DOI:10.1111/micc.12127
Original Article
Vigorous Exercise Training Improves Reactivity of Cerebral Arterioles and Reduces Brain Injury Following Transient Focal Ischemia DENISE M. ARRICK, SHU YANG, CHUN LI, SERGIO CANANZI, AND WILLIAM G. MAYHAN Department of Cellular Biology and Anatomy and the Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center-Shreveport, Shreveport, Louisiana, USA Address for correspondence: William G. Mayhan, Department of Cellular Biology and Anatomy, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA. E-mail: [email protected] Received 27 January 2014; accepted 7 March 2014.
ABSTRACT Objective: Our objective was to examine whether vigorous exercise training (VExT) could influence nitric oxide synthase (NOS)dependent vasodilation and transient focal ischemia-induced brain injury. Rats were divided into sedentary (SED) or VExT groups. Materials and Methods: Exercise was carried out 5 days/week for a period of 8–10 weeks. First, we measured responses of pial arterioles to an eNOS-dependent (ADP), an nNOS-dependent (NMDA) and a NOS-independent (nitroglycerin) agonist in SED and VExT rats. Second, we measured infarct volume in SED and VExT rats following middle cerebral artery occlusion (MCAO). Third, we measured superoxide levels in brain tissue of SED and VExT rats under basal and stimulated conditions. Results: We found that eNOS- and nNOS-dependent, but not NOS-independent vasodilation, was increased in VExT compared to SED rats, and this could be inhibited with L-NMMA in both groups.
In addition, we found that VExT reduced infarct volume following MCAO when compared to SED rats. Further, superoxide levels were similar in brain tissue from SED and VExT rats under basal and stimulated conditions. Conclusions: We suggest that VExT potentiates NOS-dependent vascular reactivity and reduces infarct volume following MCAO via a mechanism that appears to be independent of oxidative stress, but presumably related to an increase in the contribution of nitric oxide. WORDS: ADP, NMDA, pial arterioles, middle cerebral artery occlusion, ischemia/reperfusion
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Abbreviations used: ADP, Adenosine 5 diphosphate; EDHF, endo-
thelium derived hyperpolarizing factor; eNOS, endothelial nitric oxide synthase; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; NMDA, N-methyl-D-aspartic acid; NOS, nitric oxide synthase; RLU, relative light units; SED, sedentary; TTC, 2,3,5triphenyl-tetrazolium chloride; VExT, vigorous exercise training.
Please cite this paper as: Arrick DM, Yang S, Li C, Cananzi S, Mayhan WG. Vigorous exercise training improves reactivity of cerebral arterioles and reduces brain injury following transient focal ischemia. Microcirculation 21: 516–523, 2014.
INTRODUCTION Exercise training has been shown to have beneficial effects on the health of large and small blood vessels presumably via an influence on endothelial function. Studies have shown that moderate exercise training enhances eNOS-dependent reactivity of large and small peripheral blood vessels (skeletal muscle, heart, and skin) in animal models and human subjects [4, 13, 14, 16, 17, 42, 47]. Mechanisms by which exercise training potentiates eNOS-dependent vascular responses are likely to be related to increase the synthesis/ release of nitric oxide [18, 36, 37, 45], an increase in the activity of antioxidant enzymes [15, 19, 21, 26, 35, 38], and/ or via an enhancement of multiple vasodilator pathways [4]. In recent studies, we examined the influence of moderate exercise training on NOS-dependent (eNOS and nNOS)
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responses of cerebral arterioles and on brain injury following cerebral ischemia/reperfusion in nondiabetic and diabetic rats [1,30,32]. We found that moderate exercise training restored impaired eNOS- and nNOS-dependent responses of cerebral arterioles in diabetic rats toward that observed in nondiabetic rats and prevented the elevated level of brain injury observed in diabetic rats to that seen in nondiabetic rats [1,30]. Surprisingly, however, we did not observe a significant influence of moderate exercise training on eNOSor nNOS-dependent reactivity of cerebral arterioles or brain injury following cerebral ischemia/reperfusion in nondiabetic (control) rats [1,30]. Thus, although a moderate level of exercise training was beneficial in diabetic rats, it was not sufficient to provide a beneficial effect in control rats. We reasoned that this finding in control rats might be related to the intensity of exercise training. Therefore, the first goal of
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the present study was to examine the effects of VExT on eNOS- and nNOS-dependent responses of cerebral (pial) resistance arterioles. Our second goal was to examine the influence of VExT on brain injury following MCAO. Finally, in an attempt to determine mechanisms for an effect of VExT on the brain, our third goal was to determine the role of nitric oxide in vascular reactivity during VExT and determine the influence of VExT on superoxide levels in brain tissue.
MATERIALS AND METHODS Exercise Training All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Sciences Center-Shreveport. The experiments were carried out in accordance with the Code of Ethics for the use of animals in research. Male SpragueDawley rats (180–200 g body wt; two to three months of age) were randomly divided into a SED or VExT groups. All rats had access to food and water ad libitum. Rats were exercised using standard techniques similar to that which we have described previously [1,30]. Treadmill exercise was carried out five days/week until the day before the experiment. Experiments were conducted 8–10 weeks after starting treadmill exercise. The length of time on the treadmill was initially 10 min/day for the first five days at 0% grade at a speed of 20 M/min. Over the next five days, the speed was increased to 25 M/min, the time was increased by 10 minutes each day to reach a duration of 60 minutes on the treadmill and the incline remained at 0% grade. Over the next three days on the treadmill, the speed (25 M/min) and duration (60 minutes) remained the same, but the incline was increased to a 5% grade. Finally, during the next two days and continuing for the remainder of the experimental protocol, the speed (25 M/min) and duration (60 minutes) remained the same, but the incline was increase to 10%. Thus, it took 15 days to reach the maximum training regimen and this continued for the duration of the experimental period. Rats were not exercised one day prior to experiments.
Reactivity of Pial Arterioles SED (n = 9) and VExT (n = 17) rats were prepared for in vivo examination of reactivity of cerebral arterioles. Rats were anesthetized (thiobutabarbital (Inactin); 100 mg/kg i.p.) and a tracheotomy was performed. The animals were ventilated mechanically with room air and supplemental oxygen. A catheter was inserted into a femoral vein for injection of supplemental anesthesia (10–30 mg/kg). A femoral artery was cannulated for measurement of arterial pressure. After placement of all catheters, the animal was placed in a head holder and a craniotomy was made over the left parietal cortex to expose the pial microcirculation [31]. The cranial
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window was suffused with artificial cerebral spinal fluid bubbled with 95% nitrogen and 5% carbon dioxide. Temperature of the suffusate was maintained at 37 1°C. The cranial window was connected via a three-way valve to an infusion pump, which allowed for infusion of agonists into the suffusate. Arterial blood gases were monitored and maintained within normal limits. After an equilibration period of 30–45 minutes, we examined dilation of pial arterioles in SED and VExT to an eNOS-dependent agonist (ADP; 10 and 100 lM), an nNOS-dependent agonist (NMDA; 100 and 300 lM), and a NOS-independent agonist (nitroglycerin; 0.1 and 1.0 lM). Agonists were mixed in artificial cerebral spinal fluid and superfused over the cranial window preparation. The diameter of pial arterioles was measured immediately before the application of agonists and every minute for five minutes during application of the agonists. Steady-state responses were reached within two to three minutes after application, and the diameter of pial arterioles returned to baseline within five minutes after application of the agonists was stopped. After this initial test of vascular reactivity, we then started a continuous suffusion of L-NMMA (100 lM) over the cranial window preparation. Thirty minutes after starting this superfusion, we again measured responses of pial arterioles in SED and VExT rats to ADP, NMDA, and nitroglycerin.
Assessment of Brain Injury Following MCAO Transient focal cerebral ischemia was performed using methods we have described previously [44,49,50]. On the day of the experiment, SED (n = 15) and VExT (n = 16) rats were anesthetized with ketamine/xylazine (100/15 mg/kg i.p., respectively). A laser Doppler flow probe (PeriFlux System 5000, Jarfalla, Sweden) was attached to the right side of the dorsal surface of the skull (2 mm caudal and 6 mm lateral to the bregma) to observe the decrease in cerebral blood flow during MCAO. A 4-0 monofilament nylon suture was prepared by rounding its tip by heating and coating with silicon. The right common and external carotid arteries were exposed and ligated. The MCA was occluded by inserting the filament from the basal area of the external carotid artery, and advancing it cranially into the internal carotid artery to the point where the MCA branched from the internal carotid artery. Onset of MCAO was determined by a rapid drop in cerebral blood flow, as measured by the laser Doppler. After occluding the MCA for two hours, reperfusion was initiated by removing the suture. Following 24 hours of reperfusion, the SED and VExT rats were euthanized, the brains were quickly removed and placed in ice-cold saline for five minutes. The brains were then cut into six 2-mm-thick coronal sections. The sections were stained with 2% TTC (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes at 37°C. Slice images were digitalized and the infarct lesion was evaluated using Kodak Molecular Imaging Software. Com-
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plete lack of staining with TTC was defined as the infarct lesion. The infarct volume was expressed as percentage of the contralateral hemisphere. We measured the total infarct volume, cortical infarct volume, and subcortical infarct volume in SED and VExT, as described previously [44,49].
Superoxide Levels Superoxide levels were measured by lucigenin-enhanced chemiluminescence as described previously [6,7,33]. In SED (n = 19) and VExT (n = 23) rats, the brains were removed and placed in a Krebs/HEPES buffer (pH 7.4) with the following composition (in mmol/L): 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgCl2, 1.3 CaCl2, 10 HEPES, 25 NaHCO3, and 5 glucose. Samples of cortex tissue, cut from brains of SED and VExT rats, were placed in polypropylene tubes containing 5 lM lucigenin. The tubes were then read in a Sirius/ FB15 luminometer (Berthold Detections Systems, Pforzheim, Germany), which reports RLU emitted over a 30-second interval for five minutes. Levels of superoxide reported are the value of tissue plus lucigenin-containing buffer minus background (lucigenin-containing buffer without tissue) and are normalized for tissue weight (RLU/min/mg tissue). We measured basal levels of superoxide and levels of superoxide in response to NADPH, a substrate for NADPH oxidase [6].
Citrate Synthase Activity To determine the level of exercise training in the VExT rats, we obtained a sample of the soleus muscle from SED (n = 6) and VExT (n = 13) rats. Citrate synthase activity was measured using a standard kit (CS0720; Sigma-Aldrich). Citrate synthase activity was determined based on the formation of 2-nitro-5-thiobenzoic acid and measured at a wavelength of 412 nm with the aid of spectrophotometer. The background absorbance was measured, then the reaction was initiated by the addition of oxaloacetic acid and the change in absorbance was measured.
Table 1. Mean arterial pressure, baseline diameter of cerebral arterioles, blood glucose concentration, body weight, and citrate synthase activity in the soleus muscle of SED and VExT rats
SED
Mean arterial pressure (mmHg) Baseline diameter of pial arterioles (lm) Blood glucose(mg/dL) Body weight (g) Citrate synthase activity (lmol/mL/min)
133 50 126 340 56
VExT
6 3 3 19 16
127 44 105 370 116
3 (ns) 2 (ns) 6* 7 (ns) 18*
Values are means SE. ns, not significant. *p < 0.05 versus SED rats.
pial arterioles, mean arterial pressure, and body weight were similar in SED and VExT rats. However, blood glucose concentration was less in VExT rats when compared with SED rats and citrate synthase activity was significantly elevated in VExT rats when compared with SED rats.
Responses of Pial Arterioles We examined functional responses of pial arterioles to eNOS- and nNOS-dependent agonists (ADP and NMDA, respectively), and a NOS-independent agonist (nitroglycerin) in SED and VExT rats. We found that ADP- and NMDAinduced dilation of pial arterioles was significantly greater in VExT rats when compared with SED rats (Figures 1 and 2, respectively). In contrast to that observed with ADP and NMDA, nitroglycerin produced similar dose-related dilation of pial arterioles in SED and VExT rats (Figure 3). Thus, it does not appear that the effect of VExT on ADP- and
Statistical Analysis Unpaired t-tests were used to compare differences in reactivity of cerebral arterioles to the agonists, superoxide anion levels, infarct volumes and levels of citrate synthase in SED and VExT rats. A paired t-test was used to compare differences in reactivity of cerebral arterioles before and following application of L-NMMA. A p value of 0.05 or less was considered significant.
RESULTS Baseline Conditions Baseline diameter of pial arterioles, blood glucose concentration, mean arterial pressure, body weight and citrate synthase activity in the soleus muscle of SED and VExT rats are shown in Table 1. We found that baseline diameter of
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Figure 1. Response of cerebral arterioles to ADP in SED and VExT rats. Values are means SE. *p < 0.05 versus SED rats.
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NMDA-induced vasodilation is related to a nonspecific effect of VExT on vascular function. Next, we determined whether the contribution of nitric oxide to vasodilation in response to ADP and NMDA might be altered in VExT rats. We found that topical application of L-NMMA produced a similar decrease in baseline diameter of cerebral arterioles in SED (6 2%) and VExT (7 2%) rats. In addition, L-NMMA significantly decreased responses of cerebral arterioles to ADP and NMDA in both SED and VExT rats (Figures 4 and 5, respectively). Thus, it appears that the increase in vasodilation observed in the VExT rats in response to ADP and NMDA is related to the synthesis/ release of nitric oxide.
Superoxide Production We measured superoxide levels in cortex tissue in SED and VExT rats under basal conditions and during stimulation with NADPH, a substrate for NADPH oxidase. We found that the levels of superoxide in cortex tissue were similar in SED and VExT rats under basal and stimulated states (Figure 6). Thus, it appears that VExT does not influence the basal production of superoxide or the ability to produce superoxide under stimulated states.
Figure 2. Response of cerebral arterioles to NMDA in SED and VExT rats. Values are means SE. *p < 0.05 versus SED rats.
MCAO-Induced Brain Injury We measured infarct volumes in SED and VExT rats following MCAO. We found that total infarct volume produced by a two-hour MCAO followed by 24 hours of reperfusion was greater in SED than in VExT rats (Figure 7). Identification of the location of the infarct revealed that VExT significantly decreased cortical infarct volume, with subcortical infarct volume being similar in SED and VExT rats (Figure 7).
DISCUSSION There are four major new findings of the present study. First, we found that VExT increased responses of cerebral arterioles
Figure 3. Response of cerebral arterioles to nitroglycerin in SED and VExT rats. Values are means SE.
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Figure 4. Effect of L-NMMA on response of cerebral arterioles to ADP in SED (left panel) and VExT (right panel) rats. Values are meansSE. *p < 0.05 versus response before application of L-NMMA.
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Figure 5. Effect of L-NMMA on response of cerebral arterioles to NMDA in SED (left panel) and VExT (right panel) rats. Values are means SE. *p < 0.05 versus response before application of L-NMMA.
Figure 6. Superoxide levels in SED and VExT rats under baseline conditions and in response to NADPH, as substrate for NADPH oxidase. Values are means SE. *p < 0.05 versus baseline.
to eNOS- and nNOS-dependent agonists. This effect of VExT was specific since nitroglycerin produced similar dilation of cerebral arterioles in SED and VExT rats. Second, the increase in NOS-dependent reactivity of cerebral arterioles during VExT appeared to be related to the synthesis/release of nitric oxide as there was a significant reduction in responsiveness following application of L-NMMA. Third, VExT did not have a significant effect on either basal levels or agonist-induced levels of superoxide. Thus, we suggest that the effects of VExT on vascular function and brain injury following MCAO are not related to an influence of VExT on superoxide. Fourth, we found a significant protective effect of VExT on infarct volume following MCAO. Based upon these findings, we speculate that VExT may have an important therapeutic potential for reducing brain damage subsequent to a cerebral ischemic event, presumably by a mechanism that appears to involve the ability of nitric oxide to potentiate vascular function, and possibly cerebral blood flow.
Consideration of Methods In the present study, we used ADP and NMDA to examine eNOS- and nNOS-dependent responses of cerebral arterioles, respectively. Previous studies have suggested that ADP dilates
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cerebral arterioles via activation of NOS, presumably eNOS [2,9,29], whereas others [28,48] have suggested that relaxation of the rat MCA to purines is related, in part, to nitric oxide and an EDHF. In the present study, we found that LNMMA decreased responses of cerebral arterioles to ADP (10 and 100 lM) by about 85% and 76%, respectively, in SED rats, and by 80% and 74%, respectively, in VExT rats. Thus, it appears that nitric oxide plays a similar role in responses of cerebral arterioles to ADP in both SED and VExT rats. Regarding responses to NMDA, we and others have shown that NMDA dilates cerebral arterioles via the activation of nNOS and the subsequent synthesis/release of nitric oxide [10–12,43]. The findings from the present study support this conclusion. In addition, we found that L-NMMA decreased responses of cerebral arterioles to NMDA (100 and 300 lM) by about 70% and 60%, respectively, in SED rats and by about 75% and 76%, respectively, in VExT rats. Thus, it appears that nitric oxide plays a similar significant role in responses of cerebral arterioles to NMDA in SED and VExT rats. We reasoned that an increase in vascular responsiveness to NOS-dependent agonists might be related to an influence of VExT on superoxide levels, i.e., antioxidant enzyme systems
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VExT
Although we qualitatively examined changes in cerebral blood flow during MCAO in the SED and VExT rats, we did not quantify these changes. Thus, it is conceivable that the protective effect of VExT on brain infarct volume following MCAO might be related to differences in the relative level of ischemia produced in the two groups of rats by MCAO.
Infarct size (% contralateral hemisphere)
Consideration of Previous Studies
Figure 7. Top: representative 2-mm-thick TTC-stained coronal sections of the brains from SED and VExT rats subjected to two hours of MCAO and 24 hours of reperfusion. Dark stain indicates viable tissue and complete lack of staining defines the infarct region. Bottom: mean data depicting total, subcortical and cortical infarct volumes in SED and VExT rats. Values are means SE. *p < 0.05 versus SED rats.
as has been shown by others [15, 19, 21, 26, 35, 38]. We thought that if basal levels of superoxide were decreased by VExT, then this might influence the bioavailability of nitric oxide, and hence influence vascular reactivity to NOSdependent agonists. However, we found that basal levels of superoxide were similar in SED and VExT rats. This finding is similar to that which we have reported previously for moderate ExT [30], and suggests that increased responses of cerebral arterioles to NOS-dependent agonists in VExT rats are probably not related to an effect of superoxide on nitric oxide bioavailability. In addition, we found that agonistinduced stimulation of superoxide with NADPH was similar in SED and VExT rats, suggesting that the ability to produce superoxide is not altered by VExT. Thus, we suggest that levels of superoxide may not be the limiting factor in enhanced vascular responsiveness and protection against brain damage following cerebral ischemia/reperfusion by VExT.
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Many have examined the effects of exercise on NOSdependent reactivity in animals and human subjects [17, 22, 24, 25, 41, 42]. For the most part, these studies have suggested that exercise enhances NOS-dependent dilation/ relaxation of skeletal and peripheral blood arteries and arterioles. The precise mechanism(s) by which exercise potentiates NOS-dependent responses are not clear, but have been suggested to be related to an increase in eNOS [36, 41]. In previous studies, we have reported that moderate exercise training produced a small, but significant increase in eNOS and nNOS proteins in cerebral blood vessels and brain tissue from nondiabetic (control) rats [30, 32]. However, this increase in eNOS and nNOS proteins did not equate with an increase in reactivity of large (basilar artery) and small (pial arterioles) cerebral vessels to eNOS- or nNOS-dependent agonists [1, 30, 32]. The mechanisms by which exercise training produces an increase in eNOS protein may be related to changes in shear stress across endothelium [3, 18]. While we and others have shown that exercise training produces an increase in nNOS protein in the brain and other organ systems, the mechanism for this effect of exercise training on nNOS remains elusive [30, 34, 46, 51]. In the present study, we used a more vigorous level of exercise training (increased speed, greater incline, and longer duration) to determine whether this regimen might influence cerebrovascular function. We found that VExT produced a significant increase in NOS-dependent reactivity and protected the brain from ischemia/reperfusion injury following MCAO. Thus, we suggest that although a moderate level of ExT may be beneficial during disease states, a more vigorous level of ExT may be required to influence cerebrovascular function and brain injury in animals devoid of disease. However, as we increased the duration as well as the intensity of exercise training in the present study when compared with our previous study [1, 30], we cannot rule out that a longer duration of exercise training (at a similar level of intensity) would be beneficial on vascular function and brain injury following MCAO. Future studies could examine the relationship between duration and/or intensity on the protective effects of exercise training. Epidemiological studies examining the impact of preexercise training on the risk of ischemic stroke have met with mixed results. Some have reported that pre-exercise training was associated with improved functional outcome after a
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stroke, a less severe level of stroke, and/or a decrease in the actual occurrence of an ischemic stroke [5, 20, 23, 27, 39, 40]. Others have examined the influence of pre-exercise training on the pathogenesis of stroke using animal models. Endres et al. [8] found that three weeks of exercise training prior to induction of MCAO could protect against cerebral ischemic damage in eNOS wild-type mice, but not in eNOS knockout mice, suggesting that the protective effect of pre-exercise training was related to eNOS activity. The findings from the present study are in agreement and extend the findings of this previous study. We found that VExT could potentiate eNOS- and nNOS-dependent responses of cerebral arterioles and could protect the brain from cerebral ischemic damage following MCAO via a mechanism that is presumably related an increase in nitric oxide synthesis/release and not related to an influence of VExT on oxidative stress. In summary, we examined the effects of VExT on eNOSand nNOS-dependent reactivity of cerebral resistance arterioles and on brain injury following cerebral ischemia/ reperfusion. We found that VExT potentiated eNOS- and nNOS-dependent dilation of cerebral arterioles. In addition, we found that VExT alleviated brain injury following MCAO. Furthermore, basal and agonist-induced levels of superoxide were not influenced by VExT. Based upon these findings, we suggest that ExT can restore impaired eNOS- and nNOS-
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ACKNOWLEDGMENTS This study was supported by funds from the National Heart, Lung and Blood Institute Grant (HL-090657) and funds from the LSU Health Sciences Center-Shreveport.
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Original Article
Vigorous Exercise Training Improves Reactivity of Cerebral Arterioles and Reduces Brain Injury Following Transient Focal Ischemia DENISE M. ARRICK, SHU YANG, CHUN LI, SERGIO CANANZI, AND WILLIAM G. MAYHAN Department of Cellular Biology and Anatomy and the Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center-Shreveport, Shreveport, Louisiana, USA Address for correspondence: William G. Mayhan, Department of Cellular Biology and Anatomy, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA. E-mail: [email protected] Received 27 January 2014; accepted 7 March 2014.
ABSTRACT Objective: Our objective was to examine whether vigorous exercise training (VExT) could influence nitric oxide synthase (NOS)dependent vasodilation and transient focal ischemia-induced brain injury. Rats were divided into sedentary (SED) or VExT groups. Materials and Methods: Exercise was carried out 5 days/week for a period of 8–10 weeks. First, we measured responses of pial arterioles to an eNOS-dependent (ADP), an nNOS-dependent (NMDA) and a NOS-independent (nitroglycerin) agonist in SED and VExT rats. Second, we measured infarct volume in SED and VExT rats following middle cerebral artery occlusion (MCAO). Third, we measured superoxide levels in brain tissue of SED and VExT rats under basal and stimulated conditions. Results: We found that eNOS- and nNOS-dependent, but not NOS-independent vasodilation, was increased in VExT compared to SED rats, and this could be inhibited with L-NMMA in both groups.
In addition, we found that VExT reduced infarct volume following MCAO when compared to SED rats. Further, superoxide levels were similar in brain tissue from SED and VExT rats under basal and stimulated conditions. Conclusions: We suggest that VExT potentiates NOS-dependent vascular reactivity and reduces infarct volume following MCAO via a mechanism that appears to be independent of oxidative stress, but presumably related to an increase in the contribution of nitric oxide. WORDS: ADP, NMDA, pial arterioles, middle cerebral artery occlusion, ischemia/reperfusion
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Abbreviations used: ADP, Adenosine 5 diphosphate; EDHF, endo-
thelium derived hyperpolarizing factor; eNOS, endothelial nitric oxide synthase; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; NMDA, N-methyl-D-aspartic acid; NOS, nitric oxide synthase; RLU, relative light units; SED, sedentary; TTC, 2,3,5triphenyl-tetrazolium chloride; VExT, vigorous exercise training.
Please cite this paper as: Arrick DM, Yang S, Li C, Cananzi S, Mayhan WG. Vigorous exercise training improves reactivity of cerebral arterioles and reduces brain injury following transient focal ischemia. Microcirculation 21: 516–523, 2014.
INTRODUCTION Exercise training has been shown to have beneficial effects on the health of large and small blood vessels presumably via an influence on endothelial function. Studies have shown that moderate exercise training enhances eNOS-dependent reactivity of large and small peripheral blood vessels (skeletal muscle, heart, and skin) in animal models and human subjects [4, 13, 14, 16, 17, 42, 47]. Mechanisms by which exercise training potentiates eNOS-dependent vascular responses are likely to be related to increase the synthesis/ release of nitric oxide [18, 36, 37, 45], an increase in the activity of antioxidant enzymes [15, 19, 21, 26, 35, 38], and/ or via an enhancement of multiple vasodilator pathways [4]. In recent studies, we examined the influence of moderate exercise training on NOS-dependent (eNOS and nNOS)
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responses of cerebral arterioles and on brain injury following cerebral ischemia/reperfusion in nondiabetic and diabetic rats [1,30,32]. We found that moderate exercise training restored impaired eNOS- and nNOS-dependent responses of cerebral arterioles in diabetic rats toward that observed in nondiabetic rats and prevented the elevated level of brain injury observed in diabetic rats to that seen in nondiabetic rats [1,30]. Surprisingly, however, we did not observe a significant influence of moderate exercise training on eNOSor nNOS-dependent reactivity of cerebral arterioles or brain injury following cerebral ischemia/reperfusion in nondiabetic (control) rats [1,30]. Thus, although a moderate level of exercise training was beneficial in diabetic rats, it was not sufficient to provide a beneficial effect in control rats. We reasoned that this finding in control rats might be related to the intensity of exercise training. Therefore, the first goal of
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the present study was to examine the effects of VExT on eNOS- and nNOS-dependent responses of cerebral (pial) resistance arterioles. Our second goal was to examine the influence of VExT on brain injury following MCAO. Finally, in an attempt to determine mechanisms for an effect of VExT on the brain, our third goal was to determine the role of nitric oxide in vascular reactivity during VExT and determine the influence of VExT on superoxide levels in brain tissue.
MATERIALS AND METHODS Exercise Training All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Sciences Center-Shreveport. The experiments were carried out in accordance with the Code of Ethics for the use of animals in research. Male SpragueDawley rats (180–200 g body wt; two to three months of age) were randomly divided into a SED or VExT groups. All rats had access to food and water ad libitum. Rats were exercised using standard techniques similar to that which we have described previously [1,30]. Treadmill exercise was carried out five days/week until the day before the experiment. Experiments were conducted 8–10 weeks after starting treadmill exercise. The length of time on the treadmill was initially 10 min/day for the first five days at 0% grade at a speed of 20 M/min. Over the next five days, the speed was increased to 25 M/min, the time was increased by 10 minutes each day to reach a duration of 60 minutes on the treadmill and the incline remained at 0% grade. Over the next three days on the treadmill, the speed (25 M/min) and duration (60 minutes) remained the same, but the incline was increased to a 5% grade. Finally, during the next two days and continuing for the remainder of the experimental protocol, the speed (25 M/min) and duration (60 minutes) remained the same, but the incline was increase to 10%. Thus, it took 15 days to reach the maximum training regimen and this continued for the duration of the experimental period. Rats were not exercised one day prior to experiments.
Reactivity of Pial Arterioles SED (n = 9) and VExT (n = 17) rats were prepared for in vivo examination of reactivity of cerebral arterioles. Rats were anesthetized (thiobutabarbital (Inactin); 100 mg/kg i.p.) and a tracheotomy was performed. The animals were ventilated mechanically with room air and supplemental oxygen. A catheter was inserted into a femoral vein for injection of supplemental anesthesia (10–30 mg/kg). A femoral artery was cannulated for measurement of arterial pressure. After placement of all catheters, the animal was placed in a head holder and a craniotomy was made over the left parietal cortex to expose the pial microcirculation [31]. The cranial
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window was suffused with artificial cerebral spinal fluid bubbled with 95% nitrogen and 5% carbon dioxide. Temperature of the suffusate was maintained at 37 1°C. The cranial window was connected via a three-way valve to an infusion pump, which allowed for infusion of agonists into the suffusate. Arterial blood gases were monitored and maintained within normal limits. After an equilibration period of 30–45 minutes, we examined dilation of pial arterioles in SED and VExT to an eNOS-dependent agonist (ADP; 10 and 100 lM), an nNOS-dependent agonist (NMDA; 100 and 300 lM), and a NOS-independent agonist (nitroglycerin; 0.1 and 1.0 lM). Agonists were mixed in artificial cerebral spinal fluid and superfused over the cranial window preparation. The diameter of pial arterioles was measured immediately before the application of agonists and every minute for five minutes during application of the agonists. Steady-state responses were reached within two to three minutes after application, and the diameter of pial arterioles returned to baseline within five minutes after application of the agonists was stopped. After this initial test of vascular reactivity, we then started a continuous suffusion of L-NMMA (100 lM) over the cranial window preparation. Thirty minutes after starting this superfusion, we again measured responses of pial arterioles in SED and VExT rats to ADP, NMDA, and nitroglycerin.
Assessment of Brain Injury Following MCAO Transient focal cerebral ischemia was performed using methods we have described previously [44,49,50]. On the day of the experiment, SED (n = 15) and VExT (n = 16) rats were anesthetized with ketamine/xylazine (100/15 mg/kg i.p., respectively). A laser Doppler flow probe (PeriFlux System 5000, Jarfalla, Sweden) was attached to the right side of the dorsal surface of the skull (2 mm caudal and 6 mm lateral to the bregma) to observe the decrease in cerebral blood flow during MCAO. A 4-0 monofilament nylon suture was prepared by rounding its tip by heating and coating with silicon. The right common and external carotid arteries were exposed and ligated. The MCA was occluded by inserting the filament from the basal area of the external carotid artery, and advancing it cranially into the internal carotid artery to the point where the MCA branched from the internal carotid artery. Onset of MCAO was determined by a rapid drop in cerebral blood flow, as measured by the laser Doppler. After occluding the MCA for two hours, reperfusion was initiated by removing the suture. Following 24 hours of reperfusion, the SED and VExT rats were euthanized, the brains were quickly removed and placed in ice-cold saline for five minutes. The brains were then cut into six 2-mm-thick coronal sections. The sections were stained with 2% TTC (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes at 37°C. Slice images were digitalized and the infarct lesion was evaluated using Kodak Molecular Imaging Software. Com-
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plete lack of staining with TTC was defined as the infarct lesion. The infarct volume was expressed as percentage of the contralateral hemisphere. We measured the total infarct volume, cortical infarct volume, and subcortical infarct volume in SED and VExT, as described previously [44,49].
Superoxide Levels Superoxide levels were measured by lucigenin-enhanced chemiluminescence as described previously [6,7,33]. In SED (n = 19) and VExT (n = 23) rats, the brains were removed and placed in a Krebs/HEPES buffer (pH 7.4) with the following composition (in mmol/L): 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgCl2, 1.3 CaCl2, 10 HEPES, 25 NaHCO3, and 5 glucose. Samples of cortex tissue, cut from brains of SED and VExT rats, were placed in polypropylene tubes containing 5 lM lucigenin. The tubes were then read in a Sirius/ FB15 luminometer (Berthold Detections Systems, Pforzheim, Germany), which reports RLU emitted over a 30-second interval for five minutes. Levels of superoxide reported are the value of tissue plus lucigenin-containing buffer minus background (lucigenin-containing buffer without tissue) and are normalized for tissue weight (RLU/min/mg tissue). We measured basal levels of superoxide and levels of superoxide in response to NADPH, a substrate for NADPH oxidase [6].
Citrate Synthase Activity To determine the level of exercise training in the VExT rats, we obtained a sample of the soleus muscle from SED (n = 6) and VExT (n = 13) rats. Citrate synthase activity was measured using a standard kit (CS0720; Sigma-Aldrich). Citrate synthase activity was determined based on the formation of 2-nitro-5-thiobenzoic acid and measured at a wavelength of 412 nm with the aid of spectrophotometer. The background absorbance was measured, then the reaction was initiated by the addition of oxaloacetic acid and the change in absorbance was measured.
Table 1. Mean arterial pressure, baseline diameter of cerebral arterioles, blood glucose concentration, body weight, and citrate synthase activity in the soleus muscle of SED and VExT rats
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133 50 126 340 56
VExT
6 3 3 19 16
127 44 105 370 116
3 (ns) 2 (ns) 6* 7 (ns) 18*
Values are means SE. ns, not significant. *p < 0.05 versus SED rats.
pial arterioles, mean arterial pressure, and body weight were similar in SED and VExT rats. However, blood glucose concentration was less in VExT rats when compared with SED rats and citrate synthase activity was significantly elevated in VExT rats when compared with SED rats.
Responses of Pial Arterioles We examined functional responses of pial arterioles to eNOS- and nNOS-dependent agonists (ADP and NMDA, respectively), and a NOS-independent agonist (nitroglycerin) in SED and VExT rats. We found that ADP- and NMDAinduced dilation of pial arterioles was significantly greater in VExT rats when compared with SED rats (Figures 1 and 2, respectively). In contrast to that observed with ADP and NMDA, nitroglycerin produced similar dose-related dilation of pial arterioles in SED and VExT rats (Figure 3). Thus, it does not appear that the effect of VExT on ADP- and
Statistical Analysis Unpaired t-tests were used to compare differences in reactivity of cerebral arterioles to the agonists, superoxide anion levels, infarct volumes and levels of citrate synthase in SED and VExT rats. A paired t-test was used to compare differences in reactivity of cerebral arterioles before and following application of L-NMMA. A p value of 0.05 or less was considered significant.
RESULTS Baseline Conditions Baseline diameter of pial arterioles, blood glucose concentration, mean arterial pressure, body weight and citrate synthase activity in the soleus muscle of SED and VExT rats are shown in Table 1. We found that baseline diameter of
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Figure 1. Response of cerebral arterioles to ADP in SED and VExT rats. Values are means SE. *p < 0.05 versus SED rats.
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NMDA-induced vasodilation is related to a nonspecific effect of VExT on vascular function. Next, we determined whether the contribution of nitric oxide to vasodilation in response to ADP and NMDA might be altered in VExT rats. We found that topical application of L-NMMA produced a similar decrease in baseline diameter of cerebral arterioles in SED (6 2%) and VExT (7 2%) rats. In addition, L-NMMA significantly decreased responses of cerebral arterioles to ADP and NMDA in both SED and VExT rats (Figures 4 and 5, respectively). Thus, it appears that the increase in vasodilation observed in the VExT rats in response to ADP and NMDA is related to the synthesis/ release of nitric oxide.
Superoxide Production We measured superoxide levels in cortex tissue in SED and VExT rats under basal conditions and during stimulation with NADPH, a substrate for NADPH oxidase. We found that the levels of superoxide in cortex tissue were similar in SED and VExT rats under basal and stimulated states (Figure 6). Thus, it appears that VExT does not influence the basal production of superoxide or the ability to produce superoxide under stimulated states.
Figure 2. Response of cerebral arterioles to NMDA in SED and VExT rats. Values are means SE. *p < 0.05 versus SED rats.
MCAO-Induced Brain Injury We measured infarct volumes in SED and VExT rats following MCAO. We found that total infarct volume produced by a two-hour MCAO followed by 24 hours of reperfusion was greater in SED than in VExT rats (Figure 7). Identification of the location of the infarct revealed that VExT significantly decreased cortical infarct volume, with subcortical infarct volume being similar in SED and VExT rats (Figure 7).
DISCUSSION There are four major new findings of the present study. First, we found that VExT increased responses of cerebral arterioles
Figure 3. Response of cerebral arterioles to nitroglycerin in SED and VExT rats. Values are means SE.
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Figure 4. Effect of L-NMMA on response of cerebral arterioles to ADP in SED (left panel) and VExT (right panel) rats. Values are meansSE. *p < 0.05 versus response before application of L-NMMA.
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Figure 5. Effect of L-NMMA on response of cerebral arterioles to NMDA in SED (left panel) and VExT (right panel) rats. Values are means SE. *p < 0.05 versus response before application of L-NMMA.
Figure 6. Superoxide levels in SED and VExT rats under baseline conditions and in response to NADPH, as substrate for NADPH oxidase. Values are means SE. *p < 0.05 versus baseline.
to eNOS- and nNOS-dependent agonists. This effect of VExT was specific since nitroglycerin produced similar dilation of cerebral arterioles in SED and VExT rats. Second, the increase in NOS-dependent reactivity of cerebral arterioles during VExT appeared to be related to the synthesis/release of nitric oxide as there was a significant reduction in responsiveness following application of L-NMMA. Third, VExT did not have a significant effect on either basal levels or agonist-induced levels of superoxide. Thus, we suggest that the effects of VExT on vascular function and brain injury following MCAO are not related to an influence of VExT on superoxide. Fourth, we found a significant protective effect of VExT on infarct volume following MCAO. Based upon these findings, we speculate that VExT may have an important therapeutic potential for reducing brain damage subsequent to a cerebral ischemic event, presumably by a mechanism that appears to involve the ability of nitric oxide to potentiate vascular function, and possibly cerebral blood flow.
Consideration of Methods In the present study, we used ADP and NMDA to examine eNOS- and nNOS-dependent responses of cerebral arterioles, respectively. Previous studies have suggested that ADP dilates
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cerebral arterioles via activation of NOS, presumably eNOS [2,9,29], whereas others [28,48] have suggested that relaxation of the rat MCA to purines is related, in part, to nitric oxide and an EDHF. In the present study, we found that LNMMA decreased responses of cerebral arterioles to ADP (10 and 100 lM) by about 85% and 76%, respectively, in SED rats, and by 80% and 74%, respectively, in VExT rats. Thus, it appears that nitric oxide plays a similar role in responses of cerebral arterioles to ADP in both SED and VExT rats. Regarding responses to NMDA, we and others have shown that NMDA dilates cerebral arterioles via the activation of nNOS and the subsequent synthesis/release of nitric oxide [10–12,43]. The findings from the present study support this conclusion. In addition, we found that L-NMMA decreased responses of cerebral arterioles to NMDA (100 and 300 lM) by about 70% and 60%, respectively, in SED rats and by about 75% and 76%, respectively, in VExT rats. Thus, it appears that nitric oxide plays a similar significant role in responses of cerebral arterioles to NMDA in SED and VExT rats. We reasoned that an increase in vascular responsiveness to NOS-dependent agonists might be related to an influence of VExT on superoxide levels, i.e., antioxidant enzyme systems
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Although we qualitatively examined changes in cerebral blood flow during MCAO in the SED and VExT rats, we did not quantify these changes. Thus, it is conceivable that the protective effect of VExT on brain infarct volume following MCAO might be related to differences in the relative level of ischemia produced in the two groups of rats by MCAO.
Infarct size (% contralateral hemisphere)
Consideration of Previous Studies
Figure 7. Top: representative 2-mm-thick TTC-stained coronal sections of the brains from SED and VExT rats subjected to two hours of MCAO and 24 hours of reperfusion. Dark stain indicates viable tissue and complete lack of staining defines the infarct region. Bottom: mean data depicting total, subcortical and cortical infarct volumes in SED and VExT rats. Values are means SE. *p < 0.05 versus SED rats.
as has been shown by others [15, 19, 21, 26, 35, 38]. We thought that if basal levels of superoxide were decreased by VExT, then this might influence the bioavailability of nitric oxide, and hence influence vascular reactivity to NOSdependent agonists. However, we found that basal levels of superoxide were similar in SED and VExT rats. This finding is similar to that which we have reported previously for moderate ExT [30], and suggests that increased responses of cerebral arterioles to NOS-dependent agonists in VExT rats are probably not related to an effect of superoxide on nitric oxide bioavailability. In addition, we found that agonistinduced stimulation of superoxide with NADPH was similar in SED and VExT rats, suggesting that the ability to produce superoxide is not altered by VExT. Thus, we suggest that levels of superoxide may not be the limiting factor in enhanced vascular responsiveness and protection against brain damage following cerebral ischemia/reperfusion by VExT.
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Many have examined the effects of exercise on NOSdependent reactivity in animals and human subjects [17, 22, 24, 25, 41, 42]. For the most part, these studies have suggested that exercise enhances NOS-dependent dilation/ relaxation of skeletal and peripheral blood arteries and arterioles. The precise mechanism(s) by which exercise potentiates NOS-dependent responses are not clear, but have been suggested to be related to an increase in eNOS [36, 41]. In previous studies, we have reported that moderate exercise training produced a small, but significant increase in eNOS and nNOS proteins in cerebral blood vessels and brain tissue from nondiabetic (control) rats [30, 32]. However, this increase in eNOS and nNOS proteins did not equate with an increase in reactivity of large (basilar artery) and small (pial arterioles) cerebral vessels to eNOS- or nNOS-dependent agonists [1, 30, 32]. The mechanisms by which exercise training produces an increase in eNOS protein may be related to changes in shear stress across endothelium [3, 18]. While we and others have shown that exercise training produces an increase in nNOS protein in the brain and other organ systems, the mechanism for this effect of exercise training on nNOS remains elusive [30, 34, 46, 51]. In the present study, we used a more vigorous level of exercise training (increased speed, greater incline, and longer duration) to determine whether this regimen might influence cerebrovascular function. We found that VExT produced a significant increase in NOS-dependent reactivity and protected the brain from ischemia/reperfusion injury following MCAO. Thus, we suggest that although a moderate level of ExT may be beneficial during disease states, a more vigorous level of ExT may be required to influence cerebrovascular function and brain injury in animals devoid of disease. However, as we increased the duration as well as the intensity of exercise training in the present study when compared with our previous study [1, 30], we cannot rule out that a longer duration of exercise training (at a similar level of intensity) would be beneficial on vascular function and brain injury following MCAO. Future studies could examine the relationship between duration and/or intensity on the protective effects of exercise training. Epidemiological studies examining the impact of preexercise training on the risk of ischemic stroke have met with mixed results. Some have reported that pre-exercise training was associated with improved functional outcome after a
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stroke, a less severe level of stroke, and/or a decrease in the actual occurrence of an ischemic stroke [5, 20, 23, 27, 39, 40]. Others have examined the influence of pre-exercise training on the pathogenesis of stroke using animal models. Endres et al. [8] found that three weeks of exercise training prior to induction of MCAO could protect against cerebral ischemic damage in eNOS wild-type mice, but not in eNOS knockout mice, suggesting that the protective effect of pre-exercise training was related to eNOS activity. The findings from the present study are in agreement and extend the findings of this previous study. We found that VExT could potentiate eNOS- and nNOS-dependent responses of cerebral arterioles and could protect the brain from cerebral ischemic damage following MCAO via a mechanism that is presumably related an increase in nitric oxide synthesis/release and not related to an influence of VExT on oxidative stress. In summary, we examined the effects of VExT on eNOSand nNOS-dependent reactivity of cerebral resistance arterioles and on brain injury following cerebral ischemia/ reperfusion. We found that VExT potentiated eNOS- and nNOS-dependent dilation of cerebral arterioles. In addition, we found that VExT alleviated brain injury following MCAO. Furthermore, basal and agonist-induced levels of superoxide were not influenced by VExT. Based upon these findings, we suggest that ExT can restore impaired eNOS- and nNOS-
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dependent dilation of cerebral arterioles via mechanisms that may regulate the balance between oxidative stress and nitric oxide synthesis/release.
ACKNOWLEDGMENTS This study was supported by funds from the National Heart, Lung and Blood Institute Grant (HL-090657) and funds from the LSU Health Sciences Center-Shreveport.
PERSPECTIVE We report that VExT potentiated eNOS- and nNOSdependent dilation of cerebral arterioles and alleviated brain injury following MCAO. In addition, basal and agonistinduced levels of superoxide were not influenced by VExT. Based upon these findings, we suggest that VExT can restore impaired cerebrovascular function via mechanisms that may regulate the balance between oxidative stress and nitric oxide synthesis/release. We speculate that VExT may have an important therapeutic potential for reducing brain damage subsequent to a cerebral ischemic event presumably by a mechanism that appears to involve the ability of nitric oxide to potentiate vascular function, and possibly cerebral blood flow.
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