{"paper_id":"04dd7ace-9723-48d0-938f-a07be335b43d","body_text":"Protective effects and mechanisms of auricular vagus nerve stimulation in the alleviation of acute ischemic cerebral injury in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective effects and mechanisms of auricular vagus nerve stimulation in the alleviation of acute ischemic cerebral injury in mice Cher-Chia Chang, Kuo-Tong Liou, Yea-Hwey Wang, Chih-hung Hsu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2662844/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Stroke is a major cause of death and long-term disability. Although numerous treatments have shown neuroprotective effects in animal models of ischemic stroke, their clinical efficacy and safety remain to be further investigated. Previous studies have indicated that vagus nerve stimulation (VNS) may reduce cerebral damage in ischemic stroke, but the specific mechanisms by which VNS achieves this effect in murine cerebral ischemic stroke injury are unclear. In this study, an animal model was designed to investigate the therapeutic effects and mechanisms of auricular vagus nerve stimulation (aVNS) in mice with acute ischemic stroke induced by middle cerebral artery occlusion (MCAO) and reperfusion injury. We focused particularly on the involvement of α4-nicotinic acetylcholine receptor (α4-nAChR) and the molecular signaling pathways mediated by aVNS. Mobility of the animals after acute ischemic stroke, infarct volume, and expression of neuroprotective and damaging factors were analyzed to understand the therapeutic effect and protective mechanism of aVNS on ischemic stroke. Our findings demonstrate that aVNS treatment improved mobility and reduced cerebral infarct volume after acute ischemic stroke. Additionally, administration of α4-nAChR antagonists reversed the protective effects of aVNS on ischemic stroke. Our mechanistic investigations revealed that aVNS mediates protective effects against acute ischemic stroke by reducing inflammatory and apoptotic factors, while enhancing neuronal regeneration factors, through activation of α4-nAChR and the GSK3β/β-catenin pathway. Our results provide clear evidence supporting the use of aVNS as a neuroprotective treatment for ischemic stroke in mice. Glycogen synthase kinase 3 (GSK-3) Ischemic stroke Neurogenesis Nicotinic acetylcholine receptor (nAChR) Vagus nerve stimulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Stroke is a major cause of death and long-term disability. It also has a high mortality and morbidity rate, resulting in a heavy economic burden on families, society and the government. Ischemic stroke is mainly caused by obstruction of the artery vessels due to thrombus [1] and leads to a series of oxidative, biochemical and hormonal reactions triggered by loss of glucose and oxygen supply, ultimately leading to blood-brain barrier (BBB) disruption. Restoration of blood supply is considered one of the most effective treatments for ischemic stroke. Intravenous thrombolysis with recombinant tissue plasminogen activator (rt-PA) and mechanical thrombectomy have been shown to improve functional outcome. However, rt-PA is available only for some patients due to its limitations, such as a short treatment time window, potential for progression to hemorrhagic stroke and neurotoxicity [2]. And, the disadvantages of using mechanical thrombectomy include technical difficulty with the navigating wire in delicate intracranial vessels, trauma to vessels, distal embolization, vessel dissection and vasospasm leading to worsening of stroke. Therefore, there is still an urgent need to develop new, safe and effective therapies to manage acute ischemic stroke. Although many studies have demonstrated the neuroprotective effects of drug treatments on animal models of ischemic stroke in preclinical trials, their efficacy and safety have yet to be further confirmed [3]. Vagus nerve stimulation (VNS) was approved by the FDA in 1997 for the treatment of epilepsy [4]. VNS is also a potential treatment for migraine, depression, Alzheimer's disease and traumatic brain injury [5-8] and is considered to offer protection against ischemic brain injury [9]. Although VNS has become one of the most common non-pharmacological treatments for epilepsy, the effect of VNS on neurological recovery after stroke has not been clearly explained. Recently, transcutaneous electrical stimulation of the auricular branch of the vagus nerve has been shown to partially replicate the effects of traditional cervical vagus nerve stimulation on ischemic stroke. Auricular vagus nerve stimulation (aVNS) has been shown to reduce infarct volume and improve neurological function [10]. Studies have indicated that the protective effect of VNS after ischemic stroke is associated with modulation of nicotinic acetylcholine receptors (nAChRs) [11]. nAChRs are ion channels composed of five subunits that are widely expressed throughout the peripheral and central nervous systems [12]. In mammalian brain, the most predominant subtypes of nAChRs are the α4- and α7-nAChRs, which play important roles in memory, learning, pain control, anti-inflammation, and neuroprotection. Activation of nAChRs protects neurons from neurodegenerative diseases [13]. Some studies demonstrated that activation of α7-nAChR could reduce ischemic injury [14,15]. However, there is limited studies mentioned that α4-nAChR play the role in ischemic stroke. In this study, we aimed to investigate the protective effects and mechanisms of action by aVNS in a murine acute cerebral ischemic stroke injury model, with a special focus on the involvement of α4-nAChR and the possible molecular signaling mediated by aVNS. Materials And Methods Animal preparation and grouping Adult male ICR mice weighing 28-30 g (National Laboratory Animal Breeding and Research Center, Taipei, Taiwan) were used in this experiment, and the protocol was reviewed and approved by the Animal Research Committee of the National Research Institute of Chinese Medicine (approval number: NRICM-IACUC-110-912-2). All mice were fed in controlled conditions (12 h light/dark cycle with humidity of 60-70%, 21-23 °C) with free access to food and water. Mice were randomly assigned into 5 groups: (1) Sham operated group (Sham), (2) aVNS group, (3) cerebral ischemia reperfusion (CIR) group, (4) CIR + aVNS group, (5) DHβE + CIR + aVNS group, which received dihydro-β-erythroidine hydrobromide ( DHβE ), an alpha 4 nicotinic receptor antagonist. Middle cerebral artery occlusion/reperfusion (MCAO/R) model Mice were anesthetized with a mixture of 1.5-2% isoflurane and oxygen. After anesthesia, the midline of the neck was incised to expose the common carotid artery, external carotid artery, and internal carotid artery. Cerebral ischemia reperfusion (CIR) injury was induced using a heat-blunted nylon monofilament surgical suture (diameter approx. 100 μm) coated with silicone, which was introduced into the exposed external carotid artery, advanced to the internal carotid artery, and inserted into the circle of Willis to occlude the right middle cerebral artery (MCA), thereby interrupting the flow of blood through the MCA. The filament was left in place for 40 min under anesthesia. After 40 min of occlusion under anesthesia, the filament was withdrawn to cause reperfusion of the brain. After surgery, the incision was sutured and sterilized, and the mice were placed on a heating plate to maintain body temperature until they awoke and were transferred to a cage [16]. Auricular vagus nerve stimulation (aVNS) Twenty-four hours before the MCA occlusion surgery to induce ischemic stroke, the mice were anesthetized with a mixture of 1.5-2% isoflurane and oxygen, after which they were placed on a plastic plate to prevent displacement. Then two acupuncture needles (32-gauge, J.D. Acupuncture Instrument Co., Taiwan) were inserted 0.5-1.0 mm under the skin over the auricle at an interval of 0.5-1.0 mm. For the Sham group, the needles were inserted into the auricle without electrical stimulation, whereas for the CIR + aVNS and DHβE + CIR + aVNS groups, the needles were inserted into the auricle and connected to an electrical stimulator (Ching Ming Tens - D0207kl, Ching Ming Medical Device Co., Taiwan) for the first cycle of aVNS treatment. For the first cycle of aVNS, the intensity was set to 0.5 mA at a fixed frequency of 25 Hz with 0.3 millisecond square pulses for 2 minutes, which was repeated every 5 minutes for a total of 5 cycles. Twenty-four hours after the first cycle of aVNS, mice were anesthetized for MCA occlusion to induce acute ischemic stroke. The second cycle of aVNS was immediately administered after reperfusion. For the second cycle of aVNS, the intensity was set to 0.25 mA at a fixed frequency of 25 Hz with 0.3 millisecond square pulses for 2 minutes, which was repeated every 5 minutes for a total of 2 cycles. In the aVNS group, the second cycle of aVNS treatment was administered 24 hours after the first cycle of aVNS. After the electrical stimulation, the mice were placed on a heating plate to maintain body temperature until they awoke and were transferred to a clean cage [9, 17]. Assessment of neurological deficit and analysis of survival rate The neurological function of the mice was assessed by analyzing their tracking distance and tracks within 3 minutes in a behavioral observation box (50 x 50 x 50 cm 3 ) 24 h after the ischemic stroke induction. The results were then analyzed using a video-tracking system (SMART v2.5.21, Panlab, Spain). Survival rates were calculated immediately (day 0) and at 24 h (day 1) after stroke induction. Evaluation of infarct volume Twenty-four hours after ischemic stroke injury, mice were sacrificed using carbon dioxide, and the whole brain was quickly removed and sliced into five consecutive 1.5-mm-thick coronal slices (bregma 4 to -3.5 mm) for staining with 0.5% 2,3,5-triphenyltetrazoliumchloride (TTC) (Sigma-Aldrich, St. Louis, MO, USA). All brain slices were then fixed in 10% paraformaldehyde (at 4 °C for 24 hours), and the percentage of infarct volume in the whole brain was calculated in software (AlphaEaseFC software, version 4.0, Alpha Innotech Corporation, CA, USA) [16]. Western blotting Equal amounts (20-40 μg) of protein from the ischemic cerebral hemispheres (right brain) of each treatment group were subjected to 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electron transferred to hydrophobic polyvinylidene difluoride (PVDF) membranes. After blocking, the membrane was incubated overnight at 4 °C with appropriate antibodies against pP65NF-κB (1:500, BD PharMingen, San Diego, CA, USA), phospho Ser9 GSK3β (SpGSK3, 1:500, Santa Cruz Biotechnology, Europe), β-catenin (1:1000), doublecortin (DCX, 1:2000), Bcl-2 (1:1000) (all from Abcam, Cambridge, UK), a4-nAChR (1:500, Biorbyt, Cambridge, UK), phospho S473 Akt (pAkt, 1:1000, Millipore, CA, USA), and b-actin (1:5000, Sigma-Aldrich). Following the incubation with appropriate secondary antibodies, the immunoblots on the membranes were visualized after imaging with an enhanced chemiluminescence (ECL) system and quantified in imaging software (AlphaEaseFC software, version 4.0, Alpha Innotech Corporation, CA, USA) [16]. Immunohistochemical staining Twenty-four hours after the induction of ischemic stroke, the brains were prepared for immunohistochemical staining using confocal imaging equipment. Briefly, six consecutive brain slices (thickness, 20 μm) were cut and collected at the same rostrocaudal level (bregma -1.5 to -1.9 mm) from mice in different groups. After fixation, permeabilization, and blocking, the brain slices were randomly selected with appropriate first antibodies against pP65NF-κB (1:50, BD PharMingen, San Diego, CA, USA), active form caspase 3 (1:50, Santa Cruz Biotechnology, Europe), phospho Ser9 GSK3β (SpGSK3, 1:50, Novus Biologicals, CO, USA), β-catenin (1:50), doublecortin (DCX, 1:1000) (both from Abcam, Cambridge, UK), and a4-nAChR (1:50, Biorbyt, Cambridge, UK) in PBS containing 3% albumin at 4 °C overnight. Following washing, sections were incubated with AlexaFluor®488-, AlexaFluor®555- or AlexaFluor®647-conjugated secondary antibodies (Cell Signaling Technology Inc., MA, USA). All coverslips were mounted with mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) to stain the DNA in the nuclei of the cells, and sections of tissue were examined using a laser-scanning confocal microscope (Zeiss LSM780; Carl Zeiss, Jena, Germany). The distribution and numbers of immuno-positive stained cells were determined and quantified over the entire field (100X) of the selected images. Statistical analysis All results in this study are presented as the mean ± SEM, and the differences between each group were analyzed using one-way analysis of variance (ANOVA) followed by the post hoc Newman-Keuls test for multiple comparisons. Values of p < 0.05 were considered significant. Survival rates was analyzed using Log-Rank test followed by the Holm-Sidak method. Values of p < 0.05 were considered significant. Results The effect of aVNS on heart rate and salivary amylase activity In this study, we established an animal model of aVNS manipulation ( Fig. 1A ). To confirm the successful activation of the vagus nerve in this model, we measured two parameters: heart rate [18] and salivary amylase activity of mice after aVNS manipulation [19, 20]. Our results showed that the average heart rate of the mice was 441 ± 19 (bpm) before aVNS, decreased significantly to 260 ± 17 (bpm) during the 2-minute aVNS manipulation, and returned to the normal range of 448 ± 18 (bpm) immediately after cessation of aVNS ( Fig. 1B ). For salivary amylase activity assay, the mean salivary amylase activity of mice after aVNS was 2-folds higher than that before aVNS ( Fig. 1C ). These data showed that the aVNS manipulation established in this study activated the vagus nerve. The effect of aVNS on the survival rate To investigate whether the aVNS was beneficial to survival in the ischemic stroke mouse model, the survival rate of mice was assessed at 24 h after ischemic stroke. Our results showed that the survival rates at 24 h after ischemic stroke were approximately 41% in the CIR group, 60% in the CIR + aVNS group, and 42% in the DHβE + CIR + aVNS group ( Supplementary Table 1 ). It was noticed that the survival rate of ischemic stroke mice treated with aVNS manipulation was almost 20% higher at 24 h after ischemic stroke, and this effect was reversed by α 4- nAChR antagonists. The effect of aVNS on cerebral infarct volume and neurological deficits To determine the severity of brain damage and neurological deficits, the cerebral infarct volume was measured at 24 h after stroke by TTC staining and the neurological deficits were assessed by open field test to observe the tracking distance and tracks of the animals over a three-minute observation period. TTC staining showed that there was no detectable cerebral infarction in the sham and aVNS only groups ( Fig. 2A ), whereas in the CIR group, the infarct volume accounted for approximately 35% of the total brain volume, reflecting the severity of the infarction induced by MCA occlusion. In the CIR + aVNS group, a significant reduction to around 20% of the total brain volume was found ( Fig. 2B ). The results of the open field test showed that the mice from the sham and aVNS groups had a normal tracking distance and wide distribution of tracks; in contrast, the CIR group showed a reduction in tracking distance as compared to the sham and aVNS groups, and their tracks both were restricted to a certain area and rotated in a clockwise direction ( Fig. 2C ). The tracking distance of the CIR + aVNS group increased significantly, the distribution of tracks covered a wider area than that of the CIR group, and the rotation activity was lower ( Fig. 2D ). In addition, administration of DHβE to aVNS treated ischemic stroke mice reversed the protective effects of aVNS on brain damage and mobility of the mice. These results suggested that aVNS could significantly reduce the infarct volume and improve the neurological deficits in this ischemic stroke model, and this beneficial effect by aVNS could mediate the α 4 nicotinic acetylcholine receptor dependent mechanisms. The effect of aVNS on changes of inflammatory and apoptotic factors after cerebral ischemic reperfusion injury in mice Stroke-induced brain injury is closely related to the inflammatory responses and apoptosis induced by increased pP65NF-κB and active form caspase 3 [21]. In this study, we investigated the expression level of pP65NF-κB and cleaved (active) form caspase 3 in each group. Immunohistochemical staining showed that pP65NF-κB ( Fig. 3A ) and active form caspase 3 ( Fig. 3B ) were widely expressed throughout the brain in the CIR group, while the CIR + aVNS group showed lower expression levels of pP65NF-κB and active form caspase 3 as compared with the CIR group ( Fig. 3C-F ). The sham and aVNS groups did not express pP65NF-κB and active form caspase 3 due to the absence of ischemic stroke induction in these two groups. In addition, the pP65 positive cells and active form caspase 3 positive cells in the cortex or hippocampal dentate gyrus of the CIR + aVNS group were significantly lower than those in the CIR group. Western blotting also confirmed the low levels of pP65NF-κB expression in the sham and aVNS groups, while in the CIR group, it showed significantly higher levels of pP65NF-κB as compared to the CIR + aVNS group due to the severity of brain injury. Administration of DHβE to aVNS treated ischemic stroke mice reversed the effects of aVNS on inflammatory responses ( Supplementary Fig. 1 ). Based on these results, we suggest that aVNS manipulation could reduce the expression level of inflammatory factors after cerebral ischemic reperfusion injury. The effect of aVNS on changes in neuronal regeneration factors after cerebral ischemic reperfusion injury in mice To explore the effect of aVNS on the expression level of neuronal regeneration factors, the numbers of doublecortin (DCX) expression-positive (DCX+) cells [22] in the hippocampal dentate gyrus in different groups were determined. Immunohistochemical staining showed that the mean number of DCX+ cells was 58 ± 3 in the aVNS group and 31 ± 2 in the sham group ( Supplementary Fig. 2 ). The mean number of DCX+ cells in the aVNS group was significantly higher than that in the sham group. However, the CIR group showed less DCX+ cells than the sham, aVNS and CIR + aVNS groups, and the dendrites of the DCX+ cells were atrophic ( Fig. 4A ). Western blotting showed strong and comparable expression levels of DCX in both the sham and aVNS groups. However, a lower DCX expression level found in the CIR group was significantly lower than that in the CIR + aVNS group ( Fig 4B,C ). Administration of DHβE to aVNS treated ischemic stroke mice reversed the effects of aVNS on neuronal regeneration ( Supplementary Fig. 3 ). These results showed that aVNS could stimulate the expression level of neuronal regeneration factor DCX. aVNS could protect mouse against ischemic stroke by activating α4-nAChR To explore whether the activation of α4-nAChR signaling was involved in the protective effect of aVNS in this model, immunohistochemical staining of the brain tissues was performed. Our results showed that α4-nAChR were both extensively expressed around all brain regions in the sham and aVNS groups; however, ischemic stroke (CIR group) significantly reduced the expression of both α4-nAChRs, and this reduction could be considerably reversed by treatment with aVNS (CIR + aVNS group) ( Fig. 5A ). In addition, the α4-nAChR positive cells in the cortex or hippocampal dentate gyrus of the CIR + aVNS group were significantly increased as compared to the CIR group ( Fig. 5B,C ). Western blotting also confirmed the comparable results mentioned above ( Fig. 5D,E ). According to these results, we suggest that aVNS manipulation could display its powerful anti-inflammatory and protective effects in this ischemic stroke model through activation of α4-nAChR signaling. Possible signal transduction pathways involved in the aVNS protective effect on cerebral ischemic reperfusion injury in mice To further examine whether the protective effect of aVNS in mice after cerebral ischemic injury was associated with the GSK3β/β-catenin pathway [16], we assessed the expression levels of SpGSK3 (a GSK3 inhibitory form that is protective) and β-catenin in mice at 24 h after ischemic stroke. Immunohistochemical staining revealed that the CIR group showed significant reductions in SpGSK3 and β-catenin expression as compared to the sham and aVNS groups. However, aVNS treatment significantly increased SpGSK3 and β-catenin expression (CIR + aVNS group) ( Fig. 6A,B ). In addition, the SpGSK3 positive cells and β-catenin positive cells in the cortex or hippocampal dentate gyrus of the aVNS treatment group (CIR + aVNS group) were significantly increased as compared to the CIR group ( Fig. 6 C-F ). Western blotting also confirmed the comparable results mentioned above ( Fig. 6G,H ). Administration of DHβE to aVNS treated ischemic stroke mice reduced the protein expression of SpGSK3 and β-catenin expression ( Supplementary Fig. 4 ). In addition, pAkt (an upstream signal of SpGSK3) and Bcl-2 (a downstream protein of β-catenin) were expressed less in the CIR group than in the sham group, and the expression was higher in the aVNS treatment group (CIR + aVNS group). There was a significant difference between the CIR and CIR + aVNS groups for pAkt and Bcl-2 ( Supplementary Fig. 5 ). Furthermore, co-staining of SpGSK3 (protective factor) with the inflammatory factor p65NF-κB or the apoptotic protein active form caspase 3 revealed that SpGSK3 was present in areas where p65NF-κB and caspase 3 were less expressed ( Supplementary Fig. 6,7 ). These results suggested that GSK3β/β-catenin signaling might underlie the protective effects induced by aVNS manipulation in a mouse model of cerebral ischemic stroke. Discussion Recently, VNS has been reported to provide protection against cerebral ischemic injury in rats [23-25]. The outcome of VNS treatment is closely related to its specific parameter settings. Therefore, exploration of the most appropriate stimulation parameters is important. In this study, the animal model of the aVNS manipulation method was a modification and improvement based on the conditions reported in previous studies [9, 25, 26]. In our study, the two aVNS treatment time points with different stimulation intensity were set at 24 h before MCA occlusion surgery and immediately after reperfusion. During the optimization of the aVNS protocol, different treatment conditions were explored, including performing aVNS 24 h before MCA occlusion surgery (0.5 mA, 25 Hz, duration 2 minutes, repeated every 5 minutes, 5 cycles), performing aVNS immediately after reperfusion (0.5 mA, 25 Hz, duration 2 minutes, repeated every 5 minutes, 5 cycles) or two aVNS treatment time points with the same stimulation intensity (0.5 mA, 25 Hz, duration 2 minutes, repeated every 5 minutes, 5 cycles). However, these aVNS stimulation conditions did not provide effective improvements of neurological deficits in this stroke model. Although the two aVNS treatment time points with the same stimulation intensity improved neurological deficits, animals died during stimulation, possibly because animals were weaker after MCA occlusion surgery ( Supplementary Fig. 8 ). Finally, we choose that the aVNS stimulation conditions (as described method section) displaying the best protective effect to be adopted in the acute cerebral ischemic stroke mouse model. Interestingly, we found that the first session of electrostimulation is necessary to, somehow, sensitize the system to the effectiveness of the second electrostimulation session. Regardless, the reasons behind the sensitization produced by the pre-exposure to aVNS require further experimental investigation. This is the first report to demonstrate that treatment with aVNS is neuroprotective for acute ischemic brain damage in mice. Previous studies have indicated that heart rate decreases significantly when VNS is performed [18, 27] and that norepinephrine concentrations increase with continued VNS [19]. In this study, we found significant changes in heart rate. In addition, it has been reported that increased activity of norepinephrine could be associated with increases in salivary amylase activity [20]. This study confirmed utility of a change in salivary amylase activity as another indicator for detecting the activation of the vagus nerve. These two non-invasive parameters (change in heart rate and salivary amylase activity) could be used to determine the activation of the vagus nerve and avoid the influences of invasive methods on animals. After acute cerebral ischemic stroke injury, a series of cellular damage reactions occur, including the inflammatory response that plays a key role in ischemic injury. The results of this study found that aVNS reduced the expression of inflammatory factors (pP65NF-κB) and reduced the cerebral infarct volume, with results comparable to those of a previous study [24]. It has been reported that VNS could reduce the expression of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) at 24 h after reperfusion, and these anti-inflammatory and molecular mechanisms could partly be attributed to the enhanced release of acetylcholine [28], a neurotransmitter released mainly from the vagus nerve terminals. This result indicates that acetylcholine activates acetylcholine receptors to inhibit the release of pro-inflammatory cytokines, thereby preventing tissue damage. Therefore, it is reasonable to propose that aVNS manipulation could protect the brain from acute ischemic damage by activating acetylcholine receptors to inhibit inflammatory responses. Essentially, it has been reported that aVNS protects the brain from ischemic injury by activating acetylcholine receptors [28], and the most abundant nicotinic acetylcholine receptors (nAChR) in the mammalian brain are the α4- and α7-nAChRs [29], which have been shown to be important in mediating anti-inflammatory and neuroprotective actions. Activation of these receptors may enhance neuronal resistance to ischemic or other types of injury. The results of this study showed that acute cerebral ischemic stroke mice treated with aVNS manipulation had an increased survival rate, reduced neurological deficits and reduced cerebral infarct volume. However, the survival rates of the groups treated with aVNS and administration of α4-nAChR antagonist for ischemic stroke (DHβE + CIR + aVNS group) was lower than that of the CIR + aVNS group, and the neurological function and cerebral infarct volume were both severe. Although the DHβE + CIR + aVNS groups was treated with aVNS, their therapeutic effects were blocked by the antagonists of α4-nAChRs. Therefore, we propose that our aVNS manipulation is protective against acute cerebral ischemic stroke by activation of α4-nAChRs, which increases survival rate, reduces neurological deficits and reduces cerebral infarct volume. Glutamate neurotoxicity is involved in a variety of neurodegenerative diseases, including ischemic stroke, trauma, Alzheimer's disease and Parkinson's disease. In addition to neuronal death, neuroinflammatory responses are induced in these diseases. Previous studies have suggested that nicotinic acetylcholine receptors play an important role in the survival of neurons in the central nervous system during excitotoxicity and neuroinflammatory responses, and it has been suggested that the PI3K/Akt signaling pathway is involved in this protective mechanism [30, 31]. The PI3K/Akt pathway plays an important role in cell development, function and survival [32], and its downstream signal GSK3 has a neuroprotective effect on ischemic stroke by inhibiting its activity (with increased expression of SpGSK3) [33]. Furthermore, neurogenesis as an endogenous capacity for self-repair is triggered in certain pathological conditions such as ischemic injury. Ischemic stroke leads to the activation of several processes of endogenous self-repair and neuronal burst response [34, 35]. Many studies demonstrated the contribution of post‐stroke neurogenesis in functional recovery for review see [36]. Therefore, from evaluating the anti-apoptotic and neuroprotective mechanisms of aVNS in this murine model of acute cerebral ischemic stroke, we found that the expression levels of pAkt and SpGSK3 in the aVNS treated group was higher than those in the untreated CIR group, and the expression level of active form caspase 3 was lower in the aVNS treated group, while the expression level of β-catenin and its downstream proteins Bcl-2 and DCX were both higher in the aVNS treated group than in the untreated CIR group. It has been suggested that neuroblast markers such as DCX were found to be increased after ischemic injury, however, most of newborn neurons fail to survive over long‐term due to absence of trophic factors [37, 38]. In the present study, we found a reduction in DCX in the hippocampal dentate gyrus 24 h after stroke. Moreover, we found that treatment of stroke with aVNS enhanced the survival of newborn DCX and prevented them from death 24h after stroke. Therefore, we propose that aVNS may reduce the ischemic stroke-induced inflammatory responses and apoptosis and increase the expression level of neuroprotective factors (such as Bcl-2) and reduction of damaging factors (such as activated caspase 3) through activation of the Akt/GSK3/catenin pathway. Our study successfully demonstrates that the aVNS manipulation is neuroprotective in this murine model of acute ischemic stroke; however, several limitations must be noted. First, the therapeutic effect of aVNS manipulation in the later stages of ischemic stroke should be considered, and second, the time point of aVNS intervention in this study may be different from the clinical situation, as many patients are treated post-stroke in the clinic. Third, the efficacy profile of aVNS in this study may be different from that we have recently demonstrated in a migraine model in rats. These limitations will be addressed in later works. In conclusion, our results provide clear evidence showing that treatment with auricular VNS is neuroprotective for acute ischemic stroke mice by activating α4-nAChRs and inhibition of the GSK3β activity, which in turn leads to activation of β-catenin. Declarations Ethics statement The animal study was reviewed and approved by the Animal Research Committee of the National Research Institute of Chinese Medicine Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All authors declare no conflict of interest. Author Contributions Yen Jiin-Cherng, Chang Cher-Chia and Shen Yuh-Chiang participated in research design, conducted the experiments and wrote or contributed to the writing of the manuscript. Liou Kuo-Tong, Wang Yea-Hwey and Hsu Chih-hung contributed to preparation of animal surgery and chemical reagents, and analytic tools. Funding This study was supported, in part, by grants from the Ministry of Science and Technology, R.O.C.; Division of Neurovascular Disease, Neurological Institute, Taipei Veterans General Hospital; School of Medicine, National Yang-Ming University, Taiwan; and the National Research Institute of Chinese Medicine for Dr. Yen J.C., Dr. Chern C.M., Dr. Liou K.T., and Dr. Shen Y.C. (MOST 108-2320-B-077-003-MY3, MOST 110-2314-B-A49A-544, MOHW 111-NRICM-M-325-12240). Acknowledgments We would like to thank Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University for providing technical support. We also thank National Research Institute of Chinese Medicine, Ministry of Health and Welfare for technical support. References Catanese L, Tarsia J, Fisher M. Acute Ischemic Stroke Therapy Overview. Circ Res. 2017;120(3):541-558. Peña ID, Borlongan C, Shen G, Davis W. Strategies to Extend Thrombolytic Time Window for Ischemic Stroke Treatment: An Unmet Clinical Need. 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Activation of α-7 nicotinic acetylcholine receptor reduces ischemic stroke injury through reduction of pro-inflammatory macrophages and oxidative stress [published correction appears in PLoS One. 2014;9(8):e105711. Gaidhani N, Tucci FC, Kem WR, Beaton G, Uteshev VV. Therapeutic efficacy of α7 ligands after acute ischaemic stroke is linked to conductive states of α7 nicotinic ACh receptors. Br J Pharmacol. 2021;178(7):1684-1704. Chien MY, Chuang CH, Chern CM, et al. Salvianolic acid A alleviates ischemic brain injury through the inhibition of inflammation and apoptosis and the promotion of neurogenesis in mice. Free Radic Biol Med. 2016;99:508-519. Liu AF, Zhao FB, Wang J, et al. Effects of vagus nerve stimulation on cognitive functioning in rats with cerebral ischemia reperfusion. J Transl Med. 2016;14:101. Buschman HP, Storm CJ, Duncker DJ, Verdouw PD, van der Aa HE, van der Kemp P. Heart rate control via vagus nerve stimulation. Neuromodulation. 2006;9(3):214-220. Follesa P, Biggio F, Gorini G, et al. Vagus nerve stimulation increases norepinephrine concentration and the gene expression of BDNF and bFGF in the rat brain. Brain Res. 2007;1179:28-34. Warren CM, van den Brink RL, Nieuwenhuis S, Bosch JA. Norepinephrine transporter blocker atomoxetine increases salivary alpha amylase. Psychoneuroendocrinology. 2017;78:233-236. Liang J, Luan Y, Lu B, Zhang H, Luo YN, Ge P. Protection of ischemic postconditioning against neuronal apoptosis induced by transient focal ischemia is associated with attenuation of NF-κB/p65 activation. PLoS One. 2014;9(5):e96734. Biggio F, Gorini G, Utzeri C, et al. Chronic vagus nerve stimulation induces neuronal plasticity in the rat hippocampus. Int J Neuropsychopharmacol. 2009;12(9):1209-1221. Sun Z, Baker W, Hiraki T, Greenberg JH. The effect of right vagus nerve stimulation on focal cerebral ischemia: an experimental study in the rat. Brain Stimul. 2012;5(1):1-10. Ay I, Lu J, Ay H, Gregory Sorensen A. Vagus nerve stimulation reduces infarct size in rat focal cerebral ischemia. Neurosci Lett. 2009;459(3):147-151. Ay I, Napadow V, Ay H. Electrical stimulation of the vagus nerve dermatome in the external ear is protective in rat cerebral ischemia. Brain Stimul. 2015;8(1):7-12. Ma J, Zhang L, He G, Tan X, Jin X, Li C. Transcutaneous auricular vagus nerve stimulation regulates expression of growth differentiation factor 11 and activin-like kinase 5 in cerebral ischemia/reperfusion rats. J Neurol Sci. 2016;369:27-35. Morais A, Liu TT, Qin T, et al. Vagus nerve stimulation inhibits cortical spreading depression exclusively through central mechanisms. Pain. 2020;161(7):1661-1669. Jiang Y, Li L, Liu B, Zhang Y, Chen Q, Li C. Vagus nerve stimulation attenuates cerebral ischemia and reperfusion injury via endogenous cholinergic pathway in rat. PLoS One. 2014;9(7):e102342. Dani JA. Neuronal Nicotinic Acetylcholine Receptor Structure and Function and Response to Nicotine. Int Rev Neurobiol. 2015;124:3-19. Kihara T, Shimohama S, Sawada H, et al. alpha 7 nicotinic receptor transduces signals to phosphatidylinositol 3-kinase to block A beta-amyloid-induced neurotoxicity. J Biol Chem. 2001;276(17):13541-13546. Shaw S, Bencherif M, Marrero MB. Janus kinase 2, an early target of alpha 7 nicotinic acetylcholine receptor-mediated neuroprotection against Abeta-(1-42) amyloid. J Biol Chem. 2002;277(47):44920-44924. Zhang L, Qu Y, Tang J, et al. PI3K/Akt signaling pathway is required for neuroprotection of thalidomide on hypoxic-ischemic cortical neurons in vitro. Brain Res. 2010;1357:157-165. Chuang DM, Wang Z, Chiu CT. GSK-3 as a Target for Lithium-Induced Neuroprotection Against Excitotoxicity in Neuronal Cultures and Animal Models of Ischemic Stroke. Front Mol Neurosci. 2011;4:15. Lindvall O, Kokaia Z. Neurogenesis following Stroke Affecting the Adult Brain. Cold Spring Harb Perspect Biol. 2015;7(11):a019034. Lu J, Manaenko A, Hu Q. Targeting Adult Neurogenesis for Poststroke Therapy. Stem Cells Int. 2017;2017:5868632. Rahman AA, Amruta N, Pinteaux E, Bix GJ. Neurogenesis After Stroke: A Therapeutic Perspective. Transl Stroke Res. 2021;12(1):1-14. Ruan L, Wang B, ZhuGe Q, Jin K. Coupling of neurogenesis and angiogenesis after ischemic stroke. Brain Res. 2015;1623:166-173. Cuartero MI, García-Culebras A, Torres-López C, et al. Post-stroke Neurogenesis: Friend or Foe?. Front Cell Dev Biol. 2021;9:657846. Supplementary Files Supplementarymaterial.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-2662844\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":187949181,\"identity\":\"f0549c75-5e1d-4f97-b2e9-40f8acf8e04e\",\"order_by\":0,\"name\":\"Cher-Chia Chang\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYHACxgcfDP7J8bM3ANkGFkRpYTacUXHAWLLnAEiLBFFa2KQ5zhxI3HAjAcQhQot8++lkY8a2O4wzZz6/uuFHgQQDf3t3Al4tBmdyNz4ubHvGzC+dU3azB+gwiTNnN+DXwpC72XhmGzOb5OyctBs8QC0GErn4tcj3v90mzdvGzGNw80zazT/EaGG4kbtNmufMYQmDG+zHbhNli8GNt5uBgZxmINmTw3ZbxkCCh6Bf5PtzNwKj0qa+n/34s5tv/tjI8bf3EnAYAvAYgElilYMA+wNSVI+CUTAKRsEIAgAYkE3/w61IeQAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0000-0002-8300-348X\",\"institution\":\"National Yang Ming Chiao Tung University School of Medicine\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Cher-Chia\",\"middleName\":\"\",\"lastName\":\"Chang\",\"suffix\":\"\"},{\"id\":187949182,\"identity\":\"7a1a69a0-db35-4d90-94c5-921754017788\",\"order_by\":1,\"name\":\"Kuo-Tong Liou\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"National Research Institute of Chinese Medicine\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kuo-Tong\",\"middleName\":\"\",\"lastName\":\"Liou\",\"suffix\":\"\"},{\"id\":187949183,\"identity\":\"f338dc5e-1519-45ad-b93a-9fe82360c339\",\"order_by\":2,\"name\":\"Yea-Hwey Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"National Taipei University of Nursing and Health Sciences\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yea-Hwey\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":187949184,\"identity\":\"8062f317-60f6-447c-afb2-bcc42483e6c7\",\"order_by\":3,\"name\":\"Chih-hung Hsu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"National Research Institute of Chinese Medicine\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Chih-hung\",\"middleName\":\"\",\"lastName\":\"Hsu\",\"suffix\":\"\"},{\"id\":187949185,\"identity\":\"039c86be-5db0-4843-9bc7-c5b26d61ee9c\",\"order_by\":4,\"name\":\"Yuh-Chiang Shen\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0003-0895-1428\",\"institution\":\"National Research Institute of Chinese Medicine\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuh-Chiang\",\"middleName\":\"\",\"lastName\":\"Shen\",\"suffix\":\"\"},{\"id\":187949186,\"identity\":\"8aaf3271-e5a2-45ab-8fe1-ae218a89f9bf\",\"order_by\":5,\"name\":\"Jiin-Cherng Yen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"National Yang Ming Chiao Tung University School of Medicine\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jiin-Cherng\",\"middleName\":\"\",\"lastName\":\"Yen\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2023-03-07 00:00:31\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-2662844/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-2662844/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":35206727,\"identity\":\"d23b17e0-ecd4-4255-885b-cb700543e0db\",\"added_by\":\"auto\",\"created_at\":\"2023-04-03 14:07:08\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":140317,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExperimental design. \\u003cstrong\\u003e(A) \\u003c/strong\\u003eTimeline of the experimental design. After anesthesia, the mice received a total of 5 aVNS sessions of 2 minutes each with stimulation every 5 minutes. 24 hours after aVNS, ischemic stroke was induced by MCAO surgery, and after 40 minutes of occlusion, the filament was withdrawn to achieve reperfusion. After reperfusion, the mice received 2 aVNS sessions of 2 minutes each with stimulation every 5 minutes. Animal behavior was observed 24 hours after MCAO and brain tissue was extracted after sacrifice of the animals for Western blotting, TTC staining and IHC staining. \\u003cstrong\\u003e(B)\\u003c/strong\\u003eChanges in heart rate before, during and after aVNS.\\u003cstrong\\u003e (C)\\u003c/strong\\u003e Changes in salivary amylase activity before and after aVNS. Data are mean ± SEM (N=3-5). * p \\u0026lt; 0.05 versus before aVNS.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2662844/v1/1cc3774652650b648b3edfe7.jpg\"},{\"id\":35206730,\"identity\":\"16246ce9-9509-4527-8f98-1a68851a1b80\",\"added_by\":\"auto\",\"created_at\":\"2023-04-03 14:07:08\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":386101,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe effect of aVNS on the infarct volume and neurological deficits after ischemic stroke injury. \\u003cstrong\\u003e(A) \\u003c/strong\\u003eTTC staining was used to observe the infarct volume after ischemic stroke injury. \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Statistical results of TTC staining. Data are mean ± SEM (N=10 for Sham group, N=5 for aVNS group, N=4 for CIR group, N=6 for CIR + aVNS group, N=5 for DHβE + CIR + aVNS group). * p \\u0026lt; 0.05 versus CIR group. # p \\u0026lt; 0.05 versus CIR + aVNS group. \\u003cstrong\\u003e(C) \\u003c/strong\\u003eA three-minute behavior recording of the animal in the open field test was used to assess neurological deficits. \\u003cstrong\\u003e(D) \\u003c/strong\\u003eStatistical results of the neurological deficits were analyzed by measuring the tracking distance for 3 minutes. Data are mean ± SEM (N=15 for Sham group, N=6 for aVNS group, N=10 for CIR group, N=9 for CIR + aVNS group, N=4 for DHβE + CIR + aVNS). * p \\u0026lt; 0.05 versus CIR group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2662844/v1/7cc9540a8174c71e28f132a9.jpg\"},{\"id\":35207864,\"identity\":\"f812c35b-14b6-412c-8b64-565e787038a1\",\"added_by\":\"auto\",\"created_at\":\"2023-04-03 14:15:08\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":468156,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe effect of aVNS on inflammatory factors and apoptotic proteins after ischemic stroke injury. \\u003cstrong\\u003e(A)\\u003c/strong\\u003e Representative images showing comparison of pP65NF-κB (red, a marker of inflammation) and DAPI (blue, a marker of the nucleus) in the whole brain, cortex and hippocampal dentate gyrus of each group. The white arrows indicate the distribution of cells showing positive staining for pP65NF-κB (red). \\u003cstrong\\u003e(B) \\u003c/strong\\u003eQuantitative results of immunohistochemical staining in cortex and \\u003cstrong\\u003e(C) \\u003c/strong\\u003edentate gyrus. Data are mean ± SEM (N=5 for CIR group and N=3 for other groups). * p \\u0026lt; 0.05 versus CIR group. \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Representative images showing comparison of active form caspase 3 (red, marker of apoptosis) and DAPI (blue, marker of nucleus) in the whole brain, cortex and hippocampal dentate gyrus of each group. The white arrows indicate the distribution of cells showing positive staining for active form caspase 3 (red).\\u003cstrong\\u003e (E)\\u003c/strong\\u003e Quantitative results of immunohistochemical staining in cortex and \\u003cstrong\\u003e(F)\\u003c/strong\\u003e dentate gyrus. Data are mean ± SEM (N=3 for each group). * p \\u0026lt; 0.05 versus CIR group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2662844/v1/ae49d4293803d5cc379e04dc.jpg\"},{\"id\":35208258,\"identity\":\"01756e0e-0590-4b88-8170-31a7adaa36cc\",\"added_by\":\"auto\",\"created_at\":\"2023-04-03 14:23:08\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":200392,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe effect of aVNS on neurogenesis factor after ischemic stroke injury. \\u003cstrong\\u003e(A)\\u003c/strong\\u003e Representative images showing comparison of the morphologic changes of DCX (yellow) in the hippocampal dentate gyrus after ischemic stroke injury in each group. The white arrow indicates the distribution of DCX positive cell staining. \\u003cstrong\\u003e(B) \\u003c/strong\\u003eProtein expression of doublecortin (DCX) after ischemic stroke injury using Western blotting. \\u003cstrong\\u003e(C) \\u003c/strong\\u003eStatistical results of the Western blotting. Data are mean ± SEM (N=3 for aVNS group, N=4 for other groups). * p \\u0026lt; 0.05 versus CIR group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2662844/v1/2df4e248b1ba62479e8a42a2.jpg\"},{\"id\":35206728,\"identity\":\"6b252feb-438f-42d7-8866-56fc9a571a93\",\"added_by\":\"auto\",\"created_at\":\"2023-04-03 14:07:08\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":435195,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe effect of aVNS on α4 nicotinic acetylcholine receptor after ischemic stroke injury. \\u003cstrong\\u003e(A)\\u003c/strong\\u003e Representative images showing comparison of α4 nicotinic acetylcholine receptor (green) and DAPI (blue, marker of nucleus) in the whole brain, cortex and hippocampal dentate gyrus of each group. The white arrows indicate the distribution of cells showing positive staining for α4 nicotinic acetylcholine receptor (green). \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Quantitative results of immunohistochemical staining in cortex and \\u003cstrong\\u003e(C) \\u003c/strong\\u003edentate gyrus. Data are mean ± SEM (N=3 for each group). * p \\u0026lt; 0.05 versus CIR group. \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Protein expression of α4 nicotinic acetylcholine receptors after ischemic stroke injury using Western blotting. \\u003cstrong\\u003e(E)\\u003c/strong\\u003e Statistical results of the Western blotting. Data are mean ± SEM (N=3 for aVNS group, N=4 for other groups). * p \\u0026lt; 0.05 versus CIR group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2662844/v1/a121e4a51702359bf6077555.jpg\"},{\"id\":35208758,\"identity\":\"89225cab-ef2d-45ff-83b5-1a9ad0689bef\",\"added_by\":\"auto\",\"created_at\":\"2023-04-03 14:31:08\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":720465,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe effect of aVNS on SpGSK3 and β-catenin after ischemic stroke injury. \\u003cstrong\\u003e(A)\\u003c/strong\\u003e Representative images showing comparison of SpGSK3 (green) and DAPI (blue, marker of nucleus) in the whole brain, cortex and hippocampal dentate gyrus of each group. The white arrows indicate the distribution of cells showing positive staining for SpGSK3 (green). \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Quantitative results of immunohistochemical staining in cortex and\\u003cstrong\\u003e (C)\\u003c/strong\\u003e dentate gyrus. Data are mean ± SEM (N=3 for each group). * p \\u0026lt; 0.05 versus CIR group. \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Representative images showing comparison of β-catenin (yellow) and DAPI (blue, marker of nucleus) in the whole brain, cortex and hippocampal dentate gyrus of each group. The white arrows indicate the distribution of cells showing positive staining for β-catenin (yellow).\\u003cstrong\\u003e (E)\\u003c/strong\\u003e Quantitative results of immunohistochemical staining in cortex and \\u003cstrong\\u003e(F) \\u003c/strong\\u003edentate gyrus. Data are mean ± SEM (N=3 for each group). * p \\u0026lt; 0.05 versus CIR group. \\u003cstrong\\u003e(G) \\u003c/strong\\u003eProtein expression of SpGSK3 and β-catenin after ischemic stroke injury using Western blotting. \\u003cstrong\\u003e(H)\\u003c/strong\\u003e Statistical results of the Western blotting. Data are mean ± SEM (N=3 for each group of SpGSK3, N=3 for aVNS group of β-catenin, N=4 for other groups). * p \\u0026lt; 0.05 versus CIR group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2662844/v1/2bdbfea256e060b1222fff19.jpg\"},{\"id\":36296229,\"identity\":\"6468c205-d8b4-4093-8655-71e293fe1ab5\",\"added_by\":\"auto\",\"created_at\":\"2023-04-25 22:40:29\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1023794,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2662844/v1/dc0fc9b7-e46a-473b-907a-4f61d1ca74c6.pdf\"},{\"id\":35206733,\"identity\":\"d63bdc70-ea68-4984-afd3-f2776e5e8577\",\"added_by\":\"auto\",\"created_at\":\"2023-04-03 14:07:08\",\"extension\":\"pdf\",\"order_by\":34,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1130059,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarymaterial.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2662844/v1/098a8ad9a32b18db571b5270.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Protective effects and mechanisms of auricular vagus nerve stimulation in the alleviation of acute ischemic cerebral injury in mice\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eStroke is a major cause of death and long-term disability. It also has a high mortality and morbidity rate, resulting in a heavy economic burden on families, society and the government. Ischemic stroke is mainly caused by obstruction of the artery vessels due to thrombus [1] and leads to a series of oxidative, biochemical and hormonal reactions triggered by loss of glucose and oxygen supply, ultimately leading to blood-brain barrier (BBB) disruption. Restoration of blood supply is considered one of the most effective treatments for ischemic stroke. Intravenous thrombolysis with recombinant tissue plasminogen activator (rt-PA) and mechanical thrombectomy have been shown to improve functional outcome. However, rt-PA is available only for some patients due to its limitations, such as a short treatment time window, potential for progression to hemorrhagic stroke and neurotoxicity [2]. And, the disadvantages of using mechanical thrombectomy include technical difficulty with the navigating wire in delicate intracranial vessels, trauma to vessels, distal embolization, vessel dissection and vasospasm leading to worsening of stroke. Therefore, there is still an urgent need to develop new, safe and effective therapies to manage acute ischemic stroke. Although many studies have demonstrated the neuroprotective effects of drug treatments on animal models of ischemic stroke in preclinical trials, their efficacy and safety have yet to be further confirmed [3].\\u003c/p\\u003e\\n\\u003cp\\u003eVagus nerve stimulation (VNS) was approved by the FDA in 1997 for the treatment of epilepsy [4]. VNS is also a potential treatment for migraine, depression, Alzheimer\\u0026apos;s disease and traumatic brain injury [5-8] and is considered to offer protection against ischemic brain injury [9]. Although VNS has become one of the most common non-pharmacological treatments for epilepsy, the effect of VNS on neurological recovery after stroke has not been clearly explained. Recently, transcutaneous electrical stimulation of the auricular branch of the vagus nerve has been shown to partially replicate the effects of traditional cervical vagus nerve stimulation on ischemic stroke. Auricular vagus nerve stimulation (aVNS) has been shown to reduce infarct volume and improve neurological function [10].\\u003c/p\\u003e\\n\\u003cp\\u003eStudies have indicated that the protective effect of VNS after ischemic stroke is associated with modulation of nicotinic acetylcholine receptors (nAChRs) [11]. nAChRs are ion channels composed of five subunits that are widely expressed throughout the peripheral and central nervous systems [12]. In mammalian brain, the most predominant subtypes of nAChRs are the \\u0026alpha;4- and \\u0026alpha;7-nAChRs, which play important roles in memory, learning, pain control, anti-inflammation, and neuroprotection. Activation of nAChRs protects neurons from neurodegenerative diseases [13]. Some studies demonstrated that activation of \\u0026alpha;7-nAChR could reduce ischemic injury [14,15]. However, there is limited studies mentioned that \\u0026alpha;4-nAChR play the role in ischemic stroke.\\u003c/p\\u003e\\n\\u003cp\\u003eIn this study, we aimed to investigate the protective effects and mechanisms of action by aVNS in a murine acute cerebral ischemic stroke injury model, with a special focus on the involvement of \\u0026alpha;4-nAChR and the possible molecular signaling mediated by aVNS.\\u003c/p\\u003e\"},{\"header\":\"Materials And Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnimal preparation and grouping\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAdult male ICR mice weighing 28-30 g (National Laboratory Animal Breeding and Research Center, Taipei, Taiwan) were used in this experiment, and the protocol was reviewed and approved by the Animal Research Committee of the National Research Institute of Chinese Medicine (approval number: NRICM-IACUC-110-912-2). All mice were fed in controlled conditions (12 h light/dark cycle with humidity of 60-70%, 21-23 \\u0026deg;C) with free access to food and water. Mice were randomly assigned into 5 groups: (1) Sham operated group (Sham), (2) aVNS group, (3) cerebral ischemia reperfusion (CIR) group, (4) CIR + aVNS group, (5) DH\\u0026beta;E + CIR + aVNS group, which received dihydro-\\u0026beta;-erythroidine hydrobromide (\\u003cem\\u003eDH\\u0026beta;E\\u003c/em\\u003e), an \\u003cem\\u003ealpha 4\\u003c/em\\u003e\\u003cem\\u003e \\u003c/em\\u003enicotinic receptor antagonist.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMiddle cerebral artery occlusion/reperfusion (MCAO/R) model\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMice were anesthetized with a mixture of 1.5-2% isoflurane and oxygen. After anesthesia, the midline of the neck was incised to expose the common carotid artery, external carotid artery, and internal carotid artery. Cerebral ischemia reperfusion (CIR) injury was induced using a heat-blunted nylon monofilament surgical suture (diameter approx. 100 \\u0026mu;m) coated with silicone, which was introduced into the exposed external carotid artery, advanced to the internal carotid artery, and inserted into the circle of Willis to occlude the right middle cerebral artery (MCA), thereby interrupting the flow of blood through the MCA. The filament was left in place for 40 min under anesthesia. After 40 min of occlusion under anesthesia, the filament was withdrawn to cause reperfusion of the brain. After surgery, the incision was sutured and sterilized, and the mice were placed on a heating plate to maintain body temperature until they awoke and were transferred to a cage [16].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuricular vagus nerve stimulation (aVNS)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwenty-four hours before the MCA occlusion surgery to induce ischemic stroke, the mice were anesthetized with a mixture of 1.5-2% isoflurane and oxygen, after which they were placed on a plastic plate to prevent displacement. Then two acupuncture needles (32-gauge, J.D. Acupuncture Instrument Co., Taiwan) were inserted 0.5-1.0 mm under the skin over the auricle at an interval of 0.5-1.0 mm. For the Sham group, the needles were inserted into the auricle without electrical stimulation, whereas for the CIR + aVNS and DH\\u0026beta;E + CIR + aVNS groups, the needles were inserted into the auricle and connected to an electrical stimulator (Ching Ming Tens - D0207kl, Ching Ming Medical Device Co., Taiwan) for the first cycle of aVNS treatment. For the first cycle of aVNS, the intensity was set to 0.5 mA at a fixed frequency of 25 Hz with 0.3 millisecond square pulses for 2 minutes, which was repeated every 5 minutes for a total of 5 cycles. Twenty-four hours after the first cycle of aVNS, mice were anesthetized for MCA occlusion to induce acute ischemic stroke. The second cycle of aVNS was immediately administered after reperfusion. For the second cycle of aVNS, the intensity was set to 0.25 mA at a fixed frequency of 25 Hz with 0.3 millisecond square pulses for 2 minutes, which was repeated every 5 minutes for a total of 2 cycles. In the aVNS group, the second cycle of aVNS treatment was administered 24 hours after the first cycle of aVNS. After the electrical stimulation, the mice were placed on a heating plate to maintain body temperature until they awoke and were transferred to a clean cage [9, 17].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAssessment of neurological deficit and analysis of survival rate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe neurological function of the mice was assessed by analyzing their tracking distance and tracks within 3 minutes in a behavioral observation box (50 x 50 x 50 cm\\u003csup\\u003e3\\u003c/sup\\u003e) 24 h after the ischemic stroke induction. The results were then analyzed using a video-tracking system (SMART v2.5.21, Panlab, Spain). Survival rates were calculated immediately (day 0) and at 24 h (day 1) after stroke induction.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEvaluation of infarct volume \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwenty-four hours after ischemic stroke injury, mice were sacrificed using carbon dioxide, and the whole brain was quickly removed and sliced into five consecutive 1.5-mm-thick coronal slices (bregma 4 to -3.5 mm) for staining with 0.5% 2,3,5-triphenyltetrazoliumchloride (TTC) (Sigma-Aldrich, St. Louis, MO, USA). All brain slices were then fixed in 10% paraformaldehyde (at 4 \\u0026deg;C for 24 hours), and the percentage of infarct volume in the whole brain was calculated in software (AlphaEaseFC software, version 4.0, Alpha Innotech Corporation, CA, USA) [16].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eWestern blotting\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eEqual amounts (20-40 \\u0026mu;g) of protein from the ischemic cerebral hemispheres (right brain) of each treatment group were subjected to 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electron transferred to hydrophobic polyvinylidene difluoride (PVDF) membranes. After blocking, the membrane was incubated overnight at 4 \\u0026deg;C with appropriate antibodies against pP65NF-\\u0026kappa;B (1:500, BD PharMingen, San Diego, CA, USA), phospho Ser9 GSK3\\u0026beta; (SpGSK3, 1:500, Santa Cruz Biotechnology, Europe), \\u0026beta;-catenin (1:1000), doublecortin (DCX, 1:2000), Bcl-2 (1:1000) (all from Abcam, Cambridge, UK), a4-nAChR (1:500, Biorbyt, Cambridge, UK), phospho S473 Akt (pAkt, 1:1000, Millipore, CA, USA), and b-actin (1:5000, Sigma-Aldrich). Following the incubation with appropriate secondary antibodies, the immunoblots on the membranes were visualized after imaging with an enhanced chemiluminescence (ECL) system and quantified in imaging software (AlphaEaseFC software, version 4.0, Alpha Innotech Corporation, CA, USA) [16].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eImmunohistochemical staining\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwenty-four hours after the induction of ischemic stroke, the brains were prepared for immunohistochemical staining using\\u003cem\\u003e \\u003c/em\\u003econfocal imaging equipment. Briefly, six consecutive brain slices (thickness, 20 \\u0026mu;m) were cut and collected at the same rostrocaudal level (bregma -1.5 to -1.9 mm) from mice in different groups. After fixation, permeabilization, and blocking, the brain slices were randomly selected with appropriate first antibodies against pP65NF-\\u0026kappa;B (1:50, BD PharMingen, San Diego, CA, USA), active form caspase 3 (1:50, Santa Cruz Biotechnology, Europe), phospho Ser9 GSK3\\u0026beta; (SpGSK3, 1:50, Novus Biologicals, CO, USA), \\u0026beta;-catenin (1:50), doublecortin (DCX, 1:1000) (both from Abcam, Cambridge, UK), and a4-nAChR (1:50, Biorbyt, Cambridge, UK) in PBS containing 3% albumin at 4 \\u0026deg;C overnight. Following washing, sections were incubated with AlexaFluor\\u0026reg;488-, AlexaFluor\\u0026reg;555- or AlexaFluor\\u0026reg;647-conjugated secondary antibodies (Cell Signaling Technology Inc., MA, USA). All coverslips were mounted with mounting medium containing 4\\u0026apos;,6-diamidino-2-phenylindole (DAPI) to stain the DNA in the nuclei of the cells, and sections of tissue were examined using a laser-scanning confocal microscope (Zeiss LSM780; Carl Zeiss, Jena, Germany). The distribution and numbers of immuno-positive stained cells were determined and quantified over the entire field (100X) of the selected images.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStatistical analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll results in this study are presented as the mean \\u0026plusmn; SEM, and the differences between each group were analyzed using one-way analysis of variance (ANOVA) followed by the post hoc Newman-Keuls test for multiple comparisons. Values of p \\u0026lt; 0.05 were considered significant. Survival rates was analyzed using Log-Rank test followed by the Holm-Sidak method. Values of p \\u0026lt; 0.05 were considered significant.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe effect of aVNS on heart rate and salivary amylase activity\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn this study, we established an animal model of aVNS manipulation (\\u003cstrong\\u003eFig. 1A\\u003c/strong\\u003e). To confirm the successful activation of the vagus nerve in this model, we measured two parameters: heart rate [18] and salivary amylase activity of mice after aVNS manipulation [19, 20]. Our results showed that the average heart rate of the mice was 441 \\u0026plusmn; 19 (bpm) before aVNS, decreased significantly to 260 \\u0026plusmn; 17 (bpm) during the 2-minute aVNS manipulation, and returned to the normal range of 448 \\u0026plusmn; 18 (bpm) immediately after cessation of aVNS (\\u003cstrong\\u003eFig. 1B\\u003c/strong\\u003e). For salivary amylase activity assay, the mean salivary amylase activity of mice after aVNS was 2-folds higher than that before aVNS (\\u003cstrong\\u003eFig. 1C\\u003c/strong\\u003e). These data showed that the aVNS manipulation established in this study activated the vagus nerve.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe effect of aVNS on the survival rate \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo investigate whether the aVNS was beneficial to survival in the ischemic stroke mouse model, the survival rate of mice was assessed at 24 h after ischemic stroke. Our results showed that the survival rates at 24 h after ischemic stroke were approximately 41% in the CIR group, 60% in the CIR + aVNS group, and 42% in the DH\\u0026beta;E + CIR + aVNS group (\\u003cstrong\\u003eSupplementary Table 1\\u003c/strong\\u003e). It was noticed that the survival rate of ischemic stroke mice treated with aVNS manipulation was almost 20% higher at 24 h after ischemic stroke, and this effect was reversed by \\u003cem\\u003e\\u0026alpha;\\u003c/em\\u003e\\u003cem\\u003e4-\\u003c/em\\u003enAChR antagonists.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe effect of aVNS on cerebral infarct volume and neurological deficits \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo determine the severity of brain damage and neurological deficits, the cerebral infarct volume was measured at 24 h after stroke by TTC staining and the neurological deficits were assessed by open field test to observe the tracking distance and tracks of the animals over a three-minute observation period. TTC staining showed that there was no detectable cerebral infarction in the sham and aVNS only groups (\\u003cstrong\\u003eFig. 2A\\u003c/strong\\u003e), whereas in the CIR group, the infarct volume accounted for approximately 35% of the total brain volume, reflecting the severity of the infarction induced by MCA occlusion. In the CIR + aVNS group, a significant reduction to around 20% of the total brain volume was found (\\u003cstrong\\u003eFig. 2B\\u003c/strong\\u003e). The results of the open field test showed that the mice from the sham and aVNS groups had a normal tracking distance and wide distribution of tracks; in contrast, the CIR group showed a reduction in tracking distance as compared to the sham and aVNS groups, and their tracks both were restricted to a certain area and rotated in a clockwise direction (\\u003cstrong\\u003eFig. 2C\\u003c/strong\\u003e). The tracking distance of the CIR + aVNS group increased significantly, the distribution of tracks covered a wider area than that of the CIR group, and the rotation activity was lower (\\u003cstrong\\u003eFig. 2D\\u003c/strong\\u003e). In addition, administration of DH\\u0026beta;E to aVNS treated ischemic stroke mice reversed the protective effects of aVNS on brain damage and mobility of the mice. These results suggested that aVNS could significantly reduce the infarct volume and improve the neurological deficits in this ischemic stroke model, and this beneficial effect by aVNS could mediate the \\u003cem\\u003e\\u0026alpha;\\u003c/em\\u003e\\u003cem\\u003e4\\u003c/em\\u003e\\u003cem\\u003e \\u003c/em\\u003enicotinic acetylcholine receptor dependent mechanisms.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe effect of aVNS on changes of inflammatory and apoptotic factors after cerebral ischemic reperfusion injury in mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eStroke-induced brain injury is closely related to the inflammatory responses and apoptosis induced by increased pP65NF-\\u0026kappa;B and active form caspase 3 [21]. In this study, we investigated the expression level of pP65NF-\\u0026kappa;B and cleaved (active) form caspase 3 in each group. Immunohistochemical staining showed that pP65NF-\\u0026kappa;B (\\u003cstrong\\u003eFig. 3A\\u003c/strong\\u003e) and active form caspase 3 (\\u003cstrong\\u003eFig. 3B\\u003c/strong\\u003e) were widely expressed throughout the brain in the CIR group, while the CIR + aVNS group showed lower expression levels of pP65NF-\\u0026kappa;B and active form caspase 3 as compared with the CIR group (\\u003cstrong\\u003eFig. 3C-F\\u003c/strong\\u003e). The sham and aVNS groups did not express pP65NF-\\u0026kappa;B and active form caspase 3 due to the absence of ischemic stroke induction in these two groups. In addition, the pP65 positive cells and active form caspase 3 positive cells in the cortex or hippocampal dentate gyrus of the CIR + aVNS group were significantly lower than those in the CIR group. Western blotting also confirmed the low levels of pP65NF-\\u0026kappa;B expression in the sham and aVNS groups, while in the CIR group, it showed significantly higher levels of pP65NF-\\u0026kappa;B as compared to the CIR + aVNS group due to the severity of brain injury. Administration of DH\\u0026beta;E to aVNS treated ischemic stroke mice reversed the effects of aVNS on inflammatory responses (\\u003cstrong\\u003eSupplementary Fig. 1\\u003c/strong\\u003e). Based on these results, we suggest that aVNS manipulation could reduce the expression level of inflammatory factors after cerebral ischemic reperfusion injury.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe effect of aVNS on changes in neuronal regeneration factors after cerebral ischemic reperfusion injury in mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo explore the effect of aVNS on the expression level of neuronal regeneration factors, the numbers of doublecortin (DCX) expression-positive (DCX+) cells [22] in the hippocampal dentate gyrus in different groups were determined. Immunohistochemical staining showed that the mean number of DCX+ cells was 58 \\u0026plusmn; 3 in the aVNS group and 31 \\u0026plusmn; 2 in the sham group (\\u003cstrong\\u003eSupplementary Fig. 2\\u003c/strong\\u003e). The mean number of DCX+ cells in the aVNS group was significantly higher than that in the sham group. However, the CIR group showed less DCX+ cells than the sham, aVNS and CIR + aVNS groups, and the dendrites of the DCX+ cells were atrophic (\\u003cstrong\\u003eFig. 4A\\u003c/strong\\u003e). Western blotting showed strong and comparable expression levels of DCX in both the sham and aVNS groups. However, a lower DCX expression level found in the CIR group was significantly lower than that in the CIR + aVNS group (\\u003cstrong\\u003eFig 4B,C\\u003c/strong\\u003e). Administration of DH\\u0026beta;E to aVNS treated ischemic stroke mice reversed the effects of aVNS on neuronal regeneration (\\u003cstrong\\u003eSupplementary Fig. 3\\u003c/strong\\u003e). These results showed that aVNS could stimulate the expression level of neuronal regeneration factor DCX.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eaVNS could protect mouse against ischemic stroke by activating \\u0026alpha;4-nAChR\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo explore whether the activation of \\u0026alpha;4-nAChR signaling was involved in the protective effect of aVNS in this model, immunohistochemical staining of the brain tissues was performed. Our results showed that \\u0026alpha;4-nAChR were both extensively expressed around all brain regions in the sham and aVNS groups; however, ischemic stroke (CIR group) significantly reduced the expression of both \\u0026alpha;4-nAChRs, and this reduction could be considerably reversed by treatment with aVNS (CIR + aVNS group) (\\u003cstrong\\u003eFig. 5A\\u003c/strong\\u003e). In addition, the \\u0026alpha;4-nAChR positive cells in the cortex or hippocampal dentate gyrus of the CIR + aVNS group were significantly increased as compared to the CIR group (\\u003cstrong\\u003eFig. 5B,C\\u003c/strong\\u003e). Western blotting also confirmed the comparable results mentioned above (\\u003cstrong\\u003eFig. 5D,E\\u003c/strong\\u003e). According to these results, we suggest that aVNS manipulation could display its powerful anti-inflammatory and protective effects in this ischemic stroke model through activation of \\u0026alpha;4-nAChR signaling.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePossible signal transduction pathways involved in the aVNS protective effect on cerebral ischemic reperfusion injury in mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo further examine whether the protective effect of aVNS in mice after cerebral ischemic injury was associated with the GSK3\\u0026beta;/\\u0026beta;-catenin pathway [16], we assessed the expression levels of SpGSK3 (a GSK3 inhibitory form that is protective) and \\u0026beta;-catenin in mice at 24 h after ischemic stroke. Immunohistochemical staining revealed that the CIR group showed significant reductions in SpGSK3 and \\u0026beta;-catenin expression as compared to the sham and aVNS groups. However, aVNS treatment significantly increased SpGSK3 and \\u0026beta;-catenin expression (CIR + aVNS group) (\\u003cstrong\\u003eFig. \\u003c/strong\\u003e\\u003cstrong\\u003e6A,B\\u003c/strong\\u003e). In addition, the SpGSK3 positive cells and \\u0026beta;-catenin positive cells in the cortex or hippocampal dentate gyrus of the aVNS treatment group (CIR + aVNS group) were significantly increased as compared to the CIR group (\\u003cstrong\\u003eFig. 6\\u003c/strong\\u003e\\u003cstrong\\u003eC-F\\u003c/strong\\u003e). Western blotting also confirmed the comparable results mentioned above (\\u003cstrong\\u003eFig. 6G,H\\u003c/strong\\u003e). Administration of DH\\u0026beta;E to aVNS treated ischemic stroke mice reduced the protein expression of SpGSK3 and \\u0026beta;-catenin expression (\\u003cstrong\\u003eSupplementary Fig. 4\\u003c/strong\\u003e). In addition, pAkt (an upstream signal of SpGSK3) and Bcl-2 (a downstream protein of \\u0026beta;-catenin) were expressed less in the CIR group than in the sham group, and the expression was higher in the aVNS treatment group (CIR + aVNS group). There was a significant difference between the CIR and CIR + aVNS groups for pAkt and Bcl-2 (\\u003cstrong\\u003eSupplementary Fig. 5\\u003c/strong\\u003e). Furthermore, co-staining of SpGSK3 (protective factor) with the inflammatory factor p65NF-\\u0026kappa;B or the apoptotic protein active form caspase 3 revealed that SpGSK3 was present in areas where p65NF-\\u0026kappa;B and caspase 3 were less expressed (\\u003cstrong\\u003eSupplementary Fig. 6,7\\u003c/strong\\u003e). These results suggested that GSK3\\u0026beta;/\\u0026beta;-catenin signaling might underlie the protective effects induced by aVNS manipulation in a mouse model of cerebral ischemic stroke.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eRecently, VNS has been reported to provide protection against cerebral ischemic injury in rats [23-25]. The outcome of VNS treatment is closely related to its specific parameter settings. Therefore, exploration of the most appropriate stimulation parameters is important. In this study, the animal model of the aVNS manipulation method was a modification and improvement based on the conditions reported in previous studies [9, 25, 26]. In our study, the two aVNS treatment time points with different stimulation intensity were set at 24 h before MCA occlusion surgery and immediately after reperfusion. During the optimization of the aVNS protocol, different treatment conditions were explored, including performing aVNS 24 h before MCA occlusion surgery (0.5 mA, 25 Hz, duration 2 minutes, repeated every 5 minutes, 5 cycles), performing aVNS immediately after reperfusion (0.5 mA, 25 Hz, duration 2 minutes, repeated every 5 minutes, 5 cycles) or two aVNS treatment time points with the same stimulation intensity (0.5 mA, 25 Hz, duration 2 minutes, repeated every 5 minutes, 5 cycles). However, these aVNS stimulation conditions did not provide effective improvements of neurological deficits in this stroke model. Although the two aVNS treatment time points with the same stimulation intensity improved neurological deficits, animals died during stimulation, possibly because animals were weaker after MCA occlusion surgery (\\u003cstrong\\u003eSupplementary Fig. 8\\u003c/strong\\u003e). Finally, we choose that the aVNS stimulation conditions (as described method section) displaying the best protective effect to be adopted in the acute cerebral ischemic stroke mouse model. Interestingly, we found that the first session of electrostimulation is necessary to, somehow, sensitize the system to the effectiveness of the second electrostimulation session. Regardless, the reasons behind the sensitization produced by the pre-exposure to aVNS require further experimental investigation. This is the first report to demonstrate that treatment with aVNS is neuroprotective for acute ischemic brain damage in mice.\\u003c/p\\u003e\\n\\u003cp\\u003ePrevious studies have indicated that heart rate decreases significantly when VNS is performed [18, 27] and that norepinephrine concentrations increase with continued VNS [19]. In this study, we found significant changes in heart rate. In addition, it has been reported that increased activity of norepinephrine could be associated with increases in salivary amylase activity [20]. This study confirmed utility of a change in salivary amylase activity as another indicator for detecting the activation of the vagus nerve. These two non-invasive parameters (change in heart rate and salivary amylase activity) could be used to determine the activation of the vagus nerve and avoid the influences of invasive methods on animals.\\u003c/p\\u003e\\n\\u003cp\\u003eAfter acute cerebral ischemic stroke injury, a series of cellular damage reactions occur, including the inflammatory response that plays a key role in ischemic injury. The results of this study found that aVNS reduced the expression of inflammatory factors (pP65NF-\\u0026kappa;B) and reduced the cerebral infarct volume, with results comparable to those of a previous study [24]. It has been reported that VNS could reduce the expression of pro-inflammatory cytokines (TNF-\\u0026alpha;, IL-1\\u0026beta; and IL-6) at 24 h after reperfusion, and these anti-inflammatory and molecular mechanisms could partly be attributed to the enhanced release of acetylcholine [28], a neurotransmitter released mainly from the vagus nerve terminals. This result indicates that acetylcholine activates acetylcholine receptors to inhibit the release of pro-inflammatory cytokines, thereby preventing tissue damage. Therefore, it is reasonable to propose that aVNS manipulation could protect the brain from acute ischemic damage by activating acetylcholine receptors to inhibit inflammatory responses.\\u003c/p\\u003e\\n\\u003cp\\u003eEssentially, it has been reported that aVNS protects the brain from ischemic injury by activating acetylcholine receptors [28], and the most abundant nicotinic acetylcholine receptors (nAChR) in the mammalian brain are the \\u0026alpha;4- and \\u0026alpha;7-nAChRs [29], which have been shown to be important in mediating anti-inflammatory and neuroprotective actions. Activation of these receptors may enhance neuronal resistance to ischemic or other types of injury. The results of this study showed that acute cerebral ischemic stroke mice treated with aVNS manipulation had an increased survival rate, reduced neurological deficits and reduced cerebral infarct volume. However, the survival rates of the groups treated with aVNS and administration of \\u0026alpha;4-nAChR antagonist for ischemic stroke (DH\\u0026beta;E + CIR + aVNS group) was lower than that of the CIR + aVNS group, and the neurological function and cerebral infarct volume were both severe. Although the DH\\u0026beta;E + CIR + aVNS groups was treated with aVNS, their therapeutic effects were blocked by the antagonists of \\u0026alpha;4-nAChRs. Therefore, we propose that our aVNS manipulation is protective against acute cerebral ischemic stroke by activation of \\u0026alpha;4-nAChRs, which increases survival rate, reduces neurological deficits and reduces cerebral infarct volume.\\u003c/p\\u003e\\n\\u003cp\\u003eGlutamate neurotoxicity is involved in a variety of neurodegenerative diseases, including ischemic stroke, trauma, Alzheimer\\u0026apos;s disease and Parkinson\\u0026apos;s disease. In addition to neuronal death, neuroinflammatory responses are induced in these diseases. Previous studies have suggested that nicotinic acetylcholine receptors play an important role in the survival of neurons in the central nervous system during excitotoxicity and neuroinflammatory responses, and it has been suggested that the PI3K/Akt signaling pathway is involved in this protective mechanism [30, 31]. The PI3K/Akt pathway plays an important role in cell development, function and survival [32], and its downstream signal GSK3 has a neuroprotective effect on ischemic stroke by inhibiting its activity (with increased expression of SpGSK3) [33]. Furthermore, neurogenesis as an endogenous capacity for self-repair is triggered in certain pathological conditions such as ischemic injury. Ischemic stroke leads to the activation of several processes of endogenous self-repair and neuronal burst response [34, 35]. Many studies demonstrated the contribution of post‐stroke neurogenesis in functional recovery for review see [36]. Therefore, from evaluating the anti-apoptotic and neuroprotective mechanisms of aVNS in this murine model of acute cerebral ischemic stroke, we found that the expression levels of pAkt and SpGSK3 in the aVNS treated group was higher than those in the untreated CIR group, and the expression level of active form caspase 3 was lower in the aVNS treated group, while the expression level of \\u0026beta;-catenin and its downstream proteins Bcl-2 and DCX were both higher in the aVNS treated group than in the untreated CIR group. It has been suggested that neuroblast markers such as DCX were found to be increased after ischemic injury, however, most of newborn neurons fail to survive over long‐term due to absence of trophic factors [37, 38]. In the present study, we found a reduction in DCX in the hippocampal dentate gyrus 24 h after stroke. Moreover, we found that treatment of stroke with aVNS enhanced the survival of newborn DCX and prevented them from death 24h after stroke. Therefore, we propose that aVNS may reduce the ischemic stroke-induced inflammatory responses and apoptosis and increase the expression level of neuroprotective factors (such as Bcl-2) and reduction of damaging factors (such as activated caspase 3) through activation of the Akt/GSK3/catenin pathway.\\u003c/p\\u003e\\n\\u003cp\\u003eOur study successfully demonstrates that the aVNS manipulation is neuroprotective in this murine model of acute ischemic stroke; however, several limitations must be noted. First, the therapeutic effect of aVNS manipulation in the later stages of ischemic stroke should be considered, and second, the time point of aVNS intervention in this study may be different from the clinical situation, as many patients are treated post-stroke in the clinic. Third, the efficacy profile of aVNS in this study may be different from that we have recently demonstrated in a migraine model in rats. These limitations will be addressed in later works.\\u003c/p\\u003e\\n\\u003cp\\u003eIn conclusion, our results provide clear evidence showing that treatment with auricular VNS is neuroprotective for acute ischemic stroke mice by activating \\u0026alpha;4-nAChRs and inhibition of the GSK3\\u0026beta; activity, which in turn leads to activation of \\u0026beta;-catenin.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eEthics statement\\u003c/h2\\u003e\\n\\u003cp\\u003eThe animal study was reviewed and approved by the Animal Research Committee of the National Research Institute of Chinese Medicine\\u003c/p\\u003e\\n\\u003ch2\\u003eConflict of Interest\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors declare no conflict of interest.\\u003c/p\\u003e\\n\\u003ch2\\u003eAuthor Contributions\\u003c/h2\\u003e\\n\\u003cp\\u003eYen Jiin-Cherng, Chang Cher-Chia and Shen Yuh-Chiang participated in research design, conducted the experiments and wrote or contributed to the writing of the manuscript. Liou Kuo-Tong, Wang Yea-Hwey and Hsu Chih-hung contributed to preparation of animal surgery and chemical reagents, and analytic tools.\\u003c/p\\u003e\\n\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\n\\u003cp\\u003eThis study was supported, in part, by grants from the Ministry of Science and Technology, R.O.C.; Division of Neurovascular Disease, Neurological Institute, Taipei Veterans General Hospital; School of Medicine, National Yang-Ming University, Taiwan; and the National Research Institute of Chinese Medicine for Dr. Yen J.C., Dr. Chern C.M., Dr. Liou K.T., and Dr. Shen Y.C. (MOST 108-2320-B-077-003-MY3, MOST 110-2314-B-A49A-544, MOHW 111-NRICM-M-325-12240).\\u003c/p\\u003e\\n\\u003ch2\\u003eAcknowledgments\\u003c/h2\\u003e\\n\\u003cp\\u003eWe would like to thank Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University for providing technical support. We also thank National Research Institute of Chinese Medicine, Ministry of Health and Welfare for technical support.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eCatanese L, Tarsia J, Fisher M. Acute Ischemic Stroke Therapy Overview. Circ Res. 2017;120(3):541-558.\\u003c/li\\u003e\\n\\u003cli\\u003ePe\\u0026ntilde;a ID, Borlongan C, Shen G, Davis W. Strategies to Extend Thrombolytic Time Window for Ischemic Stroke Treatment: An Unmet Clinical Need. J Stroke. 2017;19(1):50-60.\\u003c/li\\u003e\\n\\u003cli\\u003eZhou Z, Lu J, Liu WW, et al. Advances in stroke pharmacology. Pharmacol Ther. 2018;191:23-42.\\u003c/li\\u003e\\n\\u003cli\\u003eArle JE, Carlson KW, Mei L. Investigation of mechanisms of vagus nerve stimulation for seizure using finite element modeling. Epilepsy Res. 2016;126:109-118.\\u003c/li\\u003e\\n\\u003cli\\u003eVonck K, Raedt R, Naulaerts J, et al. Vagus nerve stimulation\\u0026hellip;25 years later! 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Effects of vagus nerve stimulation on cognitive functioning in rats with cerebral ischemia reperfusion. J Transl Med. 2016;14:101. \\u003c/li\\u003e\\n\\u003cli\\u003eBuschman HP, Storm CJ, Duncker DJ, Verdouw PD, van der Aa HE, van der Kemp P. Heart rate control via vagus nerve stimulation. Neuromodulation. 2006;9(3):214-220. \\u003c/li\\u003e\\n\\u003cli\\u003eFollesa P, Biggio F, Gorini G, et al. Vagus nerve stimulation increases norepinephrine concentration and the gene expression of BDNF and bFGF in the rat brain. Brain Res. 2007;1179:28-34. \\u003c/li\\u003e\\n\\u003cli\\u003eWarren CM, van den Brink RL, Nieuwenhuis S, Bosch JA. Norepinephrine transporter blocker atomoxetine increases salivary alpha amylase. Psychoneuroendocrinology. 2017;78:233-236. \\u003c/li\\u003e\\n\\u003cli\\u003eLiang J, Luan Y, Lu B, Zhang H, Luo YN, Ge P. Protection of ischemic postconditioning against neuronal apoptosis induced by transient focal ischemia is associated with attenuation of NF-\\u0026kappa;B/p65 activation. PLoS One. 2014;9(5):e96734. \\u003c/li\\u003e\\n\\u003cli\\u003eBiggio F, Gorini G, Utzeri C, et al. Chronic vagus nerve stimulation induces neuronal plasticity in the rat hippocampus. Int J Neuropsychopharmacol. 2009;12(9):1209-1221. \\u003c/li\\u003e\\n\\u003cli\\u003eSun Z, Baker W, Hiraki T, Greenberg JH. The effect of right vagus nerve stimulation on focal cerebral ischemia: an experimental study in the rat. Brain Stimul. 2012;5(1):1-10. \\u003c/li\\u003e\\n\\u003cli\\u003eAy I, Lu J, Ay H, Gregory Sorensen A. Vagus nerve stimulation reduces infarct size in rat focal cerebral ischemia. Neurosci Lett. 2009;459(3):147-151. \\u003c/li\\u003e\\n\\u003cli\\u003eAy I, Napadow V, Ay H. Electrical stimulation of the vagus nerve dermatome in the external ear is protective in rat cerebral ischemia. 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Front Mol Neurosci. 2011;4:15. \\u003c/li\\u003e\\n\\u003cli\\u003eLindvall O, Kokaia Z. Neurogenesis following Stroke Affecting the Adult Brain. Cold Spring Harb Perspect Biol. 2015;7(11):a019034. \\u003c/li\\u003e\\n\\u003cli\\u003eLu J, Manaenko A, Hu Q. Targeting Adult Neurogenesis for Poststroke Therapy. Stem Cells Int. 2017;2017:5868632. \\u003c/li\\u003e\\n\\u003cli\\u003eRahman AA, Amruta N, Pinteaux E, Bix GJ. Neurogenesis After Stroke: A Therapeutic Perspective. Transl Stroke Res. 2021;12(1):1-14. \\u003c/li\\u003e\\n\\u003cli\\u003eRuan L, Wang B, ZhuGe Q, Jin K. Coupling of neurogenesis and angiogenesis after ischemic stroke. Brain Res. 2015;1623:166-173.\\u003c/li\\u003e\\n\\u003cli\\u003eCuartero MI, Garc\\u0026iacute;a-Culebras A, Torres-L\\u0026oacute;pez C, et al. Post-stroke Neurogenesis: Friend or Foe?. Front Cell Dev Biol. 2021;9:657846.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Glycogen synthase kinase 3 (GSK-3), Ischemic stroke, Neurogenesis, Nicotinic acetylcholine receptor (nAChR), Vagus nerve stimulation\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-2662844/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-2662844/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"Stroke is a major cause of death and long-term disability. Although numerous treatments have shown neuroprotective effects in animal models of ischemic stroke, their clinical efficacy and safety remain to be further investigated. Previous studies have indicated that vagus nerve stimulation (VNS) may reduce cerebral damage in ischemic stroke, but the specific mechanisms by which VNS achieves this effect in murine cerebral ischemic stroke injury are unclear. In this study, an animal model was designed to investigate the therapeutic effects and mechanisms of auricular vagus nerve stimulation (aVNS) in mice with acute ischemic stroke induced by middle cerebral artery occlusion (MCAO) and reperfusion injury. We focused particularly on the involvement of α4-nicotinic acetylcholine receptor (α4-nAChR) and the molecular signaling pathways mediated by aVNS. Mobility of the animals after acute ischemic stroke, infarct volume, and expression of neuroprotective and damaging factors were analyzed to understand the therapeutic effect and protective mechanism of aVNS on ischemic stroke. Our findings demonstrate that aVNS treatment improved mobility and reduced cerebral infarct volume after acute ischemic stroke. Additionally, administration of α4-nAChR antagonists reversed the protective effects of aVNS on ischemic stroke. Our mechanistic investigations revealed that aVNS mediates protective effects against acute ischemic stroke by reducing inflammatory and apoptotic factors, while enhancing neuronal regeneration factors, through activation of α4-nAChR and the GSK3β/β-catenin pathway. Our results provide clear evidence supporting the use of aVNS as a neuroprotective treatment for ischemic stroke in mice.\",\"manuscriptTitle\":\"Protective effects and mechanisms of auricular vagus nerve stimulation in the alleviation of acute ischemic cerebral injury in mice\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-04-03 14:07:02\",\"doi\":\"10.21203/rs.3.rs-2662844/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"b226ce10-f861-44c1-b0ff-54c6fd36bf7f\",\"owner\":[],\"postedDate\":\"April 3rd, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-04-25T22:40:21+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2023-04-03 14:07:02\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-2662844\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-2662844\",\"identity\":\"rs-2662844\",\"version\":[\"v1\"]},\"buildId\":\"WrCJVZZCHTDjtuVLN7oU0\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}