2. Rat MCAO/R Model and Treatment
4. Transmission Electron Microscopy
6. New Object Recognition Test
7. Enzyme-Linked Immunosorbent Assay
The experimental framework. Experiment 1: The study divided rats into control and MCAO/R groups to test memory impairment after modeling. The control group received no intervention, while the MCAO/R group was modeled using suturing, and tested after 28 days via the water maze or object recognition experiments. Experiment 2: This study randomly divided MCAO/R rats into different groups, with normal rats as controls, to test the sustained treatment effects. The water maze experiment was conducted 6 days after treatment, followed by IF, IHC, and TEM detection. Experiment 3: MCAO/R rats were randomly divided into different groups for ELISA detection to verify the permeability of mNGF in various brain regions. Another group of MCAO/R rats was divided into several groups to validate the p65-VEGFA-TJs signaling axis via WB and IHC, exploring the effect of SMES on hippocampal BBB opening.
(Figure 1) shows the grouping of the three experiments.
In the investigation of potential memory impairment after modeling, rats were divided into control and MCAO/R groups. Control rats received no intervention. Memory assessment was conducted using MWM or NOR experiments 28 days after modeling.
In the study aimed at confirming sustained treatment effects, MCAO/R rats were randomly divided into MCAO/R, NGF, SMES, and NGF+SMES groups, with normal rats as the control group. After intervention, a 6-day water maze task was performed, followed by IF, IHC, and TEM analysis.
To assess the permeability of mNGF in various brain regions of MCAO/R rats, MCAO/R rats were randomly divided into MCAO/R, NGF, NGF+PDTC+SMES, and NGF+SMES groups for ELISA to explore the impact of the p65-VEGFA-TJs signaling pathway on the SMES-induced opening of the hippocampal BBB to facilitate mNGF entry into the brain. Thus, MCAO/R rats were randomly divided into MCAO/R, PDTC+SMES, and SMES groups for validation of signaling pathway proteins via WB and IHC.
Quantitative and Statistical Analysis
SMES Introduces mNGF into Different Cognitive Brain Regions
In our previous experiments, we observed that SMES could enhance mNGF permeability to the rat prefrontal cortex. However, the extent of mNGF permeability to different cognition-related brain regions remains unknown. We measured and compared the mNGF permeability and specificity in cognition-related brain regions of three groups of normal rats to verify these effects in rats. Thus, SMES promoted the upregulation of mNGF in primary motor cortex (M1) and various cognition-related brain regions excluding the prefrontal cortex (PrL) (Figure Arabic Numbers A-F). A significant increase in mNGF levels was observed in the hippocampus (Figure. Arabic Numbers G).
Measured mNGF expression in M1 and various cognition-related brain regions via ELISA. A, B, C, D, E, F showed significant effects in the NGF+SMES group excluding PrL and MS. Results are presented as SEM±mean (n=5 per group). *P<0.05, **P<0.01, P<0.001, P<0.0001 compared to NGF+SMES group. G shows a significant effect on the hippocampus. Results are presented as SEM±mean (n=5 per group). **P<0.01, P<0.001, P<0.0001 compared to hippocampus group. a, b, c, d, e, f showed significant permeability effects in M1, brainstem, and hippocampus compared to MCAO/R group, NGF group, or NGF+SMES+PDTC group. Results are presented as SEM±mean (n=5 or 8 per group). *P<0.05, **P<0.01, P<0.0001 compared to NGF+SMES group. g shows a significant effect on the hippocampus. Results are presented as SEM±mean (n=6 per group). P<0.0001 vs. hippocampus group.
Given the good performance of SMES in the hippocampus, we evaluated the permeability of mNGF in the affected hemisphere of three groups of MCAO/R rats via ELISA. As expected, the NGF+SMES group increased after 28 days post-MCAO/R (Figure D). 2a, b, c, d, e, f). These findings indicate that SMES may accelerate the spread of mNGF across the BBB, especially towards the hippocampus.
Spatial Learning and Memory Remain Affected 28 Days Post-MCAO/R
We conducted NOR and MWM tests to assess the cognitive function of rats 28 days post-MCAO/R (Figure D).3A, B). There was no significant change in spatial cognitive ability of rats 28 days post-MCAO/R, and excitability showed no obvious abnormalities (Figure D).3C). The center grid stay time in open field analysis reflects spatial cognitive ability, showing no significant differences among groups (Figure D).3C1). The number of fecal pellets in the open field analysis reflects the level of stress in the animals (Figure D). The greater the number of fecal pellets, the higher the stress. The control group had significantly more fecal pellets than the MCAO/R group. The stress level of rats decreased after 28 days post-MCAO/R. In the NOR short-term memory and long-term memory tests, the main effect of group, time, group and time/group interaction was not significant (Figure D).3D-E2). The NOR experiment indicated that there were no significant differences in long-term and short-term memory between the two groups of rats, as new object recognition was attributed to prefrontal damage, indirectly demonstrating that the prefrontal cortex of MCAO/R rats was not damaged (Figure D).3D-E2). Next, we conducted the water maze test. In the water maze test prior to treatment, the main effect of group, time, group, and time/group interaction was significant (Figure D).3F). The control group exhibited strong learning ability over 5 days of the experiment, with escape latency gradually decreasing. Although the MCAO/R group showed improvement in escape latency on the 3rd and 4th days compared to the first day, the escape latency was longer on the last day (Figure D).3F). After removing the platform on the 6th day, the control group rats demonstrated superior learning bias when navigating to the target quadrant. Compared to the MCAO/R group, they showed more time spent in the target quadrant and increased frequency of crossing the platform in the target quadrant (Figure D).3F1, F2). Our results indicate that spatial learning and memory of rats remain impaired 28 days post-MCAO/R, as MWM is the most sensitive detection index for the hippocampus, highlighting the significance of SMES in opening the hippocampal BBB. Therefore, in the next experiment, we focused on improving the hippocampus through treatment.
28 days of MCAO/R modeling still resulted in impaired spatial learning ability and long-term memory in rats. A new object recognition experiment (NOR) intervention diagram. B During training, objects A1 and A2 were placed in the box, and A2 was replaced with object B after 1 hour or 24 hours. C, C1, C2 Distances traveled during habituation, center time, and number of fecal pellets. D, D1, D2, and E, E1, E2 Typical traces and heat maps of NOR test results; exploration time for A1 and A2 and recognition index representing recognition memory for both groups. F, F1, F2 Escape latency in the Morris water maze (MWM) test over 5 training days for both groups. Time and number of entries in the target quadrant during the 6th day (probe) of the MWM test. Results are presented as SEM±mean (n=7 per group). **Compared to day one, P<0.01, P<0.001, P<0.0001.
(Figure 3)
SMES-Mediated mNGF Improves Spatial Learning and Memory
This study aimed to examine the impact of spatial learning and memory on short-term memory tasks dependent on hippocampal processing. Prior to testing, we conducted continuous treatment (Figure D).1 Experiment 2). Repeated measures two-way ANOVA indicated that the learning curves among groups differed, with a major effect being the day factor (Figure D).4A). The results showed that during training while locating the hidden platform, rats in the SMES+NGF group exhibited reduced escape latency and path length, indicating enhanced learning ability following continuous SMES+NGF treatment. In contrast, rats in other groups did not show significant reductions in latency or path length (Figure D).4A, C-G). There were no significant changes in swimming speed among any experimental groups (Figure D).4 Notably, on the sixth day, rats in the NGF+SMES group demonstrated superior learning bias in navigating to the target quadrant. Compared to the MCAO/R group, they exhibited reduced time and distance traveled, with increased frequency of crossing the platform in the target quadrant (Figure D).4H-J).
Specific pattern electroacupuncture delivers mNGF to the brain, improving spatial learning ability and long-term memory in rats. A Escape latency and B swimming speed in the MWM test across 5 training days for different groups (C, D, E, F, G). Results are presented as SEM±mean (n=9 per group). *Compared to day one, P<0.05, **P<0.01, P<0.001, P<0.0001. H Time spent in the target quadrant and I number of platform crossings during the sixth day (probe) of the MWM test. J Swimming paths of rats observed post-modeling, capturing representative images for the NGF+SMES group during the water maze tests on day 5 (learning) and day 6 (memory). The removed platform is indicated by a dashed line. Results are presented as SEM±mean (n=9 per group). *P<0.05 vs. MCAO/R group.
（Figure 4）
SMES-Mediated mNGF Reduces Apoptosis and Mitochondrial Autophagy of Cholinergic Neurons in the Hippocampus
The combination of SMES and NGF has been observed to reduce apoptosis and mitochondrial autophagy in the hippocampal cells post-MCAO/R. TUNEL positive cells were detected in various hippocampal regions within each group, with lower apoptosis rates observed in the control and SMES+NGF groups. In contrast, the remaining groups exhibited more apoptotic cells than the control and SMES+NGF groups, indicating that the combination of SMES and NGF effectively alleviated apoptosis within the hippocampus (Figure D).5A-D1).
Expression of TUNEL cells in different regions of the hippocampus for each group and the mitochondrial status in the CA1 region post-treatment. 28 days post-modeling, A, B, C, DTUNEL staining of apoptotic cells remains present in various hippocampal partitions, but the SMES+NGF group expresses fewer; scale bar is 20μm. A1, B1, C1, D1 show the ratio of TUNEL positive cells to DAPI stained cells in the immunohistochemical analysis of rats post-MCAO/R. EMCAO/R and NGF groups exhibited damaged mitochondria surrounding neurons, characterized by abnormal cristae structures and vacuoles. In contrast, the remaining groups showed no significant mitochondrial damage. Results are presented as SEM±mean (n=4). Significant levels: P<0.0001 vs. control group, &&&P<0.0001 vs. SMES+NGF group.
（Figure 5）
Inspection of mitochondria in the CA1 region of the hippocampus shows normal mitochondria, with control, SMES, and SMES+NGF groups displaying intact double membranes and clear cristae. However, most mitochondria in the MCAO/R and NGF groups exhibited abnormal cristae structures, with many swollen mitochondria containing numerous small vesicles disconnected from the outer cavity. This led to disordered mitochondrial cristae structures, the appearance of blank areas, and degeneration of the outer membrane of mitochondria (Figure D).5E).
SMES and NGF Increase the Number of Cholinergic Neurons in the Infarcted Hippocampus
Post-MCAO/R, expression levels of ChAT positive cells in four regions of the hippocampus were measured to further explore the changes in ChAT positive protein expression among groups. Immunohistochemical analysis of ChAT was performed to examine whether MCAO/R and its treatment modulate protein expression in cholinergic neurons.
As shown in Figure 1.6A, immunohistochemical analysis indicated that the protein expression of ChAT positive cells in the CA1 region of the hippocampus was significantly higher in the SMES+NGF group compared to MCAO/R, NGF, and SMES groups (P<0.01; Figure.6 In the CA2 region of the hippocampus, there were no significant differences among groups (Figure D).6C). In the CA3 region of the hippocampus, the SMES+NGF group exhibited a significant increase in the number of ChAT positive cells compared to the MCAO/R group (P<0.05; Figure.6 In the DG region of the hippocampus, both SME+NGF and NGF groups showed a significant increase in ChAT positive cells compared to the MCAO/R group (P<0.05; Figure.6E).
Expression of ChAT positive cells in different regions of the hippocampus after treatment in rats. A Representative images of ChAT positive cells in the hippocampus of rats and their respective regions post-MCAO/R. B, C, D, E Immunohistochemical analysis post-ischemic top layer cortex of rats showing the number of ChAT positive cells in the ischemic top layer cortex. SEM±mean (n=5). Significant levels: *P<0.05, **P<0.01, **P<0.001 vs. MCAO/R group and ##P<0.01 vs. NGF group.
(Figure 6)
Cholinergic neurons in the hippocampus of the control group and SMES+NGF group were arranged regularly, with clear layers. Neurons in the MCAO/R, NGF, and SMES groups were irregularly arranged, with cell gaps larger than those in the normal group and SMES+NGF group, with a few neurons losing structural integrity.
SMES Promotes Activation of the p65-VEGFA-TJs Signaling Pathway
Research indicates that VEGFA can induce the activation of brain microvascular endothelial cells, while the established NF-κB/MMP-9 axis can stimulate the release of VEGF. These findings suggest that the activation of the NF-κB signaling pathway promotes angiogenesis and neurogenesis, indicating the need for further study of the downregulation of VEGFA on tight junctions.
We conducted experiments with inhibitor groups (Figure D).1 Experiment 3). We observed that SMES treatment led to the activation of hippocampal p-p65, resulting in downstream regulation of VEGFA protein, subsequently reducing the expression of TJs in the BBB. Immunohistochemistry was employed to validate the expression of phosphorylated NF-κB and VEGFA. Comparative analyses included multiple groups. The findings described in Figure 1.7A, B, and B1 indicate that the SMES group exhibited significantly higher numbers of positive cells post-treatment compared to the MCAO/R and SMES+PDTC groups, suggesting that SMES has the potential to enhance the expression of nuclear NF-κB and cytoplasmic VEGFA.
SMES altered the expression of p-p65, VEGFA, and TJs in the hippocampus. Numerous VEGFA positive cells and NF-κB nuclear staining are displayed in brown. Scale bar, 50μm. B, B1 Quantification of immunohistochemical results; n=4. C Western blot analysis of NF-κB, p-p65, VEGFA, occludin, and ZO-1 expression in the infarcted side of the hippocampus among groups. Quantification of Western blot results in D, D1, D2, D3, with the left figure showing normalized summary protein expression to β-actin; n=8. *P<0.05, **P<0.01, P<0.001, P<0.0001 vs. SMES group, #P<0.05 vs. MCAO/R group, one-way ANOVA followed by Bonferroni post hoc test.
(Figure 7)
Through WB analysis, the levels of NF-κB, p-p65, VEGFA, occludin, and ZO-1 in the infarcted side of the hippocampus among experimental groups were assessed. These findings indicate that SMES induces the activation of phosphorylated p65 and increases VEGF-A expression, thereby reducing the expression of occludin and ZO-1, which are key components of tight junction proteins (Figure D).7C-D3).
Ischemic stroke is a major cause of cognitive impairment worldwide, primarily attributed to the apoptosis of a large number of cholinergic neurons. Unfortunately, many neuroprotective agents have proven ineffective due to their inherent biotoxicity, severe adverse reactions, and limited efficacy. In this investigation, changes in hippocampal neurons in the affected hemisphere remain apparent even 28 days post-MCAO/R. This study reveals a reduction in the number of cholinergic neurons within the ipsilateral hippocampus, accompanied by increased mitochondrial autophagy and apoptosis, ultimately leading to impaired spatial learning and memory in rats. Our treatment has the potential to alleviate these conditions. Specifically, SMES promotes the activation of the p65-VEGFA-TJs pathway, significantly increasing the levels of exogenous NGF in the hippocampus of MCAO/R rats. Administering NGF to the brain enhances synaptic plasticity in the hippocampus of MCAO/R rats. Furthermore, SMES effectively opens the BBB without adversely affecting cholinergic neurons, mitochondrial integrity, or autophagy.
The central cholinergic system is closely associated with learning and memory. Acetylcholine (Ach) is an important neurotransmitter in the central cholinergic system, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Choline (Ch) and acetate synthesize ChAT, which is stored in vesicles under the action of vesicular acetylcholine transporter (VAChT) (Figure D).8). Its main function is to maintain consciousness and play a significant role in attention, learning, and memory. It is the primary neurotransmitter in the brain.BFCN is the primary target cell for NGF after it enters the brain. NGF can promote the growth, repair, and survival of BFCN. NGF can reduce damage to the cholinergic system and the impact of injury.In a normal brain, these neurotrophic factors are produced by cortical target cells projected by basal forebrain cholinergic neurons and are locally produced in the cholinergic cell body region. Therefore, the availability of neurotrophic factors, whether in the axonal terminal region or the cell body region, may contribute to the development and maintenance of the cholinergic system, and may also aid in its recovery post-injury.
Illustrates key steps in the synthesis, release, and reuptake of the neurotransmitter acetylcholine.
(Figure 8)
Our previous research found that SMES acts on the groove and Baihui to open the BBB in normal rats. The BBB opening promoted by SMES does not lead to brain edema, glial cell activation, or apoptosis. The study also confirmed that acupuncture can induce the opening of the BBB in rats by increasing cerebral blood flow and activating cortical neurons in brain tissue through SMES.
Research indicates that phosphorylation of NF-κB in brain microvascular endothelial cells leads to decreased expression of tight junction proteins occludin and ZO-1. Furthermore, studies have demonstrated that the activation of VEGFA on brain microvascular endothelial cells can mitigate brain ischemia/reperfusion injury. Additionally, the classical NF-κB/MMP-9 axis can stimulate the release of VEGF. These findings suggest that the activation of the NF-κB signaling pathway promotes angiogenesis and neurogenesis, and that downregulation of VEGFA on tight junctions warrants further investigation. Our experimental results indicate that during the repair process of brain ischemia, the activation of NF-κB phosphorylation in brain microvascular endothelial cells leads to upregulation of VEGFA expression. Consequently, this reduces the expression of tight junction proteins, resulting in the opening of the hippocampal BBB and facilitating the entry of NGF into the brain.
Future studies must delve deeper into the effects of SMES on brain ischemic injury, encompassing not only neuroregeneration but also neurodevelopment, axonal plasticity, and cerebrovascular repair.
Recent studies indicate that ischemic stroke can reduce the number of cholinergic neurons in the hippocampus of rats during the recovery phase, affecting spatial learning and memory. SMES promotes the upregulation of mNGF in M1 and various cognition-related brain regions (excluding PrL). By activating the p65-VEGFA-TJs pathway, SMES effectively opens the BBB in rats and facilitates NGF transport to the hippocampus. Post-administration, SMES demonstrates enhanced spatial learning and memory in rats, while increasing the number of cholinergic neurons in the hippocampus. This finding provides innovative evidence supporting the potential of macromolecular therapeutic agents in treating central nervous system diseases.
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