Research A combination of neostigmine and anisodamine protects against ischemic stroke by activating α7nAChR Jiao Qian1,2†, Jing-Ming Zhang2†, Li-Li Lin3, Wen-Zhe Dong2, Yan-Qiong Cheng4, Ding-Feng Su2*, and Ai-Jun Liu2* Background Increasing endogenous acetylcholine by neostigmine decreased the ischemic cerebral injury. The off-target action on muscarinic receptor produced a variety of adverse effects and limited the clinical application on stroke. Aim We combined neostigmine with anisodamine and investigated the neuroprotection and mechanism. Methods Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion. Neuroprotective action of neostigmine in combination with anisodamine at varying ratios was examined to determine the optimal combination as well as ideal therapeutic window. Potential involvement of α7 nicotinic acetylcholine receptor was examined by measuring the infarct size, the expression of proinflammatory cytokines, and the biomarkers of apoptosis in α7 nicotinic acetylcholine receptor knockout mice. A set of in vitro experiments was conducted in RAW264.7 cells to probe into potential molecular mechanisms. Results The neostigmine/anisodamine combination conferred neuroprotection. The protection was most potent at a ratio of 1 : 500. At such a ratio, the combination increased the binding of acetylcholine to α7 nicotinic acetylcholine receptor and reduced proinflammatory cytokines. The neuroprotection was evident only in wild-type and not in α7 nicotinic acetylcholine receptor knockout mice. The combination significantly decreased the expression of Bad and Bax, and increased Bcl-2 and Bcl-xl in α7 nicotinic acetylcholine receptor wild-type mice but not in knockout mice. The combination did not affect caspase-8, cleaved caspase-8, or caspase-12. Conclusions Current study identified the optimal combination of neostigmine and anisodamine against ischemic stroke, and indicated that the acetylcholine-α7 nicotinic acetylcholine receptor is involved in the protective effects. Key words: α7nAChR, anisodamine, ischemic stroke, neostigmine

Introduction Stroke is the second most common cause of death and a major cause of chronic disability in adults (1–3). Ischemic stroke is the Correspondence: Ai-Jun Liu* and Ding-Feng Su*, Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433, China. E-mail: [email protected]; [email protected] 1 Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai, China 2 Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai, China 3 Department of Pharmacology, Wuxi Higher Health Vocational Technology School, Wuxi, Jiangsu, China 4 Department of Urology, Changhai Hospital, Second Military Medical University, Shanghai, China Received: 4 August 2014; Accepted: 14 November 2014; Published online 2 March 2015 †

These authors contributed equally to this work.

Conflicts of interest: None declared. DOI: 10.1111/ijs.12458 © 2015 World Stroke Organization

largest subtype of stroke. Approved treatments for acute ischemic stroke include thrombolysis and anti-platelet therapy. Intervention focusing on neuroprotection is promising but has achieved only limited success upon translation into clinical practice (4). In a recent study, we noticed significant attenuation of ischemic cerebral injury upon increasing endogenous acetylcholine (ACh) by the anticholinesterase inhibitor neostigmine (3). Preliminary evidence in this study also indicated that the neuroprotective action of neostigmine is mediated by nicotinic ACh receptor (nAChR) and not muscarinic ACh receptor (mAChR) (3). The off-target action of neostigmine on mAChR, however, could produce a variety of adverse effects, including bradycardia, abdominal pain, hydrostomia, and bronchospasm. These sideeffects severely limited the clinical application on stroke. Anisodamine is a belladonna alkaloid that antagonizes mAChR nonselectively. Anisodamine has less toxic effects than atropine and less activity in the central nerve system than scopolamine (5). A previous study from this laboratory also suggested that anisodamine could decrease the levels of inflammatory cytokines and produce antishock effects (6,7). Because inflammation is intrinsically implicated in neuronal damage caused by stroke (8,9), we conducted a series of experiments to examine the potential benefits of combining neostigmine with anisodamine.

Methods Animals Male Sprague-Dawley rats were purchased from Sino-British SIPPR/BK Lab Animals (Shanghai, China). The α7 nicotinic acetylcholine receptor (α7nAChR) knockout (KO) mice were obtained from Jackson Laboratory (Bar Harbor, MA, USA) (B6.129S7-Chrna7tm1 Bay, Stock Number: 003232). All animals were used in accordance with the guidelines of Second Military Medical University for Animal Care. Middle cerebral artery occlusion, neurological deficit scoring, and 2, 3, 5-triphenyltetrazolium chloride staining The rats or mice were anesthetized with 2·0% isoflurane. Middle cerebral artery occlusion (MCAO) surgery was performed as described (10–12). The core temperature was maintained between 36·5°C and 37·5°C throughout the surgery by using a heating pad (Nanjing Xin Xiao Yuan Biotech, Nanjing, China). Focal ischemia was produced by intraluminal occlusion of the left middle cerebral artery (MCA) using a silicone rubber-coated nylon monofilament (Beijing Sunbio Biotech Co., Ltd, Beijing, China). Cessation of cerebral blood flow (CBF) to the area supplied by the left MCA was verified by using a laser Doppler computerized main unit (ML191, ADInstruments, New South Wales, Australia). Cerebral blood flow must be reduced by at least 70% for inclusion in further Vol 10, July 2015, 737–744

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Research experiments. Two-hours after MCAO, the occluding filament was withdrawn to allow reperfusion. Twenty-four hours after MCAO, the neurological score was measured as described (10–12). Then the animals were sacrificed for various examinations. The coronal slices of brains were prepared with brain-cutting matrix (ASI Instruments, Warren, MI, USA) and incubated in 1% 2, 3, 5-triphenyltetrazolium chloride solution (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) at 37°C for 15 min. Then the slices were photographed with a digital camera. The infarct size was traced and quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA) and expressed as a percentage of the contralateral hemisphere. The possible interference of a brain edema in assessing the infarct size was corrected with a standard method of subtracting the volume of the nonischemic ipsilateral hemisphere from that of the contralateral hemisphere. Fluorescent staining of α7nAChR by fluorescein isothiocyanate-labeled α-bungarotoxin RAW264.7 cells (Chinese Academy of Sciences, Shanghai, China) were cultured in high-glucose Dulbecco minimum essential medium (Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Gibco). Cells (1 × 105) were inoculated in 35-mm culture dish (NEST, Beijing, China) and treated with vehicle (0·9% normal saline), the combination of neostigmine and anisodamine (1 : 500: neostigmine 0·2 μg/ml + anisodamine 100 μg/ml), or ACh (750 μM, Sigma-Aldrich, MO, USA) at 37°C for 20 min prior to staining with 1·5 μg/ml of fluorescein isothiocyanate-labeled α-bungarotoxin (α-Bgt) (Sigma-Aldrich) at 4°C for 15 min. Cells were washed extensively and fixed in 4% paraformaldehyde- phosphate-buffered saline solution at 22°C for 15 min. After fixation, the cells were washed twice with phosphate-buffered saline solution, mounted in glycerol, and examined under a fluorescent confocal microscope (Leica TCSSP5, Wetzlar, Germany). The data were analyzed using ImageJ software as described previously (6). Immunoblotting Immunoblotting was performed as previously reported (3,13). Tissue lysate from ischemic penumbra was boiled, subjected to the sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and transferred onto nitrocellulose blotting membranes. The membranes were incubated with one of the following antibodies: rabbit anti-caspase 12 (BD Biosciences, San Jose, CA, USA), cleaved caspase 8 antibody, caspase-8 rabbit mAb, Bcl-xl rabbit mAb, Bad rabbit mAb, Bcl-2 rabbit mAb, and Bax antibody (six antibodies from Cell Signaling Technology, Boston, MA, USA). The images were captured by the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE, USA) and analyzed by ImageJ software (National Institutes of Health). Analysis of proinflammatory cytokines in serum The serum levels of tumour necrosis factor alpha (TNFα), interleukin-6 (IL-6), and interleukin-1α (IL-1α) were examined using an enzyme-linked immunosorbent assay system (Infinite M200; Tecan Austria GmbH, Grödig, Austria) with a commercial kit (R&D, Minneapolis, MN, USA).

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J. Qian et al. Statistical analysis The animals were randomly assigned by using the random permutations table. The investigators were blinded to the procedures when they assessed the infarct area and neurological deficient score. Data are expressed as mean ± standard deviation and analyzed with Student’s t-test or one-way analysis of variance. P < 0·05 was considered statistically significant.

Results Anisodamine, neostigmine, and their combination confer neuroprotection To investigate the protective effect and the optimal doses of anisodamine and neostigmine, male Sprague-Dawley rats were subjected to MCAO before the drugs injection by vein. Then the animals were divided into seven groups (n = 10 in each group) and treated by the following drugs: 10, 20, and 40 μg/kg of neostigmine (neostigmine methylsulfate, Wuhan Yuancheng Gong Chuang Technology Co., Ltd, Wuhan, Hubei Province, China); 10, 20, and 40 mg/kg of anisodamine (Hangzhou Hejin Technology Co., Ltd, Hangzhou, Zhejiang Province, China). The rate of death after the MCAO experiments was 13% and did not differ between the vehicle control group and the groups receiving neostigmine/ anisodamine. Cerebral blood flow reduction was 0·05 P < 0·05 P < 0·05 P > 0·05 P < 0·05 P < 0·05 P < 0·01 P < 0·01 P < 0·01 P < 0·01

Data are presented as mean ± standard deviation (SD). n = 10 in each group before middle cerebral artery occlusion (MCAO). Cessation of cerebral blood flow reduction was 0·05).

Dose range and time window In this series of experiments, 40 rats were divided into 4 groups and MCAO was performed. Five animals died after MCAO, and three animals were discarded because CBF reduction was 0·05). We also detected three proinflammatory cytokines, TNFα, IL-1α, and IL-6, at six-hours after MCAO in the α7nAChR KO mice and their littermate WT mice. In the vehicle groups, the serum levels of all these three cytokines were significantly increased in α7nAChR KO mice than in WT mice (TNFα, 36·2 ± 7·63 pg/ml vs. 24·8 ± 6·85 pg/ml, P < 0·05; IL-1α, 82·4 ± 14·8 pg/ml vs. 63·9 ± 16·2 pg/ml, P < 0·05; IL-6, 134 ± 41·2 pg/ml vs. 89·4 ± 14·9 pg/ml, P < 0·05 n = 5, Fig. 5). The neostigmine/ anisodamine combination significantly decreased the serum levels of TNFα (11·6 ± 3·86 pg/ml vs. 24·8 ± 6·85 pg/ml, P < 0·01), IL-1α (41·7 ± 11·9 pg/ml, n = 6 vs. 63·9 ± 16·2 pg/ml, P < 0·05), and IL-6 (64·9 ± 12·4 pg/ml vs. 89·4 ± 14·9 pg/ml, P < 0·01) in the α7nAChR WT mice, but not in the KO mice (Fig. 5). Taken together, these results indicate that the neuroprotection and anti-inflammation of the combination are α7nAChR dependent. © 2015 World Stroke Organization

Decreased apoptosis in the cerebral ischemic penumbra by the neostigmine/anisodamine treatment is dependent on α7nAChR To further ensure the anti-apoptosis effect of the neostigmine/ anisodamine combination and clarify the apoptosis pathway, we measured the expression of caspase-8, cleaved caspase-8 (related to death receptors pathway), caspase-12 (endoplasmic reticulumspecific pathway), and some biomarkers (Bcl-2, Bax, Bcl-xl, and Bad) involved in mitochondrial pathway (14). First, six α7nAChR WT mice (three-months old) were subjected to MCAO then administrated intravenously with the combination or the vehicle (n = 3 in each group). Twenty-four hours after MCAO, the cerebral ischemic penumbra was extracted for the following detection. The combination treatment did not affect the expression of caspase-8, cleaved caspase-8, or caspase-12 in α7nAChR WT mice (Fig. 6). The combination decreased the expression of Bad and Bax (by about 47% and 36%, respectively) and increased the expression of Bcl-2 and Bcl-xl (by 36% and 83%, respectively). These data suggest that mitochondrial pathway was involved in the protection. In α7nAChR KO mice subjected to MCAO, Bad and Bax were significantly increased. The combination treatment did not affect the expression of Bad and Bax in α7nAChR KO mice (Fig. 6).

Discussion Inflammation is an invariable pathophysiological response to ischemic stroke and contributes to secondary damage (15–17). Increases in proinflammatory cytokines and acute phase response appear to predict poor outcome after stroke (8,9,16,18). However, clinical trials (near 200 in total number) of anti-inflammatory compounds, including dexamethasone (1971), enlimomab (antiICAM-1 antibody, 1996), and FK506 (tacrolimus, 2004), have achieved limited success in translation into clinical practice (4). Vol 10, July 2015, 737–744

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Fig. 6 The inhibition of apoptosis by the combination of neostigmine and anisodamine is dependent on α7 nicotinic acetylcholine receptor (α7nAChR). The expressions of caspase-8, cleaved caspase-8, caspase-12, Bcl-2, Bad, Bcl-xl, and Bax were detected in the ischemic penumbra of α7nAChR knockout (KO) and wild-type (WT) mice on day 1 after middle cerebral artery occlusion (MCAO). Data are expressed as mean ± standard deviation (SD).* P < 0·05, ** P < 0·01, n.s., not significant. Student’s t-test, n = 3 in each group.

One of the reasons for limited success is the complicated mechanisms involved in ischemic stroke injury. Activation of efferent vagus nerve could widely decrease proinflammatory cytokines (19). In addition to the classical actions of bronchoconstriction, bradycardia, and gastrointestinal motility, vagus activation stimulates the release of ACh, which in turn inhibits the production of proinflammatory cytokines. Electrical vagus nerve stimulation could attenuate the development of shock, endotoxemia, and ischemic reperfusion injury (19). In a recent study, we showed that increasing endogenous ACh could attenuate acute cerebral ischemic injury, via a mechanism dependent on the nAChR but not mAChR (3). However, nonselective stimulation of both nAChR and mAChR by the cholinesterase inhibitor or the direct vagus nerve stimulation could produce severe side-effects. In this study, we showed that adding the mAChR antagonist anisodamine to neostigmine could attenuate the side-effects of neostigmine. More importantly, the combination of neostigmine and anisodamine with a proper ratio had a potent reaction against stroke. The present study also suggests that the activation of

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cholinergic pathway and α7nAChR play a key function against ischemic stroke. The nAChRs, particularly α7nAChR, are reported to be involved in neuronal survival and synaptic plasticity (20,21). It is well known that the α7nAChR is required for the antiinflammatory effects (19). Our results also identified α7nAChR as a potent target for stroke. In the current study, MCAO produced much larger infarct size in α7nAChR KO mice. Also, the reduction of infarct size and proinflammatory cytokines by the combination of neostigmine/anisodamine was not evident in α7nAChR KO mice. More importantly, anisodamine augmented the binding of endogenous ACh to α7nAChR through its blockade of the mAChR (22). It also helped us to understand that the protective effect of the combination was better than the drug alone (Fig. 7). Minimizing neuronal apoptosis in the ischemic penumbra is also an important strategy to manage ischemic stroke. Major pathways for apoptosis include the death receptors pathway, endoplasmic reticulum-specific pathway, and mitochondrial pathway (14,22). Cytoprotective Bcl-2 family proteins such as Bcl-2 and Bcl-XL prevent mitochondrial permeability transition © 2015 World Stroke Organization

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Fig. 7 The proposed mechanism underlying the combination of neostigmine and anisodamine against ischemic stroke. α7nAChR, α7 nicotinic acetylcholine receptor; ACh, acetylcholine; AChE, acetylcholinesterase; mAChR, muscarinic ACh receptor; nAChR, nicotinic ACh receptor.

pore opening and release of apoptogenic proteins from mitochondria, which would result in either apoptosis or necrosis. In contrast, some pro-apoptotic members, such as Bax, can induce these destructive changes in mitochondria (22). Results from the current study suggest that the neostigmine/anisodamine combination reduced the apoptosis might link to the inhibition of the mitochondrial pathway. It is noteworthy that the anti-apoptotic effects of the neostigmine/anisodamine combination are also dependent on α7nAChR. In conclusion, results from the current study indicate that, at proper ratio(s), the combination of neostigmine and anisodamine could confer significant and efficient protection against the acute ischemic cerebral injury by the cholinergic α7nAChR pathway.

Acknowledgments This study was supported by the National Natural Science Foundation of China (81273505 and 81230083) and Science and Technology Commission Project of Shanghai (14ZR1408200).

Authors’ contributions J. Q., A-J. L., and J-M. Z. performed all experiments except those specified otherwise and analyzed the data. L-L. L. performed MCAO operation and analyzed the data. W-Z. D. cultured the RAW264.7 cells. W-Z. D. and Y-Q. C. conducted Western blot © 2015 World Stroke Organization

analysis. A-J. L. and D-F. S. designed this study and revised the manuscript. A-J. L. wrote the manuscript.

References 1 Donnan GA, Fisher M, Macleod M, Davis SM. Stroke. Lancet 2008; 371:1612–23. 2 Jauch EC, Cucchiara B, Adeoye O et al. Part 11: adult stroke: 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation 2010; 122(18 Suppl. 3):S818–28. 3 Liu AJ, Zang P, Guo JM et al. Involvement of acetylcholinealpha7nAChR in the protective effects of arterial baroreflex against ischemic stroke. CNS Neurosci Ther 2012; 18:918–26. 4 Sutherland BA, Minnerup J, Balami JS, Arba F, Buchan AM, Kleinschnitz C. Neuroprotection for ischaemic stroke: translation from the bench to the bedside. Int J Stroke 2012; 7:407–18. 5 Poupko JM, Baskin SI, Moore E. The pharmacological properties of anisodamine. J Appl Toxicol 2007; 27:116–21. 6 Liu C, Shen FM, Le YY et al. Antishock effect of anisodamine involves a novel pathway for activating alpha7 nicotinic acetylcholine receptor. Crit Care Med 2009; 37:634–41. 7 Zhou JX, Ke P, Huan G, Shao BZ, Liu C. Combined treatment with anisodamine and neostigmine inhibits joint inflammation in collagen-induced arthritis mice. CNS Neurosci Ther 2014; 20:186–7. 8 Di Napoli M, Papa F. Inflammation, hemostatic markers, and antithrombotic agents in relation to long-term risk of new cardiovascular events in first-ever ischemic stroke patients. Stroke 2002; 33:1763–71. 9 Tzoulaki I, Murray GD, Lee AJ, Rumley A, Lowe GD, Fowkes FG. Relative value of inflammatory, hemostatic, and rheological factors for incident myocardial infarction and stroke: the Edinburgh Artery Study. Circulation 2007; 115:2119–27. Vol 10, July 2015, 737–744

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Research 10 Guo JM, Liu AJ, Zang P et al. ALDH2 protects against stroke by clearing 4-HNE. Cell Res 2013; 23:915–30. 11 Gui H, Guo YF, Liu X et al. Effects of combination therapy with levamlodipine and bisoprolol on stroke in rats. CNS Neurosci Ther 2013; 19:178–82. 12 Chen CH, Jiang Z, Yan JH et al. The involvement of programmed cell death 5 (PDCD5) in the regulation of apoptosis in cerebral ischemia/ reperfusion injury. CNS Neurosci Ther 2013; 19:566–76. 13 Sun Y, Gui H, Li Q et al. MicroRNA-124 protects neurons against apoptosis in cerebral ischemic stroke. CNS Neurosci Ther 2013; 19:813–9. 14 Nakagawa T, Zhu H, Morishima N et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 2000; 403:98–103. 15 Emsley HC, Tyrrell PJ. Inflammation and infection in clinical stroke. J Cereb Blood Flow Metab 2002; 22:1399–419. 16 Rost NS, Wolf PA, Kase CS et al. Plasma concentration of C-reactive protein and risk of ischemic stroke and transient ischemic attack: the Framingham study. Stroke 2001; 32:2575–9.

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J. Qian et al. 17 Gu LJ, Xiong XX, Ito T et al. Moderate hypothermia inhibits brain inflammation and attenuates stroke-induced immunodepression in rats. CNS Neurosci Ther 2014; 20:67–75. 18 Vila N, Filella X, Deulofeu R, Ascaso C, Abellana R, Chamorro A. Cytokine-induced inflammation and long-term stroke functional outcome. J Neurol Sci 1999; 162:185–8. 19 Pavlov VA, Tracey KJ. Controlling inflammation: the cholinergic antiinflammatory pathway. Biochem Soc Trans 2006; 34(Pt 6):1037–40. 20 Egleton RD, Brown KC, Dasgupta P. Nicotinic acetylcholine receptors in cancer: multiple roles in proliferation and inhibition of apoptosis. Trends Pharmacol Sci 2008; 29:151–8. 21 Parada E, Egea J, Romero A, del Barrio L, Garcia AG, Lopez MG. Poststress treatment with PNU282987 can rescue SH-SY5Y cells undergoing apoptosis via alpha7 nicotinic receptors linked to a Jak2/ Akt/HO-1 signaling pathway. Free Radic Biol Med 2010; 49:1815–21. 22 Reed JC, Jurgensmeier JM, Matsuyama S. Bcl-2 family proteins and mitochondria. Biochim Biophys Acta 1998; 1366:127–37.

© 2015 World Stroke Organization

A combination of neostigmine and anisodamine protects against ischemic stroke by activating α7nAChR.

Increasing endogenous acetylcholine by neostigmine decreased the ischemic cerebral injury. The off-target action on muscarinic receptor produced a var...
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