involvement of arterial baroreflex in the protective effect of dietary restriction against stroke

ORIGINAL ARTICLE

Involvement of arterial baroreflex in the protective effect ofdietary restriction against strokeAi-Jun Liu1,5, Jin-Min Guo1,5, Wei Liu1, Feng-Yun Su1, Qi-Wei Zhai2, Jawahar L Mehta3, Wei-Zhong Wang4 and Ding-Feng Su1

Dietary restriction (DR) protects against neuronal dysfunction and degeneration, and reduces the risk of ischemic stroke. This studyexamined the role of silent information regulator T1 (SIRT1) and arterial baroreflex in the beneficial effects of DR against stroke,using two distinct stroke models: stroke-prone spontaneously hypertensive rats (SP-SHRs) and Sprague-Dawley (SD) rats withmiddle cerebral artery occlusion (MCAO). Sirt1 knockout (KO) mice were used to examine the involvement of sirt1. Sinoaorticdenervation was used to inactivate arterial baroreflex. Dietary restriction was defined as 40% reduction of dietary intake. Briefly, DRprolonged the life span of SP-SHRs and reduced the infarct size induced by MCAO. Dietary restriction also improved the functionarterial baroreflex, decreased the release of proinflammatory cytokines, and reduced end-organ damage. The beneficial effect of DRon stroke was markedly attenuated by blunting arterial baroreflex. Lastly, the infarct area in sirt1 KO mice was significantly largerthan in the wild-type mice. However, the beneficial effect of DR against ischemic injury was still apparent in sirt1 KO mice.Accordingly, arterial baroreflex, but not sirt1, is important in the protective effect of DR against stroke.

Journal of Cerebral Blood Flow & Metabolism advance online publication, 27 February 2013; doi:10.1038/jcbfm.2013.28

Keywords: arterial baroreflex; dietary restriction; sirt1; stroke

INTRODUCTIONThe beneficial effects of dietary restriction (DR), a reduction ofenergy intake without affecting the nutritional needs, against age-related diseases are well documented. Dietary restriction prolongsthe lifespan of many organisms ranging from yeast to mammals.1

Dietary restriction also delays the occurrence of pathophysiologicalchanges including atherosclerosis, diabetes and cancer in somemammalian species,2 and lower cardiovascular disease risk.3

Dietary restriction also protects neurons from dysfunction anddegeneration, and reduces the risk of ischemic stroke.4

The benefits of DR are partly caused by induction of neuro-trophic factors, protein chaperones, antioxidant enzymes, uncou-pling proteins, and antiinflammatory cytokines.5 Among thesetargets, silent information regulator T1 (SIRT1) has attracted muchattention. Silent information regulator T1 is activated by DR andhas been implicated in various beneficial effects associated withDR.6 The beneficial effects of DR are multifold and might not beexplained by change in one gene, or one protein. Many systems,including the metabolic system,7,8 and the nervous system,9 areinvolved.

Arterial baroreflex is the most important regulator of cardio-vascular functions.10–12 Decreased baroreflex sensitivity (BRS) isoften seen with aging and in many age-related diseases,13 and animportant prognostic factor for long-term outcome in manycardiovascular diseases, including myocardial infarction, heartfailure, hypertension, and stroke.11,12 Recently, the work in thislaboratory suggested that arterial baroreflex dysfunction is animportant risk fact for the development of stroke.14,15

In the current study, we tested the hypothesis thatarterial baroreflex is involved in the protective effect of DRagainst stroke. The interaction of DR, SIRT1, and stroke was alsoevaluated.

MATERIALS AND METHODSAnimalsMale stroke-prone spontaneously hypertensive rats (SP-SHRs) wereprovided by the Animal Center of the Second Military Medical University.Male Sirt1 knockout (KO) mice, and littermate wild-type (WT) mice wereobtained from the Institute for Nutritional Sciences, Shanghai Institutes forBiological Sciences, the Chinese Academy of Sciences. Male Sprague-Dawley (SD) rats were purchased from Sino-British SIPPR/BK Lab Animals(Shanghai, China). All the animals used in this study received humane carein compliance with institutional guidelines of Second Military MedicalUniversity for health and care of experimental animals. And all experimentswere approved by Second Military Medical University for health and care ofexperimental animals.

Dietary RestrictionAll diets were purchased from Institute of Naval Medicine of China. Thediet consisted of: barley flour (14.3%), wheat flour (31.8%), corn flour (15%),bean pulp flour (14%), wheat bran (18.9%), soybean oil (4.5%), and mineralmix (1.5%, including 0.45% NaCl). Both rats and mice were placed underunrestricted diet (ad libitum, AL) or DR (food intake at 60% of the amountconsumed by AL group animals). The mean food intake of AL animals perweek was estimated by subtracting the remaining weight of food eachweek from the initial weight of the previous week.

1Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai, China; 2Institute for Nutritional Sciences, Shanghai Institutes for BiologicalSciences, Chinese Academy of Sciences, Shanghai, China; 3Department of Internal Medicine, Physiology and Biophysics, Stebbins Chair in Cardiology, Division of CardiovascularMedicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA and 4Department of Physiology, Second Military Medical University, Shanghai, China.Correspondence: W-Z Wang, Department of Physiology, Second Military Medical University, Shanghai 200433, China or D-FSU, Department of Pharmacology, School of Pharmacy,Second Military Medical University, Shanghai 200433, China.E-mail: [email protected] or [email protected] study was supported by China Basic Research Program (2009CB521901) and National Natural Science Foundation of China (30901809, 81273505, and 81230083).5These two authors contributed equally to this work.Received 23 November 2012; revised 31 December 2012; accepted 14 January 2013

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Sinoaortic DenervationSinoaortic denervation (SAD) was performed as described previously.14

Briefly, rats received a mixture of ketamine and diazepam (50 mg/kgþ 5 mg/kg, intraperitoneally), plus atropine sulfate (0.5 mg/kg, intra-peritoneally) and procaine benzylpenicillin (60,000 U, intramuscularly).The superior laryngeal nerves were transected near the vagus nerves onboth sides. The superior cervical ganglia and a small section of thesympathetic trunk were removed. The aortic depressor nerves were alsotransected on both sides. The carotid sinus baroreceptors were mecha-nically stripped at the carotid bifurcation and its branches. The external,internal, and common carotid arteries and the occipital artery were treatedwith 10% phenol (in 95% ethanol). The sham operation included a midlineneck incision and bilateral isolation of the neck muscles. Sinoaorticdenervation was carried out at 1 month before DR or other treatment.

Middle Cerebral Artery OcclusionFocal cerebral ischemia in rats or mice was produced by intraluminalocclusion of the left middle cerebral artery (MCA) for 2 hours using a siliconerubber-coated nylon monofilament.16,17 Briefly, the core temperature(rectum) was maintained at 36.51C to 37.51C by use of a temperaturecontroller pad (ML312, ADInstruments, Sydney, NSW, Australia) throughoutthe surgery. Cerebral focal ischemia was produced by intraluminal occlusionof the left MCA using a silicone rubber-coated nylon monofilament.

Achievement of ischemia was confirmed by monitoring regional cerebralblood flow in the area of the left MCA. Cerebral blood flow was monitoredthrough a laser Doppler transducer (MNP110XP, ADInstruments) to a laserDoppler computerized main unit (ML191, ADInstruments). The microtip wasattached to the skull of the animal through cyanoacrilate glue. Animals thatdid not show a cerebral blood flow reduction of at least 70% were excludedfrom the experimental group, as were animals that died after ischemiainduction. Two hours after MCA occlusion (MCAO), the occluding filamentwas withdrawn to allow reperfusion. One day after operation, neurologicdeficits was evaluated using a scoring methods as previously described.16,17

The animals were killed and the brain slices (for each brain, seven slices forrats and six for mice) were prepared with brain-cutting matrix (ASIInstruments, Warren, MI, USA). The slices were incubated in 1% TTC (2,3,5-triphenyltetrazolium chloride) solution (Sinopharm Chemical Reagents,Shanghai, China) at 371C for 30 minutes, and then mounted on dry paperand photographed with a digital camera. The infarct size was quantified withImageJ software (National Institute of Health, Bethesda, MD, USA), andexpressed as the percentage relative to the contralateral hemisphere.

Blood Pressure and Baroreflex SensitivitySystolic blood pressure (SBP), diastolic blood pressure, and heart rate werecontinuously recorded in conscious, freely moving rats as previouslydescribed.14 Noninvasive blood pressure measurements were made using

Figure 1. The effect of dietary restriction (DR) on stroke death of stroke-prone spontaneously hypertensive rats (SP-SHRs). Three-month-oldmale SP-SHRs were randomly divided into two groups and fed standard feed (Control, ad libitum (AL), n¼ 33) or placed on dietary restrictedfeed (DR, n¼ 33). (A) The amount of food in DR rats was adjusted every week according to the food intake in control rats. Body weight wasrecorded in the two groups every 2 weeks. Data are expressed as mean±s.d. *Po0.05, **Po0.01. Student’s t-test. (B) The difference inappearance between control and DR groups. (C) The brains of dead SP-SHRs with neurologic sings showed hemorrhages (top-left), ischemiaor edema (top-right). Microscopic images of brain section of SHR-SPs suffering from stroke. Hemorrhages (black arrow) or edema (red arrow)were observed in the brain. (D) Kaplan–Meier analysis was used to estimate survival probabilities in the two groups.

Dietary restriction and arterial baroreflexA-J Liu et al

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Journal of Cerebral Blood Flow & Metabolism (2013), 1 – 8 & 2013 ISCBFM

a tail-cuff system (ALCBIO, Shanghai, China). After 3 days of training, eachrat was assessed for a minimum of three times per session. Baroreflexsensitivity was measured using a previously described method.14,15

Phenylephrine (5 to 10 mg/kg, intravenously) was used to raise SBP byabout 30 mm Hg. The delay between the rise in blood pressure (stimulus)and the prolongation of heart rate (response) was B1 second. Heart periodwas plotted against SBP for linear regression analysis for 2 to 8 shifts; theslope with the largest correlation coefficient (r) was expressed as BRS (ms/mm Hg). The mean of two measurements was taken as the final result.

Behavioral Assessment and Morphological ExaminationMale SP-SHRs were placed under AL or DR, starting from 3 months of age.Behavioral signs of stroke including decreasing body weight, paralysis, seizure,bleeding from the nose and/or mouth, changes of respiratory rhythm, coma,and death, were observed daily. Upon death, the brain was removed forexamination of hemorrhage, edema, and infarction. The brains without

apparent change under gross examination were fixed in 4% paraformalde-hyde in phosphate saline buffer (pH 7.4) for 24 hours, sectioned transversely at5mm thickness with 200mm interval from the anterior to the posteriorextremity, and stained with hematoxylin and eosin.

For assessment of end-organ damage, the right kidney, thoracic aorta,and heart were dissected quickly after deep anesthesia. Gross examinationof the heart (weight and wall thickness), right kidney (appearance, weight,and thickness), and aorta (weight) was performed. The ratio of heart tobody weight (mg/g), aortic weight to length (mg/cm), kidney to bodyweight, and the thicknesses of renal cortex, and medulla were used toreflect cardiac/aortic hypertrophy and kidney damage, respectively.15

ImmunoblottingTissue/cell extract was boiled in 4� loading buffer, subjected to thesodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred

Figure 2. The effect of dietary restriction (DR) on the proinflammatory cytokines, glucose, lipids, and hemodynamic parameters of stroke-prone spontaneously hypertensive rats (SP-SHRs). Three-month-old male rats were randomly divided into two groups and fed control diet (adlibitum (AL)) or placed on DR. (A) The concentration of interleukin (IL)-1b, IL-6, and tumor necrosis factor a (TNFa) in the cerebral cortex, andthe levels of glucose, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-L), and low-density lipoproteincholesterol (LDL-L) in the serum of different group rats fed AL or placed on DR for 20 weeks. (B, C) Blood pressure, heart rate, and baroreflexsensitivity (BRS) values were measured in conscious, freely moving rats fed AL or placed on DR for 20 (B) or 10 weeks (C). Data are expressed asmean±s.d. *Po0.05, **Po0.01, Student’s t-test. n¼ 10 to 11 in each group. DBP, diastolic blood pressure; SBP, systolic blood pressure.


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& 2013 ISCBFM Journal of Cerebral Blood Flow & Metabolism (2013), 1 – 8

onto nitrocellulose blotting membranes. The membranes were incubatedwith one of the following antibodies: SIRT1, acetyl-Histone H3 and H4 (CellSignaling, Boston, MA, USA) before incubation with IRDye800CW-conjugated secondary antibody. The image was captured by the Odysseyinfrared imaging system (Li-Cor Bioscience, Lincoln, NE, USA), and analyzedusing ImageJ software (NIH). The immunoblotting experiments wereperformed in two to three representative animal subjects.

Proinflammatory CytokineThe levels of tumor necrosis factor a (TNFa), interleukin-6 (IL-6), interleukin-1a (IL-1a), or interleukin-1b (IL-1b) in the entire cerebral cortexhomogenate or serum were determined with an enzyme-linked immuno-sorbent assay using an automatic microplate reader (Infinite M200; TecanAustria GmbH, Grodig, Austria).

Serum Glucose and LipidsSerum glucose, triglyceride, total cholesterol, low-density lipoprotein-cholesterol, and high-density lipoprotein-cholesterol were measured usinga clinical chemistry analyzer (Shanghai Xunda Medical Instruments,Shanghai, China) with corresponding kits from Shanghai Fosun LongMarch Medical Sciences (Shanghai, China).

Statistical AnalysisThe investigators were blinded to the procedures when they assessed theinfarct size and neurologic deficient score of MCAO animals. The animalswere randomly assigned by using the random permutations table. Data

were expressed as the mean±s.d. Data were analyzed with two-tailedStudent’s t-test or one-way analysis of variance followed by least significantdifference t-test for pairwise comparison. Kaplan–Meier analysis was usedto estimate survival probabilities. Log-rank testing was used to evaluateequality of survival curves. Po0.05 was considered statistically significant.

RESULTSDietary Restriction Delayed the Development of Stroke inStroke-Prone Spontaneously Hypertensive RatsIn a period of 40 weeks after diet manipulation starting from3 months of age, the body weight increased by 117 and 19 g inthe AL and DR groups, respectively (Figure 1A). The animals onDR lived longer than AL group (Figure 1B). Death caused bystroke was also confirmed (Figure 1C). As shown in Figure 1D,DR prevented or delayed the development of lethal stroke.Kaplan–Meier analysis revealed significantly longer survival time inthe DR group (491±87 versus 289±84 days in AL group; log-ranktest w2¼ 54, Po0.001).

Dietary Restriction Decreased Proinflammatory Cytokines andPrevented End-Organ Damage in Stroke-Prone SpontaneouslyHypertensive RatsAt 20 weeks after the diet manipulation, the concentration ofIL-1b, IL-6, and TNFa in the cerebral cortex of rats under DR was

Figure 3. Dietary restriction (DR) reduced end-organ damage in stroke-prone spontaneously hypertensive rats (SP-SHRs). Three-month-oldmale rats were randomly divided into two groups and fed unrestricted (ad libitum (AL)) or restricted diet for 20 weeks. (A) Representativecardiac morphology of two groups of rats showing longitudinal sections of hearts. (B) DR significantly decreased cardiac hypertrophy andthickness of aorta and renal injury. The ratio of right kidney cortex thickness (RCT) to right kidney medulla thickness (RMT) represents thedegree of damage to kidney. Data are expressed as mean±s.d. *Po0.05, **Po0.01. Student’s t-test, n¼ 9 to 11 in each group. AW, aorticweight; BW, body weight; HW, heart weight; LVT, left ventricular thick; LVW, left ventricular weight; RKW, right kidney weight; RVT, rightventricular thickness; VW, ventricular weight.


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significantly lower than in the AL group (Figure 2A). Dietaryrestriction also decreased serum level of triglyceride significantly,but did not affect blood glucose, total-, high-density lipoprotein-,or low-density lipoprotein-cholesterol (Figure 2A). Hypertrophy ofthe heart/aorta and the injury of kidney were evident in all SP-SHRs, but was significantly attenuated by DR (Figures 3A and 3B).

Dietary Restriction Increased Arterial BaroreflexDietary restriction for 20 weeks decreased blood pressure (meanchange of SBP, � 8 mm Hg, P40.05) of SP-SHRs. Baroreflex sen-sitivity was increased by a much larger magnitude (0.45±0.11 versus0.23±0.09 ms/mm Hg, Po0.01; Figure 2B). Heart rate was notaffected. Similar results were obtained with 10-week DR (Figure 2C).

Sinoaortic Denervation Attenuated the Neuroprotective Effects ofDietary RestrictionMeasurement of BRS at 1 month after SAD confirmed the loss ofbaroreflex in SP-SHRs: 0.08±0.02 versus 0.38±0.08 ms/mm Hg insham-operated rats (Po0.01). Blood pressure was not affected(SBP, 211±13 versus 204±7 mm Hg, P40.05; Figure 4A). Thechange of food intake in different four groups is shown inFigure 4B. A 24-week, DR reduced the SBP by about 10 mm Hg inSP-SHRs with or without intact baroreflex (the right panel ofFigure 4B). In rats with intact arterial baroreflex, DR increased the

lifespan by about 200 days (Figure 4C, left). This prolongation wasmarkedly attenuated by SAD (about 119 days in SAD rats.Figure 4C, right panel).

Sinoaortic denervation was also successful in SD rats(Figure 5A). The infarct caused by MCAO was much larger in ratssubjected to SAD than in the sham-operated rats (40.2%±8.3%versus 25.4%±8.3%, Po0.01). Dietary restriction reduced infarctsize in SD rats with intact baroreflex (by 66%), and to a muchlesser degree in SAD rats (23%; Figure 5B). Dietary restrictionimproved neurologic function (neurologic score 1.6±0.5 versus2.6±0.7, Po0.05) in rats with intact baroreflex, but not in rats withSAD (2.8±0.7 versus 3.4±0.5, P40.05).

Dietary restriction reduced the concentration of TNFa, IL-6, andIL-1a in the serum of rats with MCAO. In contrast, SAD increasedthe concentration of IL-6 and IL-1a, and attenuated the reductionof proinflammatory cytokines induced by DR (Figure 5C).

Protective Effect of Dietary Restriction Against Stroke IsIndependent on Silent Information Regulator T1Dietary restriction increased SIRT1 expression in the cerebralcortex, hypothalamus, and medulla of brain (Figure 6A); also in theheart and kidney of SP-SHRs (Figure 6B). To investigate the effectof SIRT1 on stroke, resveratrol, an activator of SIRT1, was used intwo doses (5 and 15 mg/kg per day). The activation of SIRT1 wasconfirmed by the measurement of acetylated Histone H3 and H4

Figure 4. Arterial baroreflex involved in the neuroprotection of dietary restriction in rats. (A) Blood pressure, heart rate, and baroreflexsensitivity (BRS) were measured in stroke-prone spontaneously hypertensive rats (SP-SHRs) after 1 month recovery from sinoaorticdenervation (SAD). Student’s t-test, n¼ 5. **Po0.01. (B) Left panel, the food restriction in dietary restriction (DR) and SAD-DR rats was adjustedaccording to the intake in ad libitum (AL) and SAD-AL rats, respectively. Right panel, blood pressure was measured every 8 weeks in SP-SHRsfed with control diet or placed on DR. Student’s t-test. *Po0.05 (black asterisk versus AL, red asterisk versus SAD-AL). (C) The survival time ofSP-SHRs with intact baroreflex function (left) and baroreflex dysfunction by SAD (right). DR started at 3 months of age in SP-SHRs after1 month recovery from SAD, and lasted throughout the duration of the experiment. The timing of spontaneous lethal stroke was recorded.n¼ 20 in each SAD group; 33 in each group without SAD. Survival data were examined using Kaplan–Meier analysis (log-rank test). Po0.05was considered statistically significant.


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(Figure 6C). Resveratrol was mixed into feeding stuff, and did notincrease the lifespan of SP-SHRs (Figure 6D).

Infarct induced by MCAO was significantly larger, by B81%, insirt1 KO mice than in WT littermate controls (62%±8.5% versus34%±9.2%, Po0.01; Figure 6E). The sirt1 KO mice also hadsignificantly worse overall neurologic outcomes (neurologic score3.8±0.5 versus 2.3±0.5 in WT control mice, Po0.01). Dietaryrestriction reduced infarct size in both the sirt1 KO mice(33%±8.4% versus 62%±8.5%, decreased by 46%, Po0.01) andWT mice (15.5%±3.4% versus 34%±9.2%, decreased by 54%;Figure 6E), and significantly improved neurologic function in bothsirt1 KO mice (neurologic score 2.4±0.8 versus 3.8±0.5, Po0.05)and WT mice (1.3±0.6 versus 2.3±0.5, Po0.05).

DISCUSSIONDietary restriction delays the onset of age-associated patholo-gies.1–4 A study using rhesus monkeys showed that DR couldreduce the incidence of diabetes, cancer, cardiovascular disease,and brain atrophy, and ultimately promotes the survival.18

Reduced incidence of stroke in response to DR may be a resultof improved health of the cardiovascular system.3 A study in bothyoung and aged rhesus monkeys did not show survival benefit ofDR, but it is worthy to point out that the control subjects did not

receive true AL (to prevent obesity).19 Dietary restriction, even inslight degree, could produce beneficial effect.20 The current studydemonstrated that 20% and 40% of DR produce similar effectsagainst stroke injury in MCAO rats (data shown in SupplementaryFigure S1).

Dietary restriction reduces the risk factors for ischemic stroke,including proinflammatory cytokines, body fat, blood pressure,and serum lipid and lipoprotein levels.3,21 In the present study,hypertrophy of the heart, the thickening of aortic wall, and renalinjury all were ameliorated by DR. It should be noted that thechanges in lipids levels and blood pressure of SP-SHRs werestatistically significant but minimal. Reported effects of DR onblood pressure are variable, with DR effects ranging fromsignificant reduction of blood pressure22,23 to transient andslight reduction.24,25A recent report showed that the depressoreffect of DR wanes as pathogenesis progresses, and suggestedthat the early lasting effect on cardiac hypertrophy and function isindependent of a sustained depressor effect.25

Baroreflex sensitivity is an important determinant of manycardiovascular diseases. Patients with reduced BRS exhibit shortersurvival after myocardial infarction, heart failure,11 or stroke.12 Wepreviously reported that arterial baroreflex dysfunction promotesthe development of atherosclerosis in rats, and decreases survivaltime in lipopolysaccharide-induced lethal shock.26 Intact arterial

Figure 5. Sinoaortic denervation (SAD) attenuated the beneficial effect of dietary restriction (DR) on ischemic stroke. The animals were placed onDR for 8 weeks 1 month after SAD. (A) Measurement of blood pressure, heart rate, and baroreflex sensitivity (BRS) at 1 month after SAD inSprague-Dawley (SD) rats. Student’s t-test, n¼ 5. (B) The left panel shows representative 2,3,5-triphenyltetrazolium chloride (TTC) staining ofseven corresponding coronal brain sections 1 day after middle cerebral artery occlusion (MCAO) for 2 hours. (C) The release of interleukin (IL)-1b,IL-6, and tumor necrosis factor a (TNFa) in serum of different group rats. Data are expressed as mean±s.d. *Po0.05, **Po0.01, n.s., notsignificant. Data were analyzed by one-way analysis of variance (ANOVA) followed by least significant difference t-test. n¼ 10 to 11 in each group.


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baroreflex function is also necessary to prevent aconitine-inducedventricular arrhythmias.26 Impaired BRS has also been repeatedlyshown to be present in acute stroke.27,28 Robinson et al12 reportedpoor prognosis in poststroke patients with impaired BRS(o5.0 ms/mm Hg). We previously demonstrated that BRS is animportant factor in determining survival time in SP-SHRs. Theresult showed that death after stroke was significantly delayed inrats with high BRS than those with low BRS (time to 50% deathwas 1.47-fold longer for the high BRS group than the low BRScounterparts).15

Used MCAO rats and SP-SHRs, we now demonstrate thatbaroreflex function is important in the DR-mediated protectionagainst stroke. The release of proinflammatory cytokines is animportant mechanism involved in the development of stroke. Datashow that C-reactive protein, IL-6, leukocyte elastase, lipoprotein (a),intercellular adhesion molecule -1, E-selectin, and other hemostaticand rheological factors are consistently higher in people whodeveloped stroke compared with those who remained free of strokeand heart diseases.26 And the deficiency of E-selectin can alleviatethe cerebral injury.29 Interleukin-6 and TNFa are also reported to beassociated with risk of recurrent ischemic stroke independently ofconventional risk markers.26 So the reduction of proinflammatorycytokines might be beneficial for stroke treatment. In ourexperiments, DR significantly reduced the release of

proinflammatory cytokines, which was also confirmed by otherclinical study.21,24 We also concluded that arterial baroreflexdysfunction increased the release of proinflammatory cytokines inMCAO rats, and attenuated the beneficial effect of DR onproinflammatory cytokines. But how does arterial baroreflexcontrol these cytokines has not been fully understood.

Recently, ‘cholinergic antiinflammatory pathway’ become as acritical regulator of inflammation. Activation of cholinergic signa-ling inhibits the overproduction of TNF and other proinflammatorycytokines through a7 nicotinic acetylcholine receptor (a7nAChR).30

Our recent data showed that arterial baroreflex dysfunction leadto a decrease not only in reflex activity but also in tonic activity ofvagal nervous system.31 The endogenous ligand for a7nAChR isacetylcholine. It was found that the expression of vesicularacetylcholine transporter markedly decreased in the brain ofSAD animals. These results indicate that the dysfunction of arterialbaroreflex related an inactivation of a7nAChR.31

Dietary restriction also alleviate age-related diseases by affect-ing a variety of molecular and physiological targets beyondarterial baroreflex.1 Among these targets, sirtuins may be the mostimportant.1,6 Many studies, most performed in lower organisms,such as Saccharomyces cerevisiae and Drosophila, show thatactivation of sirtuins is a critical mechanism of DR-inducedprolongation of lifespan.32,33 In Caenorhabditis elegans, extra

Figure 6. The protective effect of dietary restriction (DR) against stroke is independent on silent information regulator T1 (SIRT1). Stroke-pronespontaneously hypertensive rats (SP-SHRs) were fed unrestricted or restricted diet. Representative Western blotting of SIRT1 in: (A) thedifferent part of brain (brain cortex, hypothalamus, and medulla), and (B) the heart and kidney. Tissue homogenate was incubated with theSIRT1 antibody, n¼ 3. (C) Representative Western blotting of Acetyl-Histone H3 and H4 in the brain of SP-SHRs after resveratrol treatment for 7days, n¼ 2. (D) SP-SHRs aged 3-month-old were fed different doses (0, 5, and 15mg/kg per day) of resveratrol. The treatment lastedthroughout the duration of the experiment. The timing of spontaneous lethal stroke was recorded. n¼ 15. Kaplan–Meier analysis was used toestimate survival probabilities in the three groups. (E) The male sirt1 knockout (KO) and wild-type (WT) littermate mice were fed unrestrictedor restricted diet for 2 months. Middle cerebral artery occlusion (MCAO) was performed after DR. The left panel shows representative 2,3,5-triphenyltetrazolium chloride (TTC) staining of six corresponding coronal brain sections on day 1 after MCAO. The ischemic infarct area andthe neurologic scores were measured, n¼ 4. Data are expressed as mean±s.d and analyzed by one-way analysis of variance (ANOVA) followedby least significant difference t-test. **Po0.01. AL, ad libitum; Con, control; Res, resveratrol.


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copies of sir-2.1 were shown to increase lifespan.34 Sinclair’s groupreported that resveratrol could extend the life in worms and fliesvia sirtuins.35,36 Mammalian species have seven sirtuins (SIRT1–SIRT7); of these, SIRT1 is activated by DR, and has been implicatedin the beneficial effects of DR.6

An earlier study demonstrated that DR could increase theexpression of SIRT1, and prevent neuropathology in an animalmodel of Alzheimer disease.37 The overexpression of SIRT1 alsoattenuated b-amyloid content in Alzheimer disease. But theauthors did not provide a direct link between the beneficial effectof DR in Alzheimer disease and SIRT1 activity. In the present study,we showed, by using sirt1 KO mice and a representative SIRT1activator, that the protective effects of DR against stroke areindependent of SIRT1. Recently, several other investigators havequestioned the role of sirtuins in lifespan prolongation or inmediating the beneficial effects of DR.38–40 Guarente’s groupreported that the overexpression of SIRT1 in mice did not increasethe lifespan, although the mice were apparently healthier uponaging.38 Burnett et al39 showed that DR could increase the lifespanof flies via a sirtuins-independent mechanism. Harrison andcolleagues40 failed to show that resveratrol could alter thelifespan in mice. It appears that the function of SIRT1 and itsrelationship with DR is more complicated than expected.

In conclusion, the present work indicates that the beneficialeffects of DR against stroke are SIRT1-independent, and the arterialbaroreflex is involved in the protective effects of DR against stroke.

DISCLOSURE/CONFLICT OF INTERESTThe authors declare no conflict interest.

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Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)


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involvement of arterial baroreflex in the protective effect of dietary restriction against stroke

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