JOURNAL OF NEUROCHEMISTRY
| 2015
doi: 10.1111/jnc.13097
Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
Abstract Vagus nerve stimulation (VNS) exerts neuroprotective effects against cerebral ischemia/reperfusion (I/R) injury and modulates redox status, potentially through the activity of miR-210, an important microRNA that is regulated by hypoxia-inducible factor and Akt-dependent pathways. The aim of this study was to determine the mechanisms of VNS- and miR-210-mediated hypoxic tolerance. Male Sprague–Dawley rats were preconditioned with a miR-210 antagomir (A) or with an antagomir control (AC), followed by middle cerebral artery occlusion and VNS treatment. The animals were divided into eight groups: sham I/R, I/R, I/R+AC, I/R+A, sham I/R+VNS, I/R+VNS, I/ R+VNS+AC, and I/R+VNS+A. Activation of the endogenous cholinergic a7 nicotinic acetylcholine receptor (a7nAchR) pathway was identified using double immunofluorescence staining. miR-210 expression was measured by PCR. Behavioral outcomes, infarct volume, and neuronal apoptosis were
Although many studies addressing neuroprotective treatments and strategies have been performed in animals, few studies have been applied in clinical settings. Reestablishment of the blood supply, which is the most effective treatment for acute cerebral ischemia, can also cause reperfusion injury (Fu et al. 2014). Therefore, it is necessary to investigate and develop more effective adjuvant treatments. Vagus nerve stimulation (VNS) – a safe and effective treatment for epilepsy (Morris et al. 2013), treatmentresistant depression (Conway et al. 2012), and circulatory shock (Guarini et al. 2003) – has also been shown to be beneficial for the treatment of ischemic stroke (Ay et al. 2009; Mravec 2010). However, the precise molecular mechanisms underlying this treatment have not been elucidated.
observed at 24 h following reperfusion. Markers of oxidative stress were detected using ELISA. Rats treated with VNS showed increased miR-210 expression as well as decreased apoptosis and antioxidant stress responses compared with the I/R group; these rats also showed increased p-Akt protein expression and significantly decreased levels of cleaved caspase 3 in the ischemic penumbra, as measured by western blot and immunofluorescence analyses, respectively. Strikingly, the beneficial effects of VNS were attenuated following miR-210 knockdown. In conclusion, our results indicate that miR-210 is a potential mediator of VNS-induced neuroprotection against I/R injury. Our study highlights the neuroprotective potential of VNS, which, to date, has been largely unexplored. Keywords: antagomir, ischemia and reperfusion, miR-210, neuroprotection, vagus nerve stimulation. J. Neurochem. (2015) 10.1111/jnc.13097
Received November 6, 2014; revised manuscript received March 7, 2015; accepted March 10, 2015. Address correspondence and reprint requests to Changqing Li, Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China. E-mail: [email protected] Abbreviations used: a7nAchR, a7 nicotinic acetylcholine receptor; A, antagomir; AC, antagomir control; ACh, acetylcholine; CC3, cleaved caspase 3; CCA, right common carotid artery; CNS, central nervous system; ECA, external carotid artery; GSH, glutathione; HIF, hypoxiainducible factor; I/R, ischemia/reperfusion; ICA, internal carotid artery; LDF, laser Doppler flowmetry; MCAO, middle cerebral artery occlusion; MDA, methane dicarboxylic aldehyde; NE, norepinephrine; NTS, nucleus tractus solitarius; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SOD, superoxide dismutase; TUNEL, TdT-mediated dUTP nick end labeling; VNS, vagus nerve stimulation.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
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MicroRNAs are highly conserved, small, noncoding RNAs that are typically 18–24 nucleotides in length, and these molecules have emerged as important regulators of many biological processes, including differentiation, proliferation, development, migration, and apoptosis. Under hypoxic conditions, miR-210 regulates the translation of multiple downstream genes and has profound effects on many disease outcomes (Hong et al. 2013). To date, it has been demonstrated that miR-210 not only influences cardiac survival by targeting apoptosis genes in vivo, but also, that it slows down mitochondrial respiration to increase cell survival (Kim et al. 2009; Zhu and Fan 2012). It is appreciated that the excessive generation of reactive oxygen species following ischemic stroke can exacerbate DNA, protein, and fatty acid damage, as well as impair tissue and cell survival. Several studies indicate that VNS decreases reactive oxygen species production in myocardial and cerebral ischemic tissues (Kong et al. 2012; Ekici et al. 2013). Moreover, miR-210 regulation is dependent on hypoxia-inducible factor and the Aktdependent pathway in both normal and transformed cells (Greco et al. 2014). Interestingly, a growing body of evidence suggests that VNS likely exerts an anti-infarct effect on ischemic heart damage through activation of the Akt cascade (Katare et al. 2009), which also has protective effects in the CNS (Chen et al. 2014). Therefore, VNSinduced neuroprotection may be associated with antioxidant and/or anti-apoptotic mechanisms. In our previous study, we demonstrated that VNS most likely exerted its neuroprotective effects against cerebral ischemic insult by activating a7 nicotinic acetylcholine receptor (a7nAchR), leading to an increase in p-Akt expression (Jiang et al. 2014). These findings prompted us to hypothesize that miR-210 may be involved in the neuroprotective effects of VNS on I/R injury in the brain. To verify this hypothesis, we first examined the stimulation efficiency of VNS by measuring the expression of a7nAchR in neurons and astrocytes, as well as by determining neurological scores, infarct volume, and neuronal apoptosis. We subsequently explored the molecular effects of miR-210 in the VNS response by assaying the levels of three oxidative stress markers and caspase 3 activity in ischemic stroke rats pretreated with a miR-210 antagomir or an antagomir control.
Materials and methods Animals and miR-210 antagomir preconditioning Male Sprague–Dawley rats (250–300 g, n = 360) were obtained from the Experimental Animal Center of Chongqing Medical University. All experimental protocols were performed according to the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Ethics Committee of Chongqing Medical University (Permit No. SCXK (Chongqing) 2007-0001) and the State Science and Technology Commission of China. The
animals were maintained in a 12/12 h light/dark cycle at 24 1°C and 60% humidity and were given an adequate supply of food and water. During the experiment, the rats were randomly divided into eight groups: sham I/R, I/R, I/R+AC, I/R+A, sham I/R+VNS, I/R+VNS, I/R+VNS+AC, and I/R+VNS+A. To better identify the function of miR-210 in the VNS-induced antioxidant stress and anti-apoptotic responses, a miR-210 antagomir (A) was used to knockdown miR-210 expression. The rats were deeply anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg) and placed in a stereotaxic frame with a head holder. The miR-210 antagomir and antagomir control (AC) (Guangzhou RiboBio Co., Ltd., Guangzhou, China) were dissolved in artificial CSF (aCSF; 119 mmol/L NaCl, 3.1 nmol/L KCl, 1.2 mmol/L CaCl2, 1 mmol/L MgSO4, 0.50 mmol/L KH2PO4, 25 mmol/L NaHCO3, 5 mmol/L D-glucose, and 2.2 mmol/L urea, pH 7.4) at a concentration of 20 nmol/mL and continuously infused at a rate of 1 lL/h into the lateral ventricles of the rat cohorts (n = 8/ group). The solution was infused using a minipump (Alza Co., Palo Alto, CA, USA) connected to an Alzet brain infusion stainless steel cannula via peristaltic tubing, according to a previous description (Dharap et al. 2009) (coordinates: 0.8 mm posterior, 4.8 mm dorsoventral, and 1.5 mm lateral to Bregma). The surgery to implant both the cannula and pump was also performed under anesthesia, according to a previous study (Dharap et al. 2009). After 3 days, the rats were subjected to middle cerebral artery occlusion (MCAO) surgery for 2 h, and reperfusion and VNS treatment were subsequently initiated.
Transient middle cerebral artery occlusion model and VNS Three days after administration of the miR-210 antagomir or antagomir control, transient MCAO was induced in the rats for 2 h using the intraluminal suture technique, as previously described (Koizumi et al. 1986), which was followed by reperfusion. Briefly, the rats were anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg) during the surgery, and 24 G catheters (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) were inserted into the caudal ventral artery of the tail. Blood pressure was continuously monitored during the experiment. The right common and external carotid arteries (CCA and ECA) were exposed and ligated; the ECA was then cut at the distal portion. A 30 monofilament nylon suture (Beijing Sunbio Biotech Co. Ltd., Beijing, China) coated with silica gel was inserted into the CCA and advanced into the internal carotid artery. In addition, the cerebral blood flow changes of the right middle cerebral artery were monitored by continuous laser Doppler flowmetry (Perimed, North Royalton, OH, USA), as previously described (Hiraki et al. 2012). Decreases in average cerebral blood flow to < 20–30% of the baseline rate were considered as successful MCAO models. Blood gas levels and heart rate were also monitored during these procedures, and body temperature was maintained at normal physiological levels using a heating pad, as previously described (STARR Life Sciences, Allison Park, PA, USA) (Goyagi et al. 2011). After 30 min of ischemia, the animals were maintained in an anesthetized state and received right cervical VNS delivered using a Grass Model S48 stimulator and constant current unit. The stimulating electrodes were self-constructed according to the method used by Smith (Ay et al. 2011), and they comprised two polyethylene-coated curved silver wires held 1.5 mm apart by a solid bar.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
Ischemia/reperfusion injury in rats
The electrodes were sutured to the sternocleidomastoid muscle and wrapped around the right cervical nerve using a microscope, as performed in our previous study (Jiang et al. 2014). The stimulation pulses consisted of repeated square pulses of 0.5 mA in 30-s trains (0.5 ms length at 20 Hz), which were repeated every 5 min and lasted for 60 min (Ay et al. 2011). Two hours after occlusion, reperfusion was initiated, and blood flow restoration was confirmed by laser Doppler flowmetry. Rats from the sham I/R group underwent CCA and ECA surgical exposure but did not undergo filament insertion; rats from the sham I/R+VNS group only received electrical stimulation without occlusion. Assessment of neurological deficit scores and infarct volumes Neurological deficit scores after 24 h of reperfusion were evaluated using a scoring system that ranged from 0 to 4, which was modified as previously described (Sch€abitz et al. 1996). The scores were determined as follows: 0, no deficit; 1, failure to extend the left forepaw; 2, decreased grip strength of the left forepaw; 3, circling to the left by pulling the tail; and 4, spontaneous circling (n = 8/ group). Infarct volume was assessed using 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St Louis, MO, USA) staining, as previously described (Burnett et al. 2006). The brains (n = 8/group) were rapidly removed and sliced into five serial 2-mm-thick sections using a rodent brain matrix. The slices were stained with 1% TTC at 37°C for 30 min, fixed in 4% paraformaldehyde, photographed, and quantified for ischemic damage using Image J Software, as previously described (Won et al. 2014) (NIH Image, Bethesda, MD, USA). miRNA real-time reverse transcriptase polymerase chain reaction The ischemic penumbra regions of the right cerebral cortex were collected and quickly frozen in liquid nitrogen (n = 8/group). Total RNA was extracted from isolated cerebral cortices using the acid guanidinium thiocyanate–phenol–chloroform method (TRIzol reagent), and the RNA was resuspended in RNase-free water. The A260/A280 ratios of the purified RNA samples were between 1.8 and 2.0. For the miR-210 expression analysis, total RNA was transcribed using a TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. miR-210 expression was assessed using real-time PCR according to the TaqMan MicroRNA assay protocol (Applied Biosystems). Amplifications were performed at 95°C followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 70°C for 1 s. The levels of miR-210 were normalized to the endogenous levels of U6 in each triplicate sample, and average expression levels were calculated. The data were analyzed using the 2DCt method (Livak and Schmittgen 2001), and p < 0.05 were considered statistically significant. The miR-210 sequence was 50 -CUGUG CGUGUGACAGCGGCUGA-30 . The miR-210 antagomir was synthesized by Ribobio Co. (Guangzhou RiboBio Co., Ltd.); this antagomir was a single-stranded RNA analog complementary to mature miR-210. Double immunofluorescence for a7nAChR and NeuN/GFAP and immunofluorescence for caspase 3 The rats (n = 8/group) were deeply anesthetized and transcardially perfused with saline and 4% phosphate-buffered paraformaldehyde.
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The brains were sliced into 10-lm-thick coronal sections, incubated with 0.4% Triton X-100 for 15 min, and washed with phosphatebuffered saline (PBS) for 15 min. After blocking with donkey serum for 30 min at 37°C, the sections were incubated with primary antibodies diluted in PBS [rabbit anti-a7nAchR, 1 : 500, mouse antiNeuN, 1 : 200, rabbit anti-cleaved caspase 3 (1 : 100; Abcam, Cambridge, UK) or mouse anti-glial fibrillary acidic protein (1 : 100; Cell Signaling Technology, Danvers, MA, USA)] at 4°C overnight. The sections were subsequently treated with Alexa Fluor-555 donkey anti-rabbit Ig (H+G) (1 : 200) or IFKine Green Donkey anti-mouse IgG (H+G) (1 : 200) for 90 min at 37°C. Three areas in the cortex of the ischemic penumbra were imaged using laser-scanning confocal microscopy (Nikon TE2000, Tokyo, Japan and LEICA TCS SP2, Solms, Germany). Caspase 3-positive cells were quantitatively measured using ImageJ software. All analyses were performed by investigators who were blinded to the treatment conditions. TdT-mediated dUTP nick end labeling assay To examine brain histopathology at 24 h post-I/R, brain slices (n = 5/group) were stained using an in situ cell death detection kit, according to the manufacturer’s instructions (Yang et al. 2000). Briefly, the sections were permeabilized with 1% proteinase K for 15 min, rinsed with PBS, exposed to the TdT-mediated dUTP nick end labeling (TUNEL) reaction for 1 h at 37°C, and then washed again in PBS for 5 min. Five randomly selected regions within the ischemic penumbra in the right cerebral hemisphere were counted under high-power magnification (4009). Three slices per brain were counted by a blinded investigator, and the average number of TUNEL-positive cells was recorded. Enzyme-linked immunosorbent assay Twenty-four hours after surgery, the rats (n = 8/group) were sacrificed, and the brains were immediately removed. Brain tissues were isolated and homogenized in potassium phosphate buffer. After centrifugation, the supernatant, which contained the crude membranes, was prepared for measuring methane dicarboxylic aldehyde (MDA); the remainder of the supernatant was centrifuged once again and used to estimate superoxide dismutase (SOD) and glutathione (GSH) levels. The levels of MDA, SOD, and GSH in the ischemic penumbra cortex tissue homogenates were analyzed using commercially available kits (Nanjing Jiancheng Biological Technology, Nanjing, China) according to the manufacturer’s instructions. The experiments were performed in triplicate. Western blotting The rats (n = 8/group) were decapitated at 24 h after reperfusion. The brain tissues of the ipsilateral penumbra were dissected on ice. The samples were homogenized with protein extraction reagent (Pierce, Illinois, USA) supplemented with protease inhibitors. The supernatants were collected, and the total protein concentrations were analyzed with a Bio-Rad Dc protein assay (Bio-Rad, Hercules, CA, USA). Equivalent amounts of total protein were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), blocked with a 5% skim milk solution, washed in Tris-buffered saline containing 0.1% Tween-20 and probed with primary antibodies, including a rabbit anti-phospho Akt (1 : 1000; Cell Signaling Technology), a rabbit anti-cleaved caspase
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3 antibody (1 : 1000; Abcam) and a mouse anti-b-actin antibody (1 : 5000; Sigma, St Louis, MO, USA), at 4°C overnight. The membranes were then incubated with horseradish peroxidaseconjugated anti-rabbit or anti-mouse antibodies for 2 h at 37°C. The western blot signals were quantified using an enhanced chemiluminescence kit and examined using the Bio-Image Analysis System (Bio-Rad). The expression ratio of each target protein was normalized against the expression of b-actin. Statistical analyses All values are expressed as the mean SEM of at least three independent experiments performed in triplicate. Comparisons between multiple groups were conducted using one-way ANOVA, followed by a post hoc Tukey’s multiple-comparisons test. The neurological scores were analyzed using the Kruskal–Wallis test. p < 0.05 was considered significant. All statistical analyses were performed using Statistical Product and Service Solutions (SPSS) (Version 17.0).
Results Physiological parameters The mean values for blood pressure, heart rate, and blood gas were not significantly different among the groups, and all values were within the normal range (Table 1). The results indicated that VNS treatment had no significant effect on these parameters during the experiment, which was consistent with previous study. (Sun et al. 2012). Activation of a7nAChR in the ischemic penumbra following VNS treatment To determine the stimulation effectiveness in ischemic stroke rats, we used a double immunofluorescence staining assay to detect the induction of a7nAchR expression. As shown in Fig. 1, ischemic insult caused a significant reduction in a7nAchR expression at the surfaces of neurons and astrocytes, whereas VNS treatment mitigated this effect. Our results demonstrate that VNS treatment affects brain tissues through the activation of a7nAchR, the expression of which plays an important role in the activation of intracellular signaling pathways via a7nAchR expression.
miR-210 levels under various experimental conditions As miR-210 is robustly and uniquely increased during hypoxia, we confirmed the expression of miR-210 using realtime PCR at 24 h following reperfusion near the ischemic boundary in rats. As shown in Fig. 2, compared with the I/R group, miR-210 level was not significantly different from that in the I/R+AC group; however, it was significantly decreased following miR-210 blockade (p < 0.05). A further increase in miR-210 expression was identified in the I/ R+VNS and I/R+VNS+AC groups (p < 0.05), whereas the level of miR-210 was reduced in the I/R+VNS+A group, albeit to a lower extent (p < 0.05). These results suggested that as a protective factor, miR-210 activity could be blocked using the miR-210 antagomir. Moreover, VNS treatment could continue to enhance the expression of miR-210 following ischemic stroke, whereas in the I/R+VNS+A group, the level of miR-210 was down-regulated, which indicates that miR-210 was likely involved in the response induced by VNS. Furthermore, the levels of miR-210 were not different between the I/R+VNS+AC and I/R+VNS groups and decreased in the I/R+VNS+A group compared with the I/R+VNS group. These findings further support the relationship between miR-210 and VNS treatment. Incidentally, the miR-210 level was increased to a greater extent in the I/R and I/R+VNS groups compared with the sham I/R and sham I/R+VNS groups, respectively (p < 0.05). These findings indicate that miR-210 is rapidly up-regulated in response to hypoxia, consistent with previous studies (Qiu et al. 2013). Blockade of miR-210 impairs the improvements in neurological deficit scores and infarct volumes induced by VNS As shown in Fig. 3(a and b), we evaluated neurological scores and infarct volumes in rats pretreated with the miR210 antagomir to determine the effects of silencing miR-210 expression following I/R and VNS treatment. There was no significant difference between the I/R+AC and I/R groups. Increased neurological deficit scores and infarct volume were identified in the I/R+A group compared with the I/R group.
Table 1 The physiological parameters during the experiment Group
Mean blood pressure (mmHg)
sham I/R I/R I/R+AC I/R+A sham I/R+VNS I/R+VNS I/R+VNS+AC I/R+VNS+A
90 83 84 87 89 81 80 78
4.0 5.8 6.0 6.1 7.3 8.8 9.5 10.4
Heart rate (bp/min) 376 369 370 373 370 363 365 362
11 9 9 10 11 11 13 14
PH 7.41 7.39 7.39 7.41 7.38 7.39 7.37 7.39
PCO2 (mmHg)
0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.01
47.0 46.8 47.1 46.9 45.6 46.2 46.7 47.2
1.2 0.9 1.3 1.2 1.3 0.9 1.2 1.0
PO2 (mmHg) 99.7 105.3 98.2 108.4 112.3 107.9 108.5 98.9
8.1 9.2 9.9 10.1 10.3 10.8 11.1 8.4
All data are shown as the mean SEM.
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Fig. 1 The expression of a7nAchR on the surface of representative neurons and astrocytes revealed by double immunofluorescence staining for a7nAChR (red) and NeuN/astrocyte (green) in the ischemia penumbra of the three groups. Sham I/R group, I/R group, and I/R+VNS group. GFAP, glial fibrillary acidic protein (astrocyte marker). Scale bars = 50 lm.
Fig. 2 miRNA RT-PCR analysis of miR-210 expression with an antagomir control or with an antagomir following ischemic stroke and vagus nerve stimulation (VNS) treatment in the rat cerebral cortex. Data are expressed as the relative miR-210 levels normalized to the U6 endogenous control. Data are expressed as the mean SEM. ∇ p < 0.05 versus the sham I/R group. +p < 0.05 versus the sham I/ R+VNS group. #,&,*,@p < 0.05 versus the I/R group. ●p < 0.05 versus the I/R+VNS group.
Consistent with previous studies, VNS exerted significant neuroprotective effects, including improvements in neurological function and reductions in infarct volume compared with ischemic stroke rats; similar effects were also observed in the I/R+VNS+AC group. By contrast, these results appeared more severe in the I/R+VNS+A group compared with the I/R group. No changes were observed between the I/ R+VNS+AC and I/R+VNS groups. The improvements in neurological deficit scores and infarct volume induced by VNS treatment were significantly reduced as a result of miR210 knockdown, which was demonstrated via the comparison of the I/R+VNS+A and I/R+VNS groups. Based on our findings, we conclude that miR-210 exerts protection against ischemic infarct and mediates the neuroprotective effect of VNS in cerebral ischemia rats. Blockade of miR-210 via its antagomir attenuates the VNS-mediated protection against I/R injury As shown in Fig. 4, to further explore the role of miR-210 in mediating the protection induced by VNS against I/R injury in rats, we measured neuronal apoptotic markers after blocking miR-210. Compared with the I/R group, the number of TUNEL-positive cells was not remarkably different from
that of the I/R+AC group and was significantly increased compared with the I/R+A group, whereas it decreased after VNS treatment (p < 0.05). By contrast, an increased number of TUNEL-positive cells was identified in the ischemic penumbra in the I/R+VNS+A group compared with the I/R group. These findings indicated that miR-210 silencing exacerbated neuronal damage under hypoxic conditions. VNS treatment attenuated the apoptotic response, whereas this protection was decreased following miR-210 blockade preconditioning. Furthermore, there was no significant difference between the I/R+VNS+AC and I/R+VNS groups. However, the number of TUNEL-positive cells in the I/R+VNS+A group was greater than in the I/R+VNS group (p < 0.05). The results indicated that miR-210 down-regulation could markedly influence the VNS-mediated suppression of neuronal apoptosis. Thus, it appeared that miR-210 played a crucial role in the VNS-mediated anti-apoptosis effect against ischemic injury in rats. miR-210 is involved in the VNS-regulated oxidative stress response following cerebral ischemia in rats Cerebral ischemia results in the generation of excessive free radicals and lipid peroxidation. The three tested markers did not show significant changes in the I/R+AC group compared with the I/R group. MDA was significantly unregulated, whereas SOD and GSH were both down-regulated in the I/R+A group compared with the I/R group. Consistent with previous studies, we verified that VNS could decrease MDA levels and increase SOD and GSH levels to prevent further damage to brain tissues. No significant difference was identified between the I/R+VNS+AC and I/R+VNS groups. After miR-210 knockdown, the MDA level was higher in the I/R+VNS+A group compared with the I/R and I/R+VNS groups, whereas GSH and SOD activity levels remained lower (p < 0.05) (Fig. 5). In addition, ischemic stroke led to the up-regulation of MDA and the down-regulation of SOD and GSH activity; however, these effects were reversed following VNS treatment. These results also indicate that increased miR-210 expression reduces oxidative stress and that the antioxidant stress effects of VNS treatment are inhibited by miR-210 blockade.
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Fig. 3 The effects of vagus nerve stimulation (VNS) treatment on neurological deficit scores (a) and infarct volume (b) following miR-210
knockdown. Data are expressed as the mean SEM. #,&,*,@p < 0.05 versus the I/R group. ●p < 0.05 versus the I/R+VNS group.
Fig. 4 Neuronal apoptosis was measured by TdT-mediated dUTP nick end labeling (TUNEL) staining 24 h after I/R in rats pretreated with miR-210 inhibitor and vagus nerve stimulation (VNS) treatment. Sham I/R, I/R, I/R+AC, I/R+A, sham I/R+VNS, I/R+VNS, I/R+VNS+AC, and
I/R+VNS. Scale bar = 100 lm. Data are expressed as the mean SEM. #,&,*,@p < 0.05 versus the I/R group. ●p < 0.05 versus the I/R+VNS group.
Suppression of postischemic apoptosis induced by VNS is attenuated following miR-210 knockdown preconditioning To examine the role of miR-210 mediation in the antiapoptotic effect induced by VNS, the protein levels of p-Akt and activated caspase 3 were measured by western blot analysis. There were no significant differences in p-Akt protein expression among the I/R+AC, I/R+A and I/R groups, whereas there was an increase in p-Akt expression in the I/R+VNS, I/R+VNS+AC, and I/R+VNS+A groups compared with the I/R group. No significant differences were detected among the I/R+VNS, I/R+VNS+AC and I/R+VNS+A groups. Therefore, VNS caused an increase in p-Akt protein expression following ischemia and was not significantly affected by miR-210 knockdown. Caspase 3 activity did not significantly change in the I/R+AC group, but it was markedly up-regulated in the I/R+A group compared with the I/R group. Importantly, the activity of caspase 3 was suppressed following VNS, whereas this inhibition was diminished in the I/R+VNS+A group compared with the I/R and I/R+VNS groups (p < 0.05) (Fig. 6a and b). Furthermore, caspase 3 protein expression in the I/R and I/R+VNS groups was significantly higher compared with the sham I/R
and sham I/R+VNS groups, respectively (p < 0.05). These results also suggested that VNS suppressed the apoptotic response following I/R. During this process, the action of miR-210 appears to be a protective factor that augments brain tissue survival. As expected, the anti-apoptotic effect induced by VNS treatment was decreased after miR-210 antagomir preconditioning. Therefore, miR-210 could mediate the antiapoptotic response induced by VNS in postischemic rat brains.
Discussion Recent studies demonstrated that VNS initiated 30 min after cerebral middle artery occlusion in rats reduced ischemic lesion volume, improved the neurological outcomes, reduced brain water content, and suppressed inflammatory cytokines (Mravec 2010). The benefits of VNS are associated with its central projections to various brain structures. The afferents of the vagus nerve fibers project bilaterally to the nucleus tractus solitarius in the medulla oblongata, which then projects to the locus coeruleus, the major source of norepinephrine in the brain. Furthermore, polysynaptic
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
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Fig. 5 miR-210 mediates the vagus nerve stimulation (VNS)-induced antioxidant response. Changes in the levels of MDA (methane dicarboxylic aldehyde), SOD (superoxide dismutase), and GSH (glutathione) in the ischemic penumbra in each group. Data are expressed
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as the mean SEM. ∇p < 0.05 versus the sham I/R group. +p < 0.05 versus the sham I/R+VNS group. #,&,*,@p < 0.05 versus the I/R group. ● p < 0.05 versus the I/R+VNS group.
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(b)
Fig. 6 miR-210 knockdown attenuates vagus nerve stimulation (VNS)induced protection against I/R injury in vivo. (a) Western blot analysis of p-Akt and cleaved caspase 3 with or without miR-210 knockdown in the VNS-treated group (b). Immunostaining reveals changes the number of cleaved caspase 3-positive cells following intervention. Scale
bar = 75 lm. Data are expressed as the mean SEM. ∇p < 0.05 versus the sham I/R group. +p < 0.05 versus the sham I/R+VNS group. #,&, *p < 0.05 versus the I/R group. ●p < 0.05 versus the I/R+VNS group.
projections from the nucleus tractus solitarius also innervate many other brain areas, including the thalamus, hypothalamus, limbic system, and cerebral cortex. It is well established that norepinephrine can stimulate 5-HT and acetylcholine
(ACh) release to exert potent anti-inflammatory effects in the brain (Cheyuo et al. 2011). Abundant evidence suggests that a7nAchR, which is an effector receptor that can be activated by drugs or electrical stimulation of the vagus nerve, plays an
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important role in the protective effects of VNS toward I/R injury in the brain. For example, the activation and up-regulation of Akt phosphorylation is involved in donepezil-dependent neuroprotection, which counteracts glutamate-induced neurotoxicity via a7nAchR activation (Oda et al. 2007). In our study, we measured various physiological parameters during the experimental process, all of which remained within the normal range. The simultaneous activation of a7nAchR on neurons and astrocytes was identified via immunofluorescence after VNS treatment, providing visual evidence of the effectiveness of electrical stimulation in the rat brain following ischemia. miR-210 is a highly conserved, small, noncoding RNA that participates in hypoxia-dependent disease states, including apoptosis, inflammation, and tumorigenesis. miR-210 exhibits neuroprotective effects via the inhibition of apoptosis in a murine model of hypoxic-ischemic encephalopathy (Qiu et al. 2013). Similarly, a recent study demonstrated that the introduction of a minicircle vector carrying a miR-210 precursor into an ischemic heart could dramatically improve heart function by the promotion of angiogenesis and the inhibition of apoptosis (Zhu and Fan 2012). In addition, Yang et al. (2012) demonstrated that miR-210 down-regulation significantly increased the apoptotic rate and radiosensitivity of human hepatoma cells. Our results indicate that miR-210 expression was up-regulated following VNS compared with the corresponding I/R groups. Furthermore, the up-regulation of miR-210 was positively correlated with lower neurological scores and smaller infarct volumes. Following miR-210 blockade, the neuroprotective effects of VNS were decreased. These results suggest that miR-210 is involved in the VNS-mediated neuroprotection. miR-210 is not only a potential stroke marker, but it may also be a key factor in the repression of mitochondrial metabolism in many cell types under hypoxic conditions. In one study, miR-210 overexpression increased the protection of mesenchymal stem cells against oxidative stress-induced injury; in another study, miR-210 knockdown in myoblasts significantly elevated myotube sensitivity to oxidative stress and resulted in mitochondrial dysfunction (Greco et al. 2014). VNS also confers protection against the accumulation of free radicals during myocardial and cerebral ischemia/ reperfusion (I/R) in rats (Kong et al. 2012; Ekici et al. 2013). Therefore, we attempted to determine whether miR210 was also involved in the VNS-mediated antioxidant signaling pathway. Our results indicate that VNS can significantly modulate the oxidative stress response, concomitant with an increase in miR-210. Indeed, the antioxidant effect of VNS induction was greatly decreased by miR210 blockade. Therefore, we infer that miR-210 plays an important role in the VNS-mediated antioxidant response, which also leads to an anti-apoptotic effect. Kim et al. (2009) demonstrated that rat mesenchymal stem cell survival was improved by increasing the expres-
sion of miR-210, which led to decreased levels of CASP8AP2, a direct target of miR-210 that was negatively correlated with cell survival. Increased miR-210 levels could have an effect on the activity of caspase 3/7 in hypoxia. Some, but not all, studies have suggested that miR-210 may be induced by p-Akt, which is another regulator of the hypoxia response (Chan and Loscalzo 2010). Kawada and coworkers confirmed that VNS significantly enhanced myocardial interstitial ACh levels in the ischemic region compared with baseline levels, triggering a cascade of survival signals through Akt phosphorylation and the inhibition of proapoptotic cascades (Kawada et al. 2007). These findings indicate that miR-210 and p-Akt could be correlated with the VNS-mediated antiapoptotic effects against ischemic damage. Consistent with this hypothesis, our results indicate that VNS exerts an anti-apoptotic effect, which is accompanied by an increase in p-Akt protein levels in the rat brain following I/R and a concomitant increase in miR-210 expression. Therefore, our findings suggest that VNS plays a critical role in facilitating the adaptation of brain tissues to hypoxic stress. To further evaluate the role of miR-210 in the anti-apoptotic effect of VNS, a miR-210 antagomir was administered to the lateral ventricles prior to MCAO surgery and VNS treatment. The anti-apoptotic effect induced by VNS was decreased after miR-210 knockdown pretreatment, and brain damage was not reduced under these conditions. These results indicate that miR-210 mediates the VNS-induced antiapoptotic effects in ischemic stroke in rats. Although the results of the miRNA array analysis indicated that there are also many other endogenous miRNAs involved in cerebral I/R processes, when considering our previous findings, we chose to focus on miR-210, which emerged as a major potential regulator of the VNS-mediated neuroprotective effect. Additional investigations of other miRNAs are required in the future. Taken together, these results indicate that miR-210 may be involved in the antioxidant and anti-apoptotic responses induced by VNS against I/R damage. To the best of our knowledge, this is the first report regarding the neuroprotective mechanism of VNS at the mRNA level. The downregulation of miR-210 attenuated the antiapoptotic and antioxidant effects induced by VNS in I/R rats. In summary, our findings provide valuable insight into the association between miR-210 and VNS-induced neuroprotection.
Acknowledgments and conflict of interest disclosure This work was supported by the National Nature and Science Foundation of China (Grant No. 81271306). There are no conflicts of interest in this manuscript. All experiments were conducted in compliance with the ARRIVE guidelines.
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References Ay I., Lu J., Ay H. and Sorensen A. G. (2009). Vagus nerve stimulation reduces infarct size in rat focal cerebral ischemia. Neurosci. Lett. 459, 147–151. Ay I., Sorensen A. G. and Ay H. (2011) Vagus nerve stimulation reduces infarct size in rat focal cerebral ischemia: an unlikely role for cerebral blood flow. Brain Res. 1392, 110–115. Burnett M. G., Shimazu T., Szabados T., Muramatsu H., Detre J. A. and Greenberg J. H. (2006) Electrical forepaw stimulation during reversible forebrain ischemia decreases infarct volume. Stroke 37, 1327–1331. Chan S. Y. and Loscalzo J. (2010) MicroRNA-210: a unique and pleiotropic hypoxamir. Cell Cycle 9, 1072–1083. Chen L., Wei X., Hou Y., Liu X., Li S., Sun B., Liu X. and Liu H. (2014) Tetramethylpyrazine analogue CXC195 protects against cerebral ischemia/reperfusion-induced apoptosis through PI3K/ Akt/GSK3. Neurochem. Int. 66, 27–32. Cheyuo C., Jacob A., Wu R., Zhou M., Coppa G. F. and Wang P. (2011) The parasympathetic nervous system in the quest for stroke therapeutics. J. Cereb. Blood Flow Metab. 31, 1187–1195. Conway C. R., Sheline Y. I., Chibnall J. T., Bucholz R. D., Price J. L., Gangwani S. and Mintun M. A. (2012) Brain blood-flow change with acute vagus nerve stimulation in treatment-refractory major depressive disorder. Brain Stimul. 5, 163–171. Dharap A., Bowen K., Place R., Li L. C. and Vemuganti R. (2009) Transient focal ischemia induces extensive temporal changes in rat cerebral microRNAome. J. Cereb. Blood Flow Metab. 29, 675– 687. Ekici F., Karson A., Dillioglugil M. O., Gurol G., Kir H. M. and Ates N. (2013) The effects of vagal nerve stimulation in focal cerebral ischemia and reperfusion model. Turk. Neurosurg. 23, 451–457. Fu S. H., Zhang H. F., Yang Z. B., Li T. B., Liu B. and Lou Z. (2014) Alda-1 reduces cerebral ischemia/reperfusion injury in rat through clearance of reactive aldehydes. Naunyn Schmiedebergs Arch. Pharmacol. 387, 87–94. Goyagi T., Nishikawa T. and Tobe Y. (2011) Neuroprotective effects and suppression of ischemia-induced glutamate elevation by b1adrenoreceptor antagonists administered before transient focal ischemia in rats. J. Neurosurg. Anesthesiol. 23, 131–137. Greco S., Gaetano C. and Martelli F. (2014) HypoxamiR regulation and function in ischemic cardiovascular diseases. Antioxid. Redox Signal. 21, 1202–1219. Guarini S., Altavilla D., Cainazzo M. M. et al. (2003) Efferent vagal fibre stimulation blunts nuclear factor-kappaB activation and protects against hypovolemic hemorrhagic shock. Circulation 107, 1189–1194. Hiraki T., Baker W. and Greenberg J. H. (2012) Effect of vagus nerve stimulation during transient focal cerebral ischemia on chronic outcome in rats. J. Neurosci. Res. 90, 887–894. Hong L., Han Y., Zhang H., Zhao Q. and Qiao Y. (2013) miR-210: a therapeutic target in cancer. Expert Opin. Ther. Targets 17, 21–28. Jiang Y., Li L., Liu B., Zhang Y., Chen Q. and Li C. (2014) Vagus nerve stimulation attenuates cerebral ischemia and reperfusion injury via endogenous cholinergic pathway in rat. PLoS ONE 9, e102342. Katare R. G., Ando M., Kakinuma Y., Arikawa M., Handa T., Yamasaki F. and Sato T. (2009) Vagal nerve stimulation prevents reperfusion injury through inhibition of opening of mitochondrial permeability
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transition pore independent of the bradycardiac effect. J. Thorac. Cardiovasc. Surg. 137, 223–231. Kawada T., Yamazaki T., Akiyama T., Li M., Zheng C., Shishido T., Mori H. and Sugimachi M. (2007) Angiotensin II attenuates myocardial interstitial acetylcholine release in response to vagal stimulation. Am. J. Physiol. Heart Circ. Physiol. 293, 2516–2522. Kim H. W., Haider H. K., Jiang S. and Ashraf M. (2009) Ischemic preconditioning augments survival of stem cells via miR-210 expression by targeting caspase-8-associated protein 2. J. Biol. Chem. 284, 33161–33168. Koizumi J., Yoshida Y., Nakazawa T. and Ooneda G. (1986) Experimental studies of ischemic brain edema: 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn. J. Stroke 8, 1–8. Kong S. S., Liu J. J., Yu X. J., Lu Y. and Zang W. J. (2012) Protection against ischemia-induced oxidative stress conferred by vagal stimulation in the rat heart: involvement of the AMPK-PKC pathway. Int. J. Mol. Sci. 13, 14311–14325. Livak K. J. and Schmittgen T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. Morris G. L., 3rd, Gloss D., Buchhalter J., Mack K. J., Nickels K. and Harden C. (2013) Evidence-based guideline update: vagus nerve stimulation for the treatment of epilepsy: report of the guideline development subcommittee of the american academy of neurology. Epilepsy Curr. 13, 297–303. Mravec B. (2010) The role of the vagus nerve in stroke. Auton Neurosci. 158, 8–12. Oda T., Kume T., Katsuki H., Niidome T., Sugimoto H. and Akaike A. (2007) Donepezil potentiates nerve growth factor-induced neurite outgrowth in PC12 cells. J. Pharmacol. Sci. 104, 349–354. Qiu J., Zhou X. Y., Zhou X. G., Cheng R., Liu H. Y. and Li Y. (2013) Neuroprotective effects of microRNA-210 on hypoxic-ischemic encephalopathy. Biomed Res. Int. 2013, 350419. Sch€abitz W. R., Weber J., Takano K., Sandage B. W., Locke K. W. and Fisher M. (1996) The effects of prolonged treatment with citicoline in temporary experimental focal ischemia. J. Neurol. Sci. 138, 21– 25. Sun Z., Baker W., Hiraki T. and Greenberg J. H. (2012) The effect of right vagus nerve stimulation on focal cerebral ischemia: an experimental study in the rat. Brain Stimul. 5, 1–10. Won S., Lee J. H., Wali B., Stein D. G. and Sayeed I. (2014) Progesterone attenuates hemorrhagic transformation after delayed tPA treatment in an experimental model of stroke in rats: involvement of the VEGF-MMP pathway. J. Cereb. Blood Flow Metab. 34, 72–80. Yang S. H., Shi J., Day A. L. and Simpkins J. W. (2000) Estradiol exerts neuroprotective effects when administered after ischemic insult. Stroke 31, 745–749. Yang W., Sun T., Cao J., Liu F., Tian Y. and Zhu W. (2012) Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro. Exp. Cell Res. 318, 944–954. Zhu H. and Fan G. C. (2012) Role of MicroRNAs in the reperfused myocardium towards post-infarct remodelling. Cardiovasc. Res. 94, 284–292.
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| 2015
doi: 10.1111/jnc.13097
Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China
Abstract Vagus nerve stimulation (VNS) exerts neuroprotective effects against cerebral ischemia/reperfusion (I/R) injury and modulates redox status, potentially through the activity of miR-210, an important microRNA that is regulated by hypoxia-inducible factor and Akt-dependent pathways. The aim of this study was to determine the mechanisms of VNS- and miR-210-mediated hypoxic tolerance. Male Sprague–Dawley rats were preconditioned with a miR-210 antagomir (A) or with an antagomir control (AC), followed by middle cerebral artery occlusion and VNS treatment. The animals were divided into eight groups: sham I/R, I/R, I/R+AC, I/R+A, sham I/R+VNS, I/R+VNS, I/ R+VNS+AC, and I/R+VNS+A. Activation of the endogenous cholinergic a7 nicotinic acetylcholine receptor (a7nAchR) pathway was identified using double immunofluorescence staining. miR-210 expression was measured by PCR. Behavioral outcomes, infarct volume, and neuronal apoptosis were
Although many studies addressing neuroprotective treatments and strategies have been performed in animals, few studies have been applied in clinical settings. Reestablishment of the blood supply, which is the most effective treatment for acute cerebral ischemia, can also cause reperfusion injury (Fu et al. 2014). Therefore, it is necessary to investigate and develop more effective adjuvant treatments. Vagus nerve stimulation (VNS) – a safe and effective treatment for epilepsy (Morris et al. 2013), treatmentresistant depression (Conway et al. 2012), and circulatory shock (Guarini et al. 2003) – has also been shown to be beneficial for the treatment of ischemic stroke (Ay et al. 2009; Mravec 2010). However, the precise molecular mechanisms underlying this treatment have not been elucidated.
observed at 24 h following reperfusion. Markers of oxidative stress were detected using ELISA. Rats treated with VNS showed increased miR-210 expression as well as decreased apoptosis and antioxidant stress responses compared with the I/R group; these rats also showed increased p-Akt protein expression and significantly decreased levels of cleaved caspase 3 in the ischemic penumbra, as measured by western blot and immunofluorescence analyses, respectively. Strikingly, the beneficial effects of VNS were attenuated following miR-210 knockdown. In conclusion, our results indicate that miR-210 is a potential mediator of VNS-induced neuroprotection against I/R injury. Our study highlights the neuroprotective potential of VNS, which, to date, has been largely unexplored. Keywords: antagomir, ischemia and reperfusion, miR-210, neuroprotection, vagus nerve stimulation. J. Neurochem. (2015) 10.1111/jnc.13097
Received November 6, 2014; revised manuscript received March 7, 2015; accepted March 10, 2015. Address correspondence and reprint requests to Changqing Li, Department of Neurology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China. E-mail: [email protected] Abbreviations used: a7nAchR, a7 nicotinic acetylcholine receptor; A, antagomir; AC, antagomir control; ACh, acetylcholine; CC3, cleaved caspase 3; CCA, right common carotid artery; CNS, central nervous system; ECA, external carotid artery; GSH, glutathione; HIF, hypoxiainducible factor; I/R, ischemia/reperfusion; ICA, internal carotid artery; LDF, laser Doppler flowmetry; MCAO, middle cerebral artery occlusion; MDA, methane dicarboxylic aldehyde; NE, norepinephrine; NTS, nucleus tractus solitarius; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SOD, superoxide dismutase; TUNEL, TdT-mediated dUTP nick end labeling; VNS, vagus nerve stimulation.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
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MicroRNAs are highly conserved, small, noncoding RNAs that are typically 18–24 nucleotides in length, and these molecules have emerged as important regulators of many biological processes, including differentiation, proliferation, development, migration, and apoptosis. Under hypoxic conditions, miR-210 regulates the translation of multiple downstream genes and has profound effects on many disease outcomes (Hong et al. 2013). To date, it has been demonstrated that miR-210 not only influences cardiac survival by targeting apoptosis genes in vivo, but also, that it slows down mitochondrial respiration to increase cell survival (Kim et al. 2009; Zhu and Fan 2012). It is appreciated that the excessive generation of reactive oxygen species following ischemic stroke can exacerbate DNA, protein, and fatty acid damage, as well as impair tissue and cell survival. Several studies indicate that VNS decreases reactive oxygen species production in myocardial and cerebral ischemic tissues (Kong et al. 2012; Ekici et al. 2013). Moreover, miR-210 regulation is dependent on hypoxia-inducible factor and the Aktdependent pathway in both normal and transformed cells (Greco et al. 2014). Interestingly, a growing body of evidence suggests that VNS likely exerts an anti-infarct effect on ischemic heart damage through activation of the Akt cascade (Katare et al. 2009), which also has protective effects in the CNS (Chen et al. 2014). Therefore, VNSinduced neuroprotection may be associated with antioxidant and/or anti-apoptotic mechanisms. In our previous study, we demonstrated that VNS most likely exerted its neuroprotective effects against cerebral ischemic insult by activating a7 nicotinic acetylcholine receptor (a7nAchR), leading to an increase in p-Akt expression (Jiang et al. 2014). These findings prompted us to hypothesize that miR-210 may be involved in the neuroprotective effects of VNS on I/R injury in the brain. To verify this hypothesis, we first examined the stimulation efficiency of VNS by measuring the expression of a7nAchR in neurons and astrocytes, as well as by determining neurological scores, infarct volume, and neuronal apoptosis. We subsequently explored the molecular effects of miR-210 in the VNS response by assaying the levels of three oxidative stress markers and caspase 3 activity in ischemic stroke rats pretreated with a miR-210 antagomir or an antagomir control.
Materials and methods Animals and miR-210 antagomir preconditioning Male Sprague–Dawley rats (250–300 g, n = 360) were obtained from the Experimental Animal Center of Chongqing Medical University. All experimental protocols were performed according to the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Ethics Committee of Chongqing Medical University (Permit No. SCXK (Chongqing) 2007-0001) and the State Science and Technology Commission of China. The
animals were maintained in a 12/12 h light/dark cycle at 24 1°C and 60% humidity and were given an adequate supply of food and water. During the experiment, the rats were randomly divided into eight groups: sham I/R, I/R, I/R+AC, I/R+A, sham I/R+VNS, I/R+VNS, I/R+VNS+AC, and I/R+VNS+A. To better identify the function of miR-210 in the VNS-induced antioxidant stress and anti-apoptotic responses, a miR-210 antagomir (A) was used to knockdown miR-210 expression. The rats were deeply anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg) and placed in a stereotaxic frame with a head holder. The miR-210 antagomir and antagomir control (AC) (Guangzhou RiboBio Co., Ltd., Guangzhou, China) were dissolved in artificial CSF (aCSF; 119 mmol/L NaCl, 3.1 nmol/L KCl, 1.2 mmol/L CaCl2, 1 mmol/L MgSO4, 0.50 mmol/L KH2PO4, 25 mmol/L NaHCO3, 5 mmol/L D-glucose, and 2.2 mmol/L urea, pH 7.4) at a concentration of 20 nmol/mL and continuously infused at a rate of 1 lL/h into the lateral ventricles of the rat cohorts (n = 8/ group). The solution was infused using a minipump (Alza Co., Palo Alto, CA, USA) connected to an Alzet brain infusion stainless steel cannula via peristaltic tubing, according to a previous description (Dharap et al. 2009) (coordinates: 0.8 mm posterior, 4.8 mm dorsoventral, and 1.5 mm lateral to Bregma). The surgery to implant both the cannula and pump was also performed under anesthesia, according to a previous study (Dharap et al. 2009). After 3 days, the rats were subjected to middle cerebral artery occlusion (MCAO) surgery for 2 h, and reperfusion and VNS treatment were subsequently initiated.
Transient middle cerebral artery occlusion model and VNS Three days after administration of the miR-210 antagomir or antagomir control, transient MCAO was induced in the rats for 2 h using the intraluminal suture technique, as previously described (Koizumi et al. 1986), which was followed by reperfusion. Briefly, the rats were anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg) during the surgery, and 24 G catheters (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) were inserted into the caudal ventral artery of the tail. Blood pressure was continuously monitored during the experiment. The right common and external carotid arteries (CCA and ECA) were exposed and ligated; the ECA was then cut at the distal portion. A 30 monofilament nylon suture (Beijing Sunbio Biotech Co. Ltd., Beijing, China) coated with silica gel was inserted into the CCA and advanced into the internal carotid artery. In addition, the cerebral blood flow changes of the right middle cerebral artery were monitored by continuous laser Doppler flowmetry (Perimed, North Royalton, OH, USA), as previously described (Hiraki et al. 2012). Decreases in average cerebral blood flow to < 20–30% of the baseline rate were considered as successful MCAO models. Blood gas levels and heart rate were also monitored during these procedures, and body temperature was maintained at normal physiological levels using a heating pad, as previously described (STARR Life Sciences, Allison Park, PA, USA) (Goyagi et al. 2011). After 30 min of ischemia, the animals were maintained in an anesthetized state and received right cervical VNS delivered using a Grass Model S48 stimulator and constant current unit. The stimulating electrodes were self-constructed according to the method used by Smith (Ay et al. 2011), and they comprised two polyethylene-coated curved silver wires held 1.5 mm apart by a solid bar.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
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The electrodes were sutured to the sternocleidomastoid muscle and wrapped around the right cervical nerve using a microscope, as performed in our previous study (Jiang et al. 2014). The stimulation pulses consisted of repeated square pulses of 0.5 mA in 30-s trains (0.5 ms length at 20 Hz), which were repeated every 5 min and lasted for 60 min (Ay et al. 2011). Two hours after occlusion, reperfusion was initiated, and blood flow restoration was confirmed by laser Doppler flowmetry. Rats from the sham I/R group underwent CCA and ECA surgical exposure but did not undergo filament insertion; rats from the sham I/R+VNS group only received electrical stimulation without occlusion. Assessment of neurological deficit scores and infarct volumes Neurological deficit scores after 24 h of reperfusion were evaluated using a scoring system that ranged from 0 to 4, which was modified as previously described (Sch€abitz et al. 1996). The scores were determined as follows: 0, no deficit; 1, failure to extend the left forepaw; 2, decreased grip strength of the left forepaw; 3, circling to the left by pulling the tail; and 4, spontaneous circling (n = 8/ group). Infarct volume was assessed using 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St Louis, MO, USA) staining, as previously described (Burnett et al. 2006). The brains (n = 8/group) were rapidly removed and sliced into five serial 2-mm-thick sections using a rodent brain matrix. The slices were stained with 1% TTC at 37°C for 30 min, fixed in 4% paraformaldehyde, photographed, and quantified for ischemic damage using Image J Software, as previously described (Won et al. 2014) (NIH Image, Bethesda, MD, USA). miRNA real-time reverse transcriptase polymerase chain reaction The ischemic penumbra regions of the right cerebral cortex were collected and quickly frozen in liquid nitrogen (n = 8/group). Total RNA was extracted from isolated cerebral cortices using the acid guanidinium thiocyanate–phenol–chloroform method (TRIzol reagent), and the RNA was resuspended in RNase-free water. The A260/A280 ratios of the purified RNA samples were between 1.8 and 2.0. For the miR-210 expression analysis, total RNA was transcribed using a TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. miR-210 expression was assessed using real-time PCR according to the TaqMan MicroRNA assay protocol (Applied Biosystems). Amplifications were performed at 95°C followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 70°C for 1 s. The levels of miR-210 were normalized to the endogenous levels of U6 in each triplicate sample, and average expression levels were calculated. The data were analyzed using the 2DCt method (Livak and Schmittgen 2001), and p < 0.05 were considered statistically significant. The miR-210 sequence was 50 -CUGUG CGUGUGACAGCGGCUGA-30 . The miR-210 antagomir was synthesized by Ribobio Co. (Guangzhou RiboBio Co., Ltd.); this antagomir was a single-stranded RNA analog complementary to mature miR-210. Double immunofluorescence for a7nAChR and NeuN/GFAP and immunofluorescence for caspase 3 The rats (n = 8/group) were deeply anesthetized and transcardially perfused with saline and 4% phosphate-buffered paraformaldehyde.
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The brains were sliced into 10-lm-thick coronal sections, incubated with 0.4% Triton X-100 for 15 min, and washed with phosphatebuffered saline (PBS) for 15 min. After blocking with donkey serum for 30 min at 37°C, the sections were incubated with primary antibodies diluted in PBS [rabbit anti-a7nAchR, 1 : 500, mouse antiNeuN, 1 : 200, rabbit anti-cleaved caspase 3 (1 : 100; Abcam, Cambridge, UK) or mouse anti-glial fibrillary acidic protein (1 : 100; Cell Signaling Technology, Danvers, MA, USA)] at 4°C overnight. The sections were subsequently treated with Alexa Fluor-555 donkey anti-rabbit Ig (H+G) (1 : 200) or IFKine Green Donkey anti-mouse IgG (H+G) (1 : 200) for 90 min at 37°C. Three areas in the cortex of the ischemic penumbra were imaged using laser-scanning confocal microscopy (Nikon TE2000, Tokyo, Japan and LEICA TCS SP2, Solms, Germany). Caspase 3-positive cells were quantitatively measured using ImageJ software. All analyses were performed by investigators who were blinded to the treatment conditions. TdT-mediated dUTP nick end labeling assay To examine brain histopathology at 24 h post-I/R, brain slices (n = 5/group) were stained using an in situ cell death detection kit, according to the manufacturer’s instructions (Yang et al. 2000). Briefly, the sections were permeabilized with 1% proteinase K for 15 min, rinsed with PBS, exposed to the TdT-mediated dUTP nick end labeling (TUNEL) reaction for 1 h at 37°C, and then washed again in PBS for 5 min. Five randomly selected regions within the ischemic penumbra in the right cerebral hemisphere were counted under high-power magnification (4009). Three slices per brain were counted by a blinded investigator, and the average number of TUNEL-positive cells was recorded. Enzyme-linked immunosorbent assay Twenty-four hours after surgery, the rats (n = 8/group) were sacrificed, and the brains were immediately removed. Brain tissues were isolated and homogenized in potassium phosphate buffer. After centrifugation, the supernatant, which contained the crude membranes, was prepared for measuring methane dicarboxylic aldehyde (MDA); the remainder of the supernatant was centrifuged once again and used to estimate superoxide dismutase (SOD) and glutathione (GSH) levels. The levels of MDA, SOD, and GSH in the ischemic penumbra cortex tissue homogenates were analyzed using commercially available kits (Nanjing Jiancheng Biological Technology, Nanjing, China) according to the manufacturer’s instructions. The experiments were performed in triplicate. Western blotting The rats (n = 8/group) were decapitated at 24 h after reperfusion. The brain tissues of the ipsilateral penumbra were dissected on ice. The samples were homogenized with protein extraction reagent (Pierce, Illinois, USA) supplemented with protease inhibitors. The supernatants were collected, and the total protein concentrations were analyzed with a Bio-Rad Dc protein assay (Bio-Rad, Hercules, CA, USA). Equivalent amounts of total protein were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), blocked with a 5% skim milk solution, washed in Tris-buffered saline containing 0.1% Tween-20 and probed with primary antibodies, including a rabbit anti-phospho Akt (1 : 1000; Cell Signaling Technology), a rabbit anti-cleaved caspase
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3 antibody (1 : 1000; Abcam) and a mouse anti-b-actin antibody (1 : 5000; Sigma, St Louis, MO, USA), at 4°C overnight. The membranes were then incubated with horseradish peroxidaseconjugated anti-rabbit or anti-mouse antibodies for 2 h at 37°C. The western blot signals were quantified using an enhanced chemiluminescence kit and examined using the Bio-Image Analysis System (Bio-Rad). The expression ratio of each target protein was normalized against the expression of b-actin. Statistical analyses All values are expressed as the mean SEM of at least three independent experiments performed in triplicate. Comparisons between multiple groups were conducted using one-way ANOVA, followed by a post hoc Tukey’s multiple-comparisons test. The neurological scores were analyzed using the Kruskal–Wallis test. p < 0.05 was considered significant. All statistical analyses were performed using Statistical Product and Service Solutions (SPSS) (Version 17.0).
Results Physiological parameters The mean values for blood pressure, heart rate, and blood gas were not significantly different among the groups, and all values were within the normal range (Table 1). The results indicated that VNS treatment had no significant effect on these parameters during the experiment, which was consistent with previous study. (Sun et al. 2012). Activation of a7nAChR in the ischemic penumbra following VNS treatment To determine the stimulation effectiveness in ischemic stroke rats, we used a double immunofluorescence staining assay to detect the induction of a7nAchR expression. As shown in Fig. 1, ischemic insult caused a significant reduction in a7nAchR expression at the surfaces of neurons and astrocytes, whereas VNS treatment mitigated this effect. Our results demonstrate that VNS treatment affects brain tissues through the activation of a7nAchR, the expression of which plays an important role in the activation of intracellular signaling pathways via a7nAchR expression.
miR-210 levels under various experimental conditions As miR-210 is robustly and uniquely increased during hypoxia, we confirmed the expression of miR-210 using realtime PCR at 24 h following reperfusion near the ischemic boundary in rats. As shown in Fig. 2, compared with the I/R group, miR-210 level was not significantly different from that in the I/R+AC group; however, it was significantly decreased following miR-210 blockade (p < 0.05). A further increase in miR-210 expression was identified in the I/ R+VNS and I/R+VNS+AC groups (p < 0.05), whereas the level of miR-210 was reduced in the I/R+VNS+A group, albeit to a lower extent (p < 0.05). These results suggested that as a protective factor, miR-210 activity could be blocked using the miR-210 antagomir. Moreover, VNS treatment could continue to enhance the expression of miR-210 following ischemic stroke, whereas in the I/R+VNS+A group, the level of miR-210 was down-regulated, which indicates that miR-210 was likely involved in the response induced by VNS. Furthermore, the levels of miR-210 were not different between the I/R+VNS+AC and I/R+VNS groups and decreased in the I/R+VNS+A group compared with the I/R+VNS group. These findings further support the relationship between miR-210 and VNS treatment. Incidentally, the miR-210 level was increased to a greater extent in the I/R and I/R+VNS groups compared with the sham I/R and sham I/R+VNS groups, respectively (p < 0.05). These findings indicate that miR-210 is rapidly up-regulated in response to hypoxia, consistent with previous studies (Qiu et al. 2013). Blockade of miR-210 impairs the improvements in neurological deficit scores and infarct volumes induced by VNS As shown in Fig. 3(a and b), we evaluated neurological scores and infarct volumes in rats pretreated with the miR210 antagomir to determine the effects of silencing miR-210 expression following I/R and VNS treatment. There was no significant difference between the I/R+AC and I/R groups. Increased neurological deficit scores and infarct volume were identified in the I/R+A group compared with the I/R group.
Table 1 The physiological parameters during the experiment Group
Mean blood pressure (mmHg)
sham I/R I/R I/R+AC I/R+A sham I/R+VNS I/R+VNS I/R+VNS+AC I/R+VNS+A
90 83 84 87 89 81 80 78
4.0 5.8 6.0 6.1 7.3 8.8 9.5 10.4
Heart rate (bp/min) 376 369 370 373 370 363 365 362
11 9 9 10 11 11 13 14
PH 7.41 7.39 7.39 7.41 7.38 7.39 7.37 7.39
PCO2 (mmHg)
0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.01
47.0 46.8 47.1 46.9 45.6 46.2 46.7 47.2
1.2 0.9 1.3 1.2 1.3 0.9 1.2 1.0
PO2 (mmHg) 99.7 105.3 98.2 108.4 112.3 107.9 108.5 98.9
8.1 9.2 9.9 10.1 10.3 10.8 11.1 8.4
All data are shown as the mean SEM.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
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Fig. 1 The expression of a7nAchR on the surface of representative neurons and astrocytes revealed by double immunofluorescence staining for a7nAChR (red) and NeuN/astrocyte (green) in the ischemia penumbra of the three groups. Sham I/R group, I/R group, and I/R+VNS group. GFAP, glial fibrillary acidic protein (astrocyte marker). Scale bars = 50 lm.
Fig. 2 miRNA RT-PCR analysis of miR-210 expression with an antagomir control or with an antagomir following ischemic stroke and vagus nerve stimulation (VNS) treatment in the rat cerebral cortex. Data are expressed as the relative miR-210 levels normalized to the U6 endogenous control. Data are expressed as the mean SEM. ∇ p < 0.05 versus the sham I/R group. +p < 0.05 versus the sham I/ R+VNS group. #,&,*,@p < 0.05 versus the I/R group. ●p < 0.05 versus the I/R+VNS group.
Consistent with previous studies, VNS exerted significant neuroprotective effects, including improvements in neurological function and reductions in infarct volume compared with ischemic stroke rats; similar effects were also observed in the I/R+VNS+AC group. By contrast, these results appeared more severe in the I/R+VNS+A group compared with the I/R group. No changes were observed between the I/ R+VNS+AC and I/R+VNS groups. The improvements in neurological deficit scores and infarct volume induced by VNS treatment were significantly reduced as a result of miR210 knockdown, which was demonstrated via the comparison of the I/R+VNS+A and I/R+VNS groups. Based on our findings, we conclude that miR-210 exerts protection against ischemic infarct and mediates the neuroprotective effect of VNS in cerebral ischemia rats. Blockade of miR-210 via its antagomir attenuates the VNS-mediated protection against I/R injury As shown in Fig. 4, to further explore the role of miR-210 in mediating the protection induced by VNS against I/R injury in rats, we measured neuronal apoptotic markers after blocking miR-210. Compared with the I/R group, the number of TUNEL-positive cells was not remarkably different from
that of the I/R+AC group and was significantly increased compared with the I/R+A group, whereas it decreased after VNS treatment (p < 0.05). By contrast, an increased number of TUNEL-positive cells was identified in the ischemic penumbra in the I/R+VNS+A group compared with the I/R group. These findings indicated that miR-210 silencing exacerbated neuronal damage under hypoxic conditions. VNS treatment attenuated the apoptotic response, whereas this protection was decreased following miR-210 blockade preconditioning. Furthermore, there was no significant difference between the I/R+VNS+AC and I/R+VNS groups. However, the number of TUNEL-positive cells in the I/R+VNS+A group was greater than in the I/R+VNS group (p < 0.05). The results indicated that miR-210 down-regulation could markedly influence the VNS-mediated suppression of neuronal apoptosis. Thus, it appeared that miR-210 played a crucial role in the VNS-mediated anti-apoptosis effect against ischemic injury in rats. miR-210 is involved in the VNS-regulated oxidative stress response following cerebral ischemia in rats Cerebral ischemia results in the generation of excessive free radicals and lipid peroxidation. The three tested markers did not show significant changes in the I/R+AC group compared with the I/R group. MDA was significantly unregulated, whereas SOD and GSH were both down-regulated in the I/R+A group compared with the I/R group. Consistent with previous studies, we verified that VNS could decrease MDA levels and increase SOD and GSH levels to prevent further damage to brain tissues. No significant difference was identified between the I/R+VNS+AC and I/R+VNS groups. After miR-210 knockdown, the MDA level was higher in the I/R+VNS+A group compared with the I/R and I/R+VNS groups, whereas GSH and SOD activity levels remained lower (p < 0.05) (Fig. 5). In addition, ischemic stroke led to the up-regulation of MDA and the down-regulation of SOD and GSH activity; however, these effects were reversed following VNS treatment. These results also indicate that increased miR-210 expression reduces oxidative stress and that the antioxidant stress effects of VNS treatment are inhibited by miR-210 blockade.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
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(a)
(b)
Fig. 3 The effects of vagus nerve stimulation (VNS) treatment on neurological deficit scores (a) and infarct volume (b) following miR-210
knockdown. Data are expressed as the mean SEM. #,&,*,@p < 0.05 versus the I/R group. ●p < 0.05 versus the I/R+VNS group.
Fig. 4 Neuronal apoptosis was measured by TdT-mediated dUTP nick end labeling (TUNEL) staining 24 h after I/R in rats pretreated with miR-210 inhibitor and vagus nerve stimulation (VNS) treatment. Sham I/R, I/R, I/R+AC, I/R+A, sham I/R+VNS, I/R+VNS, I/R+VNS+AC, and
I/R+VNS. Scale bar = 100 lm. Data are expressed as the mean SEM. #,&,*,@p < 0.05 versus the I/R group. ●p < 0.05 versus the I/R+VNS group.
Suppression of postischemic apoptosis induced by VNS is attenuated following miR-210 knockdown preconditioning To examine the role of miR-210 mediation in the antiapoptotic effect induced by VNS, the protein levels of p-Akt and activated caspase 3 were measured by western blot analysis. There were no significant differences in p-Akt protein expression among the I/R+AC, I/R+A and I/R groups, whereas there was an increase in p-Akt expression in the I/R+VNS, I/R+VNS+AC, and I/R+VNS+A groups compared with the I/R group. No significant differences were detected among the I/R+VNS, I/R+VNS+AC and I/R+VNS+A groups. Therefore, VNS caused an increase in p-Akt protein expression following ischemia and was not significantly affected by miR-210 knockdown. Caspase 3 activity did not significantly change in the I/R+AC group, but it was markedly up-regulated in the I/R+A group compared with the I/R group. Importantly, the activity of caspase 3 was suppressed following VNS, whereas this inhibition was diminished in the I/R+VNS+A group compared with the I/R and I/R+VNS groups (p < 0.05) (Fig. 6a and b). Furthermore, caspase 3 protein expression in the I/R and I/R+VNS groups was significantly higher compared with the sham I/R
and sham I/R+VNS groups, respectively (p < 0.05). These results also suggested that VNS suppressed the apoptotic response following I/R. During this process, the action of miR-210 appears to be a protective factor that augments brain tissue survival. As expected, the anti-apoptotic effect induced by VNS treatment was decreased after miR-210 antagomir preconditioning. Therefore, miR-210 could mediate the antiapoptotic response induced by VNS in postischemic rat brains.
Discussion Recent studies demonstrated that VNS initiated 30 min after cerebral middle artery occlusion in rats reduced ischemic lesion volume, improved the neurological outcomes, reduced brain water content, and suppressed inflammatory cytokines (Mravec 2010). The benefits of VNS are associated with its central projections to various brain structures. The afferents of the vagus nerve fibers project bilaterally to the nucleus tractus solitarius in the medulla oblongata, which then projects to the locus coeruleus, the major source of norepinephrine in the brain. Furthermore, polysynaptic
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
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Fig. 5 miR-210 mediates the vagus nerve stimulation (VNS)-induced antioxidant response. Changes in the levels of MDA (methane dicarboxylic aldehyde), SOD (superoxide dismutase), and GSH (glutathione) in the ischemic penumbra in each group. Data are expressed
7
as the mean SEM. ∇p < 0.05 versus the sham I/R group. +p < 0.05 versus the sham I/R+VNS group. #,&,*,@p < 0.05 versus the I/R group. ● p < 0.05 versus the I/R+VNS group.
(a)
(b)
Fig. 6 miR-210 knockdown attenuates vagus nerve stimulation (VNS)induced protection against I/R injury in vivo. (a) Western blot analysis of p-Akt and cleaved caspase 3 with or without miR-210 knockdown in the VNS-treated group (b). Immunostaining reveals changes the number of cleaved caspase 3-positive cells following intervention. Scale
bar = 75 lm. Data are expressed as the mean SEM. ∇p < 0.05 versus the sham I/R group. +p < 0.05 versus the sham I/R+VNS group. #,&, *p < 0.05 versus the I/R group. ●p < 0.05 versus the I/R+VNS group.
projections from the nucleus tractus solitarius also innervate many other brain areas, including the thalamus, hypothalamus, limbic system, and cerebral cortex. It is well established that norepinephrine can stimulate 5-HT and acetylcholine
(ACh) release to exert potent anti-inflammatory effects in the brain (Cheyuo et al. 2011). Abundant evidence suggests that a7nAchR, which is an effector receptor that can be activated by drugs or electrical stimulation of the vagus nerve, plays an
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
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important role in the protective effects of VNS toward I/R injury in the brain. For example, the activation and up-regulation of Akt phosphorylation is involved in donepezil-dependent neuroprotection, which counteracts glutamate-induced neurotoxicity via a7nAchR activation (Oda et al. 2007). In our study, we measured various physiological parameters during the experimental process, all of which remained within the normal range. The simultaneous activation of a7nAchR on neurons and astrocytes was identified via immunofluorescence after VNS treatment, providing visual evidence of the effectiveness of electrical stimulation in the rat brain following ischemia. miR-210 is a highly conserved, small, noncoding RNA that participates in hypoxia-dependent disease states, including apoptosis, inflammation, and tumorigenesis. miR-210 exhibits neuroprotective effects via the inhibition of apoptosis in a murine model of hypoxic-ischemic encephalopathy (Qiu et al. 2013). Similarly, a recent study demonstrated that the introduction of a minicircle vector carrying a miR-210 precursor into an ischemic heart could dramatically improve heart function by the promotion of angiogenesis and the inhibition of apoptosis (Zhu and Fan 2012). In addition, Yang et al. (2012) demonstrated that miR-210 down-regulation significantly increased the apoptotic rate and radiosensitivity of human hepatoma cells. Our results indicate that miR-210 expression was up-regulated following VNS compared with the corresponding I/R groups. Furthermore, the up-regulation of miR-210 was positively correlated with lower neurological scores and smaller infarct volumes. Following miR-210 blockade, the neuroprotective effects of VNS were decreased. These results suggest that miR-210 is involved in the VNS-mediated neuroprotection. miR-210 is not only a potential stroke marker, but it may also be a key factor in the repression of mitochondrial metabolism in many cell types under hypoxic conditions. In one study, miR-210 overexpression increased the protection of mesenchymal stem cells against oxidative stress-induced injury; in another study, miR-210 knockdown in myoblasts significantly elevated myotube sensitivity to oxidative stress and resulted in mitochondrial dysfunction (Greco et al. 2014). VNS also confers protection against the accumulation of free radicals during myocardial and cerebral ischemia/ reperfusion (I/R) in rats (Kong et al. 2012; Ekici et al. 2013). Therefore, we attempted to determine whether miR210 was also involved in the VNS-mediated antioxidant signaling pathway. Our results indicate that VNS can significantly modulate the oxidative stress response, concomitant with an increase in miR-210. Indeed, the antioxidant effect of VNS induction was greatly decreased by miR210 blockade. Therefore, we infer that miR-210 plays an important role in the VNS-mediated antioxidant response, which also leads to an anti-apoptotic effect. Kim et al. (2009) demonstrated that rat mesenchymal stem cell survival was improved by increasing the expres-
sion of miR-210, which led to decreased levels of CASP8AP2, a direct target of miR-210 that was negatively correlated with cell survival. Increased miR-210 levels could have an effect on the activity of caspase 3/7 in hypoxia. Some, but not all, studies have suggested that miR-210 may be induced by p-Akt, which is another regulator of the hypoxia response (Chan and Loscalzo 2010). Kawada and coworkers confirmed that VNS significantly enhanced myocardial interstitial ACh levels in the ischemic region compared with baseline levels, triggering a cascade of survival signals through Akt phosphorylation and the inhibition of proapoptotic cascades (Kawada et al. 2007). These findings indicate that miR-210 and p-Akt could be correlated with the VNS-mediated antiapoptotic effects against ischemic damage. Consistent with this hypothesis, our results indicate that VNS exerts an anti-apoptotic effect, which is accompanied by an increase in p-Akt protein levels in the rat brain following I/R and a concomitant increase in miR-210 expression. Therefore, our findings suggest that VNS plays a critical role in facilitating the adaptation of brain tissues to hypoxic stress. To further evaluate the role of miR-210 in the anti-apoptotic effect of VNS, a miR-210 antagomir was administered to the lateral ventricles prior to MCAO surgery and VNS treatment. The anti-apoptotic effect induced by VNS was decreased after miR-210 knockdown pretreatment, and brain damage was not reduced under these conditions. These results indicate that miR-210 mediates the VNS-induced antiapoptotic effects in ischemic stroke in rats. Although the results of the miRNA array analysis indicated that there are also many other endogenous miRNAs involved in cerebral I/R processes, when considering our previous findings, we chose to focus on miR-210, which emerged as a major potential regulator of the VNS-mediated neuroprotective effect. Additional investigations of other miRNAs are required in the future. Taken together, these results indicate that miR-210 may be involved in the antioxidant and anti-apoptotic responses induced by VNS against I/R damage. To the best of our knowledge, this is the first report regarding the neuroprotective mechanism of VNS at the mRNA level. The downregulation of miR-210 attenuated the antiapoptotic and antioxidant effects induced by VNS in I/R rats. In summary, our findings provide valuable insight into the association between miR-210 and VNS-induced neuroprotection.
Acknowledgments and conflict of interest disclosure This work was supported by the National Nature and Science Foundation of China (Grant No. 81271306). There are no conflicts of interest in this manuscript. All experiments were conducted in compliance with the ARRIVE guidelines.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13097
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References Ay I., Lu J., Ay H. and Sorensen A. G. (2009). Vagus nerve stimulation reduces infarct size in rat focal cerebral ischemia. Neurosci. Lett. 459, 147–151. Ay I., Sorensen A. G. and Ay H. (2011) Vagus nerve stimulation reduces infarct size in rat focal cerebral ischemia: an unlikely role for cerebral blood flow. Brain Res. 1392, 110–115. Burnett M. G., Shimazu T., Szabados T., Muramatsu H., Detre J. A. and Greenberg J. H. (2006) Electrical forepaw stimulation during reversible forebrain ischemia decreases infarct volume. Stroke 37, 1327–1331. Chan S. Y. and Loscalzo J. (2010) MicroRNA-210: a unique and pleiotropic hypoxamir. Cell Cycle 9, 1072–1083. Chen L., Wei X., Hou Y., Liu X., Li S., Sun B., Liu X. and Liu H. (2014) Tetramethylpyrazine analogue CXC195 protects against cerebral ischemia/reperfusion-induced apoptosis through PI3K/ Akt/GSK3. Neurochem. Int. 66, 27–32. Cheyuo C., Jacob A., Wu R., Zhou M., Coppa G. F. and Wang P. (2011) The parasympathetic nervous system in the quest for stroke therapeutics. J. Cereb. Blood Flow Metab. 31, 1187–1195. Conway C. R., Sheline Y. I., Chibnall J. T., Bucholz R. D., Price J. L., Gangwani S. and Mintun M. A. (2012) Brain blood-flow change with acute vagus nerve stimulation in treatment-refractory major depressive disorder. Brain Stimul. 5, 163–171. Dharap A., Bowen K., Place R., Li L. C. and Vemuganti R. (2009) Transient focal ischemia induces extensive temporal changes in rat cerebral microRNAome. J. Cereb. Blood Flow Metab. 29, 675– 687. Ekici F., Karson A., Dillioglugil M. O., Gurol G., Kir H. M. and Ates N. (2013) The effects of vagal nerve stimulation in focal cerebral ischemia and reperfusion model. Turk. Neurosurg. 23, 451–457. Fu S. H., Zhang H. F., Yang Z. B., Li T. B., Liu B. and Lou Z. (2014) Alda-1 reduces cerebral ischemia/reperfusion injury in rat through clearance of reactive aldehydes. Naunyn Schmiedebergs Arch. Pharmacol. 387, 87–94. Goyagi T., Nishikawa T. and Tobe Y. (2011) Neuroprotective effects and suppression of ischemia-induced glutamate elevation by b1adrenoreceptor antagonists administered before transient focal ischemia in rats. J. Neurosurg. Anesthesiol. 23, 131–137. Greco S., Gaetano C. and Martelli F. (2014) HypoxamiR regulation and function in ischemic cardiovascular diseases. Antioxid. Redox Signal. 21, 1202–1219. Guarini S., Altavilla D., Cainazzo M. M. et al. (2003) Efferent vagal fibre stimulation blunts nuclear factor-kappaB activation and protects against hypovolemic hemorrhagic shock. Circulation 107, 1189–1194. Hiraki T., Baker W. and Greenberg J. H. (2012) Effect of vagus nerve stimulation during transient focal cerebral ischemia on chronic outcome in rats. J. Neurosci. Res. 90, 887–894. Hong L., Han Y., Zhang H., Zhao Q. and Qiao Y. (2013) miR-210: a therapeutic target in cancer. Expert Opin. Ther. Targets 17, 21–28. Jiang Y., Li L., Liu B., Zhang Y., Chen Q. and Li C. (2014) Vagus nerve stimulation attenuates cerebral ischemia and reperfusion injury via endogenous cholinergic pathway in rat. PLoS ONE 9, e102342. Katare R. G., Ando M., Kakinuma Y., Arikawa M., Handa T., Yamasaki F. and Sato T. (2009) Vagal nerve stimulation prevents reperfusion injury through inhibition of opening of mitochondrial permeability
9
transition pore independent of the bradycardiac effect. J. Thorac. Cardiovasc. Surg. 137, 223–231. Kawada T., Yamazaki T., Akiyama T., Li M., Zheng C., Shishido T., Mori H. and Sugimachi M. (2007) Angiotensin II attenuates myocardial interstitial acetylcholine release in response to vagal stimulation. Am. J. Physiol. Heart Circ. Physiol. 293, 2516–2522. Kim H. W., Haider H. K., Jiang S. and Ashraf M. (2009) Ischemic preconditioning augments survival of stem cells via miR-210 expression by targeting caspase-8-associated protein 2. J. Biol. Chem. 284, 33161–33168. Koizumi J., Yoshida Y., Nakazawa T. and Ooneda G. (1986) Experimental studies of ischemic brain edema: 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn. J. Stroke 8, 1–8. Kong S. S., Liu J. J., Yu X. J., Lu Y. and Zang W. J. (2012) Protection against ischemia-induced oxidative stress conferred by vagal stimulation in the rat heart: involvement of the AMPK-PKC pathway. Int. J. Mol. Sci. 13, 14311–14325. Livak K. J. and Schmittgen T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. Morris G. L., 3rd, Gloss D., Buchhalter J., Mack K. J., Nickels K. and Harden C. (2013) Evidence-based guideline update: vagus nerve stimulation for the treatment of epilepsy: report of the guideline development subcommittee of the american academy of neurology. Epilepsy Curr. 13, 297–303. Mravec B. (2010) The role of the vagus nerve in stroke. Auton Neurosci. 158, 8–12. Oda T., Kume T., Katsuki H., Niidome T., Sugimoto H. and Akaike A. (2007) Donepezil potentiates nerve growth factor-induced neurite outgrowth in PC12 cells. J. Pharmacol. Sci. 104, 349–354. Qiu J., Zhou X. Y., Zhou X. G., Cheng R., Liu H. Y. and Li Y. (2013) Neuroprotective effects of microRNA-210 on hypoxic-ischemic encephalopathy. Biomed Res. Int. 2013, 350419. Sch€abitz W. R., Weber J., Takano K., Sandage B. W., Locke K. W. and Fisher M. (1996) The effects of prolonged treatment with citicoline in temporary experimental focal ischemia. J. Neurol. Sci. 138, 21– 25. Sun Z., Baker W., Hiraki T. and Greenberg J. H. (2012) The effect of right vagus nerve stimulation on focal cerebral ischemia: an experimental study in the rat. Brain Stimul. 5, 1–10. Won S., Lee J. H., Wali B., Stein D. G. and Sayeed I. (2014) Progesterone attenuates hemorrhagic transformation after delayed tPA treatment in an experimental model of stroke in rats: involvement of the VEGF-MMP pathway. J. Cereb. Blood Flow Metab. 34, 72–80. Yang S. H., Shi J., Day A. L. and Simpkins J. W. (2000) Estradiol exerts neuroprotective effects when administered after ischemic insult. Stroke 31, 745–749. Yang W., Sun T., Cao J., Liu F., Tian Y. and Zhu W. (2012) Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro. Exp. Cell Res. 318, 944–954. Zhu H. and Fan G. C. (2012) Role of MicroRNAs in the reperfused myocardium towards post-infarct remodelling. Cardiovasc. Res. 94, 284–292.
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