did not change significantly than that in the other groups (P > 0.05) (Figure 4).

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Figure 4 Synaptophysin expression by flow cytometry assay. Hippocampal brain specimens and neural stem cells were inoculated in the Transwell co-culture system. On the 4^th, 7^th, and 10^th d, flow cytometry was used to detect the expression of synaptophysin-positive cells. A: Synaptophysin expression; B: Proportion of cells with synaptophysin expression. ^aP < 0.05 when compared to senescence-accelerated mouse resistant 1 control group, ^bP < 0.05 when compared to senescence-accelerated mouse prone 8 (SAMP8) control group, ^cP < 0.05 when compared to SAMP8 sham operation group, ^dP < 0.05 when compared to SAMP8 neural stem cells transplantation with non-acupoint group. RC: Senescence-accelerated mouse resistant 1 control group; PC: Senescence-accelerated mouse prone 8 control group; PS: Senescence-accelerated mouse prone 8 sham operation group; PT: Senescence-accelerated mouse prone 8 neural stem cells transplantation group; PTA: Senescence-accelerated mouse prone 8 neural stem cells transplantation with acupuncture group; PTN: Senescence-accelerated mouse prone 8 neural stem cells transplantation with non-acupoint group.

Alzheimer's disease (AD) is a chronic, progressive, age-related degenerative disease of the central nervous system, which seriously affects the quality of life of the elderly. Synaptic changes are closely related to cognitive impairment of AD. The effects of acupuncture on cognitive impairment may be related to the regulation of the neural stem cell (NSC) microenvironment.

Previous studies have shown that the improvement of senescence-accelerated mouse prone 8 (SAMP8) cognitive impairment by acupuncture is related to the regulation of NSCs microenvironment. This study aimed to observe the effects of acupuncture on nerve regeneration and synapse after regulating the microenvironment of NSCs, and further explore the anti-dementia mechanism of acupuncture.

To observe the mechanism of acupuncture promoting nerve regeneration and synaptophysin expression in dementia mice after NSC transplantation.

Senescence-accelerated mice (SAM) were divided into six groups: SAMR1 (RC), SAMP8 (PC), sham transplantation (PS), NSC transplantation (PT), NSC transplantation with acupuncture (PTA), and NSC transplantation with non-acupoint acupuncture (PTN). The behavior changes of mice after NSC transplantation were observed by the Morris water maze test. The histological changes of the hippocampus, NSC proliferation, as well as synaptophysin production in the hippocampal microenvironment were studied by hematoxylin-eosin staining, immunofluorescence staining, and flow cytometry.

Escape latency of all the NSC transplantation groups increased obviously. The behavioral change in the PTA group was stronger than those of the other two groups (P < 0.05). After acupuncture, the hippocampal structure in the PTA group was clear, the cell arrangement was orderly, and the necrosis of cells in CA1 and CA3 areas was significantly reduced. The number of BrdU-positive proliferating cells increased significantly in the PTA group compared to those in the PT and PTN groups (P < 0.05). The synaptophysin expression in the PC group decreased in comparison to the RC group, that in PT, PTA, and PTN groups increased as compared to the PC group, and that in the PTA group increased significantly as compared to the PTN group with acupoint-related specificity (P < 0.05).

Acupuncture accelerates nerve regeneration and synaptophysin production in SAMP8 mice by regulating the hippocampal microenvironment after NSCs transplantation.

These findings provide an experimental basis for the treatment of nerve regeneration in AD.