Electroacupuncture reverses endothelial cell death and promotes angiogenesis through the VEGF/Notch signaling pathway after focal cerebral ischemia‐reperfusion injury

Abstract Background Angiogenesis is an important mechanism of recovery from ischemic stroke. Recent studies have found that there is a close relationship between the VEGF/Notch pathway and angiogenesis. It is unknown whether EA can exert a brain protection effect and promote angiogenesis by acting on the VEGF/Notch signaling pathway after focal cerebral ischemia‐reperfusion injury (CIRI). Methods The Middle Cerebral Artery occlusion/Reperfusion (MCAo/R) model was established, in which rats were subjected to occlusion with ischemic intervention for 30 min, followed by reperfusion for 8 h, 1 day, 3 days, and 7 days. The first EA treatment was performed 90 min after the animal model was successfully established, and then EA treatments were performed once a day for 7 days. The 2,3,5‐triphenyltetrazolium chloride staining and neurological deficit examination were performed to assess the level of CIRI and neuroprotection by EA. Expression levels of VEGFA, Notch1, and Hes1 proteins were measured via western blotting, while the morphological changes of ECs and microvasculature in the cortex were determined using an ultrastructural observation method. Results EA treatment of PC6, GV26, and SP6 can significantly improve the neurological function of MCAO/R rats, reduce the volume of cerebral infarction, and modulate the ultrastructure of ECs and microvessels in pathological states. Western blotting revealed that EA increased VEGFA protein expression at 8 h and 3 days after CIRI, as well as Notch1 protein expression at 1 and 7 days. Subsequently, EA activated the VEGF/Notch pathway, increasing the expression of the downstream target protein Hes1, reversing EC death, and promoting angiogenesis. Conclusion Our findings showed that EA plays a role in promoting angiogenesis following focal CIRI, and we hypothesized that this was due to the regulation of ECs by the EA‐activated VEGF/Notch signaling pathway.


INTRODUCTION
Cerebral Ischemic Stroke (CIS) is the major cause of disability and death worldwide, putting human health at considerable risk (Virani et al., 2021). After CIS, the quick reestablishment of blood flow boosts the level of tissue oxygenation, resulting in the second burst of cell damage (Amani et al., 2019). This is called cerebral ischemia-reperfusion injury (CIRI) (Sharma et al., 2020). Endothelial cells (ECs) are the primary constituents of the blood-brain barrier, and their dysfunction and death contribute significantly to the pathological progression of CIRI (Li et al., 2020a,b;Zhang et al., 2021;Saberianpour et al., 2021;Nemcovsky et al., 2019;Nitzsche et al., 2021). Although CIRI is a complex pathological process involving multiple cytokines and signaling pathways, it is a deterioration of brain tissue that can be partially repaired through interventions (Khamchai et al., 2022;Tuo et al., 2022). Secondary brain injuries occur after focal CIRI. Due to the increasing brain edema, the blood-brain barrier (BBB) is severely disrupted, leading to the infiltration of peripheral immune cells and exacerbating the inflammatory response of the central nervous system (CNS) (Kawabori & Yenari, 2015). Accumulating evidence shows that angiogenesis not only protects brain tissue (Zhang et al., 2000), but also contributes to neuronal recovery . It has been reported that in the rodent MCAO model, EC began to proliferate as early as 12−24 h after ischemia, but the cerebral microvessels in the core ischemic area and ischemic penumbra recombined within 4−7 days (Hayashi et al., 2003;Kanazawa et al., 2017). At the same time, neuronal remodeling and axonal growth occurred 14 days after the stroke (Kanazawa et al., 2017). Therefore, promoting angiogenesis may become a promising therapeutic strategy for the treatment of ischemic stroke. Angiogenesis (AG) is an important process of neurovascular unit remodeling after ischemic stroke Seet et al., 2012). It not only increases oxygen and nutrition supply to the ischemic penumbra (Zhang et al., 2002), but also regulates the growth of axons and neurogenesis (Liu et al., 2014;Widenfalk et al., 2003), which can protect brain tissue from cerebral ischemic damage. Previous studies have demonstrated that increased microvessel density in the periinfarct region contributes to prolonging the survival time of stroke patients, suggesting that angiogenesis is important for brain tissue repair after stroke (Krupinski et al., 1994). In addition, angiogenesis also plays a key role in the recovery of neurological function after ischemic stroke, as it facilitates the establishment of collateral circulation and increases the migration of oxygen, glucose, nutrients, and neural stem cells (NSCs) to the ischemic area (Beck & Plate, 2009;Ruan et al., 2015). It is considered one of the most promising strategies to treat cerebral infarction. However, the self-repair ability of cerebral ischemic tissue is limited and cannot effectively improve the blood supply and neurological function recovery after cerebral ischemia. Therefore, it is very important for us to explore an effective method as soon as possible to restore the blood supply in the ischemic area, promote angiogenesis, and help the damaged brain tissue and neurological function recover in time.
VEGF and Notch signaling pathways are important parts of many signaling pathways that are related to angiogenesis after cerebral ischemia (Liang et al., 2020(Liang et al., , 2021. VEGF (vascular endothelial growth factor) is a specific mitogen for ECs and a potent regulator of angiogenesis, capable of initiating a complex cascade of events including EC activation, assembly of new vascular structures, mural cell recruitment, and vascular stabilization (Gianni-Barrera et al., 2011). Lobov et al. (2007 found that VEGF is located upstream of the Notch signaling pathway. Studies have shown that there is a complex coregulatory relationship between VEGF and Notch pathways (Jin et al., 2014).
After VEGF binds to related receptors, it can activate the Notch signaling pathway by increasing the expression level of Notch ligands such as DLL4. This indicates that the regulation of angiogenesis by the Notch signaling pathway is accompanied by the close participation of VEGF. The Notch pathway is mainly composed of Notch receptors, ligands, and target genes, which determine the differentiation of cells and the occurrence of tissues. When the ligand binds to the receptor, the Notch molecule is activated, releasing Notch1 (NICD) and initiating its downstream transcriptional targets, such as Hes1, to play a regulatory role. Some researchers used the immunofluorescence double staining method to observe that the expression of NICD in vascular ECs in the ischemic cortex was significantly upregulated after 3 days of cerebral ischemia compared with the control group (Lobov et al., 2007). It was still expressed at a high level after 3 days, indicating that Notch1 may participate in the regulation of angiogenesis after cerebral ischemia by upregulating its target gene, the Hes1 protein. Even though the articles on the VEGF-Notch signaling pathway and angiogenesis have clues that can be looked into, most studies go into detail about the role of a certain protein in one of the signaling pathways, such as a single study on the role of the VEGF pathway in angiogenesis after CIRI or a single study on the role of the Notch pathway in angiogenesis after CIRI. However, based on previous evidence, there is a complete link between VEGF and Notch signaling pathways. The innovation of our research is to focus on the cascade reaction of the expression of angiogenic factors in the VEGF/Notch pathway caused by CIRI, including the dynamic changes of angiogenesis in the upstream, middle, and downstream target proteins of this pathway. We are dedicated to investigating the molecular mechanism of EA intervention on the dynamic expression of VEGF/Notch pathway angiogenic factors and precise regulation of angiogenesis as they relate to morphological changes in ECs, thus revealing the brain protective effect of EA.
Electroacupuncture (EA) can influence cell electrophysiology and has a high therapeutic potential for angiogenesis, neurogenesis, and cerebral circulation reconstruction in the ischemic area following a stroke . Furthermore, EA can also affect certain signaling pathways and play a role in reducing cerebral edema and antiapoptosis after focal CIRI (Long et al., 2021;Zou et al., 2015). The role of EA in promoting angiogenesis after cerebral ischemia is clear, but its ability to regulate the VEGF/Notch signaling pathway is still limited. However, it is worth noting that the latest evidence shows that EA can activate the HIF-1/VEGF/Notch1 signaling pathway and promote angiogenesis after ischemic stroke through exon miR-210 . This study aims to analyze the particular mechanism of EA as an intervention method for supporting angiogenesis at various time periods after CIRI and to offer a scientific basis for the clinical treatment of cerebral ischemia disorders.

Model preparation
The middle cerebral artery occlusion/reperfusion (MCAo/R) model was made according to Zea-Longa's suture method (Longa et al., 1989). The sham surgery group were the same as those in the MCAo/R group, except that the nylon filament was not inserted. After waking up, animals with scores of 1, 2, and 3 on the Longa 5-point method (Longa et al., 1989) were chosen as experimental subjects.

Electroacupuncture treatment
We kept the rats awake during the EA treatment. All rats wearing rat clothes were fixed on the table with adhesive tape. According to "Experimental Acupuncture Science" (Guo, 2016), "Shuigou" (GV26), "Neiguan" (PC6), and "Sanyinjiao" (SP6) were selected. GV26 was located in the middle of the cleft lip, 1 mm below the tip of the nose.
PC6 was located 3 mm above the wrist joint on the medial side of the forelimb between the ulna and the radius and was punctured vertically to a depth of 1 mm; SP6 was located 1 cm above the tip of the medial malleolus of the hind limb. Each acupoint was needled for 20 min. During the needle retention period, the filiform needle handles at PC6 and SP6 acupoints on the affected side were respectively connected to the HANS nerve acupoint stimulator, and the EA waveform was set as a distant dense wave. The frequency was 2/15 Hz (adjusted to the muscle twitch threshold), and the current was 1 mA. The first EA treatment was performed 90 min after the animal model was successfully established, and then EA treatments were performed once a day for seven days. The rats in the blank group, sham surgery group, and MCAo/R model group received the same grasping but were not given EA treatment.

Effect index and measurement method
2.4.1 Neurological deficit score in MCAo/R rats The neurological function was assessed by professional staff who did not participate in MCAo/R surgical and EA treatment in accordance with the Zea-Longa 5-point method (Longa et al., 1989) at 8 h, 1 day, 3 days, and 7 days after MCAo/R surgery. The higher the score, the more serious the neurological deficit. The specific methods and scoring standards are shown in the figure below ( Figure 1).

2,3,5-Triphenyltetrazolium chloride (TTC) staining
The volume of cerebral infarction in rats was detected by using the TTC staining method (Cheng et al., 2015). Place the brain tissue in the refrigerator at −20 • C for 15 min until the brain tissue becomes hard. Slice the brain from the frontal to occipital poles to make coronal slices. Each slice is about 2 mm thick and cut into six pieces. Put the brain slices in 1% TTC phosphate buffer (pH = 7.4) (Nasi Biotechnology Co., Ltd., Shanghai, China) and cover them with tin foil. Place them in a 37 • C incubator for 10−15 min. Turn the brain slices with a brush to ensure even staining. TTC reacts with dehydrogenase in normal tissues and turns red, but in ischemic tissues, dehydrogenase activity decreases and fails to respond, so cerebral infarct tissue appears pale. Use Image-Pro Plus software (version 6.0, Media Cybernetics, Rockville, MD, USA) to calculate cerebral infarction volume. Infarct vol-

Statistical analysis
The data are analyzed by SPSS 18.0 system software (SPSS, Chicago, IL, USA), and the inspection level is (α = 0.05). All data are tested by one sample Kolmogorov-Smirnov test (K-S test). For those with a normal distribution (p > .05), the statistical description is expressed by mean ± SD, and the homogeneity of variance test is performed.
One-way ANOVA is used for multigroup comparison of the normal distribution data. The least significant difference method (LSD test) was used for the pairwise comparison of each experimental group. A nonparametric Kruskal-Wallis test is used for nonnormal distribution data.
p < .05 is statistically significant.

Focal CIRI caused edema, necrosis, and apoptosis in MCAo/R ECs
After MCAo/R surgery, the infarct volume of the MCAo/R rats expanded in 8 h, 1 day, 3 days, and 7 days (all p < .01), indicating that the model was successful (Figure 4a (Figure 3a and b). One day after focal CIRI, more vacuoles appear around ECs, organelles are swollen, endothelial nuclei are fragmented, the nucleus membrane is no longer intact, and lysosomes increase. Cell edema is more intense in this phase than in the initial stages, and early vascular EC necrosis may occur (Figure 3c-h).
The obvious transition occurred 3 days after the focal CIRI. The ultrastructure showed the characteristics of ECs in preapoptosis: A deepening of nucleus chromatin accompanies the nucleus shrinkage of ECs. There are multiple vacuoles of different sizes around ECs-the formation of plasma membrane blebs, and the nucleus membrane is sunken inward. The inside of the nucleus is fragmented, but the nucleus membrane is still intact-a typical feature before cell apoptosis. Cell edema at this stage is not the primary manifestation (Figure 3i-l).
Notably, 7 days after focal CIRI, the ultrastructure displayed signs of the last stage of EC apoptosis, including endothelial nucleus fragmentation and nucleus dissolution. The nucleus membrane disappearance, organelle damage, and apoptotic cell debris fall off into the lumen ( Figure 3m).

3.2
The cerebral infarction volume of MCAo/R rats changed, the expression of Hes1 protein dropped, and the neurological function deteriorated We continued to detect the cerebral infarction volume changes at 8 h, 1 day, 3 days, and 7 days after focal CIRI in rats and found that they were related to the pathological changes of ECs. As shown in Figure 4a, the volume of cerebral infarction 7 days after MCAo/R surgery was significantly lower than it was 8 h and 3 days later (p < .05 and p < .01), suggesting that as the infarction time continues to prolong, rats show a certain degree of self-regulation ability. The ECs develop from severe swelling and necrosis at 8 h and 1 day in the early stage to shrinkage and apoptosis of the ECs at 3 days and 7 days. Brain edema will gradually decrease under the self-regulation of brain tissue ECs, and the infarct volume will also shrink accordingly (Figure 4b

EA activated the VEGF/Notch pathway, regulated EC lesions, relieved brain edema 8 h and 1 day after CIRI, and reduced cerebral infarction volume
Based on the above findings, we further explored the mechanism of EA treatment. The protein levels of VEGFA, Notch1, and Hes1 were quantified by western blot analysis. The results indicated that VEGFA in the EA group for 8 h and Notch1 in the EA group for 1 day were significantly higher compared with the MCAo/R group at the same time 8 h after MCAo/R surgery (p < .01) (Figure 4a and c), suggesting that the application of EA at 8 h and 1 day after focal CIRI can reduce the brain edema and promote the absorption of edema in cerebral infarction tissue. This is related to the activation of the VEGF/Notch pathway by EA and the elevation of VEGFA, Notch1, and Hes1 protein expression in the infarcted brain area of the MCAo/R rats.

EA upregulated VEGFA, Notch1, and Hes1 protein levels, promoted angiogenesis 3 and 7 days after focal CIRI, and inhibited EC apoptosis
In order to clarify the cascade reaction of VEGF-Notch1-Hes1 expression caused by EA treatment and the molecular mechanism of the VEGF/Notch signaling pathway involved in regulating angiogenesis, we further detected the expression of the above proteins in the EA group for 3 and 7 days. We found that the VEGFA of the EA group for 3 days and Notch1 of the EA group for 7 days were significantly higher compared with the MCAo/R group at the same time (p < .05 and p < .01), and Hes1 of the EA group at 3 and 7 days were significantly higher compared with the MCAo/R group at the same time (p < .01) (Figure 5a and c). In combination with the electron microscope, it was detected that the morphology of ECs in the EA group at 3 days was normal and the phagosomes were clearing the damaged mitochondria ( Figure 6e and d). Microvascular proliferation can be seen under the electron microscope at this stage (two microvessels can be seen in one ultramicrofield) (Figure 6e). The endothelial nuclei migrate and proliferate, and the cytoplasm of the EC fills the whole EC (Figure 6e).
Because the activation of the VEGF/Notch pathway after EA treatment causes the upregulation of VEGFA, Notch1, and Hes1 proteins, EA can reduce the accumulation of damaged mitochondria, promote the self-renewal of damaged brain tissue, and reduce the occurrence of apoptosis. Compared with the MCAo/R group at the same time, the neurological deficit score of rats in the EA group decreased at each time point, especially at 3 days (p < .01) (Figure 4b), and the volume of cerebral infarction in the EA group decreased significantly at 3 days (p < .01) (Figure 4a and c). The morphology of ECs in the EA group at 7 days of angiogenesis can be seen under the electron microscope: an EC differentiated into a new microvessel (Figure 6c①). The EC and newborn microvessel morphology are healthy (Figure 6c and e). Taken together,

F I G U R E 3 Ultrastructure observation of MCAo/R group.
because the angiogenesis regulators VEGFA, Notch1, and Hes1 act on the upstream, midstream, and downstream of the VEGF/Notch signaling pathway, respectively, the effect of angiogenesis after focal CIRI was fully demonstrated after EA treatment (Figures 5 and 6).

DISCUSSION
EA has been reported to be actively involved in various pathophysiological cascades following ischemic stroke, including angiogenesis and neurogenesis (Chavez et al., 2017;. A combination of related studies suggests that EA may act on ECs and exert a protective effect on the brain by activating the VEGF/Notch signaling pathway (Fu et al., 2021;Lee et al., 2015;Xu et al., 2022;Zhang et al., 2018;. In this study, we hypothesized that EA can promote angiogenesis in the ischemic area by activating the VEGF/Notch pathway after focal CIRI, thereby reducing brain injury and promoting neurological recovery. What is the specific molecular biological mechanism? In order to study the neurovascular protection mecha-nism of EA after ischemic stroke, we created a rat model of MCAo/R and administered EA treatment at different times of reperfusion. As we estimated, the enlarged infarct volume and neurological deterioration in rats could be ameliorated by EA. What surprised us even more was that, under the ultrastructural observation of ECs, EA could not only reverse the necrosis and apoptosis of ECs after focal CIRI but also promote EC migration and proliferation. Microangiogenesis was also observed under ultrastructural microscopy. To further corroborate our findings, we also detected proteins in the VEGF/Notch pathway that are closely related to angiogenesis.

Vascular endothelial growth factor A (VEGF-A) is a central medi-
ator in postischemic proangiogenic molecules in rodent models and humans (Alrafiah et al., 2021). Currently, emerging evidence demonstrates that VEGFA mediates directed angiogenesis in ECs and appears to be one of the most potent mediators in both physiological and pathological angiogenesis (Geiseler & Morland, 2018;Li et al., 2021;Shibuya, 2011). Furthermore, VEGFA-mediated neurovascular protection and Notch-1-triggered antiapoptotic effects in endothelium and other neural cells may synergistically contribute to neurovascular protection

F I G U R E 4 Changes of the cerebral infarction volume (%) and neurological function scores in each group of rats at different times. (a)
Quantitative results of the cerebral infarction volume. Data are presented as the mean ± SD (n = 5 rats). Compared with the sham surgery group at the same time, **p < .01. Compared with the MCAo/R group at the same time, ## p < .01. Compared with 8 h in the MCAo/R group, ΔΔ P < 0.01, compared with 3 days in the MCAo/R group, ▲ p < .05. (b) Quantitative results of Zea-Longa neurological function scores (n = 8 rats). Compared with the sham surgery group at the same time, **p < .01; compared with the MCAo/R group at the same time, ## p < .01; compared with 8 h after MCAo/R surgery in the EA group, ΔΔ p < .01, compared with 1 day after MCAo/R surgery in the EA group, ▲ p < .05, ▲▲ p < .01. (c) The cerebral infarction areas were measured by 2,3,5-triphenyltetrazolium chloride staining. The white represents the ischemic area, and the red shows the normal area.
during the acute stage of ischemic stroke (Chen et al., 2020;Li et al., 2022). Our findings show that after focal CIRI, an 8-h EA treatment can begin to mobilize VEGFA protein (Figure 5a). At 8 h, the level of VEGFA protein in the EA group was significantly higher than in the MCAo/R group. (p < .01), indicating that an 8-h EA treatment activates the central mediator of the VEGF/Notch pathway after focal CIRI.
One-day EA treatment after reperfusion, the expression of Notch1 protein increased. Compared with the MCAo/R group, the expression level of Notch1 in the EA group increased significantly (p < .01) (Figure 5c), which is consistent with the conclusion of Takeshita et al. (2007) that Notch1 is a downstream protein of VEGF. Under an ultrastructural microscope, the swelling of ECs was reduced after EA treatment for 8 h and 1 day, and the brain tissue and microvessels of the ischemic area were restored to a homeostatic state. The VEGF/Notch signaling pathway may have been activated in this period after EA intervention. Hes1, the downstream target gene of the Notch pathway, was transcribed in the EA group for 8 h, 1 day, 3 days, and 7 days, and the Hes1 protein level of the EA group was significantly higher than that in the MCAo/R group (p < .05 or p < .01) (Figure 5e). After 3 and 7 days of EA treatment, the levels of VEGFA and Notch1 proteins continued to increase after reperfusion (Figure 5a and c), and EC pro-liferation and microangiogenesis were observed under ultrastructural microscopy ( Figure 6c and  it has been reported that EA treatment significantly enhances the expression levels of Notch1 and VEGFA after ischemic injury and exerts a repairing effect on cerebral blood vessels and brain tissue Li et al., 2022), whether the antiapoptotic effect to EC under the ultrastructure is related to the activation of the VEGF/Notch pathway by EA remains to be further explored.
Hairy and enhancer of split (Hes1), a basic helixloop-helix transcription factor, is a downstream effector gene of the Notch receptor. Studies have shown that Hes1 deficiency in human and mouse cell lines induces apoptosis (Takeshita et al., 2007). Furthermore, Hes1 knockdown exacerbates cerebral infarction and neurological deficits after MCAO (Li et al., 2020a,b). Our study found that the transcriptional level of Hes1 was significantly down-regulated (p < .01) in brain tissue after reperfusion for 3 and 7 days, compared with sham surgery (Figure 5e).
In addition, ultrastructural analysis showed that ECs showed apoptosis at 3 and 7 days of reperfusion (Figure 3i, k, and m), and the cerebral infarction volume and neurological function scores of MCAo/R rats were also worse than those of the sham surgery group (p < .01) (Figure 4a and b). These findings suggest that the decreased protein level of Hes1 after focal CIRI is closely related to EC apoptosis. It provides useful clues for future research into the ability of EA to promote Hes1 protein transcription as well as activate the VEGF/Notch pathway to exert neurovascular protection after focal CIRI.
After EA treatment, VEGFA, Notch1, and Hes1 increased correspondingly at each period after reperfusion ( Figure 5). EA group for 3 and 7 days, the structure of EC proliferation and microangiogenesis was observed (Figure 6e and c), and no apoptotic cells were found. The above results suggest that after EA activates the VEGF/Notch pathway, it not only promotes angiogenesis but also reduces the apoptosis of ECs. Compared with the model group, the volume of cerebral infarction decreased at 3 days in the EA group (p < .01) (Figure 4a and c), and the neurological function score also significantly improved at 3 days (p < .01) (Figure 4b). The above results suggest that there is a close relationship between neurons and ECs in the process of angiogenesis, and angiogenesis often promotes the recovery of neurological function after cerebral ischemia. Moreover, studies report that postischemic angiogenesis follows a temporal course (Bi et al., 2022;Zhang et al., 2009). ECs begin mitosis as early as 1 day after mouse cerebral reperfusion after 30 min of MCAO, and the number of vessels begins to significantly increase on the 3 days before peaking at 7 days after stroke (Yang et al., 2016). In our study, under the ultrastructural microscope, ECs migrated and proliferated at 3 and 7 days after EA intervention, and angiogenesis was observed under the microscope. It is not difficult to discover that EA has the greatest effect on promoting angiogenesis three and 7 days after focal CIRI.
In terms of experimental design, our research follows the design idea of complementing and supporting each other from macro to micro, from morphology to pathology, from ultrastructure to molecular biology. To demonstrate the effective role of EA after CIRI, electron microscope images of ultrastructural analysis were used as the F I G U R E 6 Ultrastructure observation of EA group. main line, molecular biology-related proteins were used as important evidence, and cerebral infarction volume and behavioral scores were used as supporting evidence. Not only that, in the next step, we will transform the knowledge of medical biology gained from the laboratory research-that is, the proangiogenic effect of EA-into improved therapeutic measures for patients with focal CIRI. In clinical practice, it is possible to begin EA treatment 8 h after CIRI. Application of EA 3 and 7 days after CIRI may encourage angiogenesis and aid in the better recovery of ischemic stroke patients. After the results of clinical trials are confirmed, the transition from the lab to stroke clinical practice will be realized. This will make it possible for ischemic stroke patients and clinicians to get better treatment and rehabilitation programs.
Several limitations of our research merit discussion. There are various explanations for the mechanism of cell pathological damage and angiogenesis in cerebral ischemia reperfusion at present (Jurcau & Simion, 2021). The occurrence of cell necrosis and apoptosis is the pathological result of the interaction of receptors and signal pathways at the molecular biological level (Saberianpour et al., 2021). Angiogenesis is also a complex and strictly controlled process . Both of them play an important role in the process of ischemic stroke. In our study, it has been confirmed that EA can help encourage angiogenesis after CIRI, which is closely related to the activation of the VEGF/Notch signaling pathway. During this process, it can be found from morphological observation that the pathological changes of EC necrosis and apoptosis have decreased, but this is only a speculation based on the ultrastructural analysis and molecular biology levels.
The exact impact of EA on brain cell necrosis and apoptosis still needs further evidence from related detection methods.
Another limitation is the time window for EA to promote angiogenesis after CIRI. Since our EA intervention started 90 min after the successful modeling, but the first observation node was set 8 h after the EA intervention, it cannot be proved whether EA can promote angiogenesis at the superearly stage of CIRI. The discovery of this problem can help us find the direction of improvement for the next stage of experimental research. It is critical to investigate the target of angiogenesis intervention in order to improve the clinical efficacy of stroke. After the onset of an ischemic stroke, early intervention with EA for rehabilitation purposes is recommended. However, the timing mechanism of EA regulating angiogenesis and nerve function reconstruction after CIS is relatively complicated. At present, we still need more work to further prove the therapeutic window of EA and promote the timely repair of brain tissue damage and the reconstruction of neurological function in patients with ischemic stroke during clinical treatment.
Finally, I would want to emphasize that the CIRI investigated in this study-a focal one-is fundamentally distinct from a global one.
In this study, the MCAo/R model simulated the most common pathological type of ischemic stroke in neurology, while global cerebral ischemia-reperfusion injury (GCI/R) is commonly seen in emergency medicine, and cardiac arrest (CA) and return of spontaneous circulation (ROSC) are the most common types of GCI/R in experiments in which rats received the 4-VO model . In our research, EA plays an effective role in promoting angiogenesis in focal CIRI.

CONCLUSIONS
In summary, except for demonstrating that EA can activate the VEGF/Notch signaling pathway to promote angiogenesis after focal CIRI, the more important discovery is that EA can reduce the pathological changes of edema, necrosis, and apoptosis by regulating the morphological changes of ECs and promoting the recovery of neurological function after ischemic stroke. This also confirmed our hypothesis that EA promoting angiogenesis after focal CIRI is an important mechanism for ischemic stroke treatment.