Exercise training is shown to enhance brain functions by increasing neuron survival, activity, and plasticity (9). Early gait-related exercise training is suggested to be well tolerated and feasible for stroke patients, with their functional improvements strongly correlating with early training (28,29). Our previous studies show that 2 wk of treadmill training reduced infarct volume and improved neurological outcomes and motor function in brain ischemic rats (7,37,38). These results suggest that early exercise training has neuroprotective effects after brain ischemia. However, the mechanisms of exercise-induced neuroprotective effects are not fully known.
All types of exercise stimulate the secretion of growth hormones; the insulin-like growth factor I (IGF-I) is a primary downstream mediator of growth hormone actions (13). Studies indicate that exercise enhances the circulating IGF-I to enter the normal hippocampus, thereby increasing its neuronal activity, neurogenesis, and spine growth (4,14,36). These results suggest that IGF-I mediates exercise-induced improvements in cognitive functions such as learning and memory. Other studies suggest that IGF-I is a biomarker for health status and is a useful predictor for outcomes in patients who experienced stroke (2,19,25). In addition, IGF-I is found to reduce the extent of ischemic brain injury, improve somatosensory function, and promote exercise-induced protective effects in some neurodegenerative animal models (5,15,16). Some studies suggest that IGF-I–activating phosphoinositide 3-kinase/Akt signaling pathway plays an important role in regulating cell survival, differentiation, and metabolism (3,10). Collectively, these results indicate that IGF-I might be involved in exercise-induced neuroprotection after brain ischemia. However, whether IGF-I is involved in exercise-induced neuroprotection after brain ischemia needs to be examined.
Previous studies using anti–IGF-I antiserum to block exercise-induced brain uptake of circulating IGF-I show that IGF-I is important in exerting the effects of exercise on brain functions (4,5,36). However, the exercise ability of animals after IGF-I inhibition is not reported in these studies. Brain ischemia causes damage not only to the hippocampus but also the motor cortex and striatum, which are known as the two major motor control areas (38). Therefore, whether IGF-I signaling also participates in regulating the motor activity after brain ischemia also needs to be investigated. We hypothesized that 2 wk of treadmill training will enhance the IGF-I entrance and the expression of its downstream signaling molecules (phosphorylated Aktser473, p-Aktser473) in ischemic brain, leading to improved neuroprotection and motor function. We examined the expression of IGF-I concentration in the plasma, affected motor cortex, and striatum as well as the expression of its downstream signaling molecules in the affected motor cortex and striatum. In addition, we used the IGF-I receptor inhibitor to downregulate IGF-I signaling in the ischemic brain to determine the effects of IGF-I signaling on motor activity.
Adult Sprague-Dawley rats (8 wk of age, body weight = 300–350 g) were used. All experimental procedures were approved by the Institutional Animal Care and Use Committee of National Yang-Ming University, Taipei, Taiwan (IACUC 941147). This procedure is in accordance with the policy statement of the American College of Sports Medicine on research with experimental animals. The rats were housed in a temperature-controlled room (22°C ± 1°C) with daily artificial illumination for 12 h (7:00 a.m. to 7:00 p.m.). All animals received food and water ad libitum. Rats were randomly assigned to one of four groups: middle cerebral artery occlusion (MCAO) without exercise training (MC), MCAO with exercise training (ME), MCAO with IGF-I receptor inhibitor and without exercise training (MAg), and MCAO with IGF-I receptor inhibitor and exercise training (MEAg). There were two subgroups in each group (n = 8 for each subgroup). Rats in one subgroup were used to examine the following: IGF-I concentration in the plasma, affected motor cortex, and striatum; expression of p-Aktser473 in the affected motor cortex and striatum; and motor function. Rats in the other subgroup were used to measure the infarct volume.
The MCAO procedures leading to focal brain ischemia were conducted under pentobarbital anesthesia (50 mg·kg−1). After anesthesia, a 2-cm vertical skin incision was made midway between the right eye and ear, and the temporalis muscle was split; then a 2-mm burr hole was drilled at the junction of the zygomatic arch and the squamous bone to expose the middle cerebral artery (MCA) trunk under the microscope. The right MCA trunk was ligated with a 10-0 suture. According to previous studies, the occlusion of bilateral common carotid arteries (CCAs) in addition to the MCA is essential to induce brain ischemia in rats because of the abundant communicative circulation between cerebral arteries (8,20,23). Bilateral CCAs were occluded using nontraumatic aneurysm clips. Complete interruption of blood flow was confirmed under the microscope. After a predetermined ischemic duration (60 min), the aneurysm clips and the 10-0 suture were removed from bilateral CCAs and MCA. The restoration of blood flow in all three arteries was observed directly under the microscope. During surgery, the rectal temperature was monitored and maintained at 37.0°C ± 0.5°C by using a heating blanket with an electronic temperature controller (050100C1; WATLOW, Bowdoinham, ME). After suturing and recovery, the rats were returned to their cages and were allowed free access to food and water. Neurological examinations were performed 24 h after MCAO by using a neurological grading system with a five-point scale (from 0 to 4) as described by Menzies et al. (23). Median neurological scores were 2 (range = 1–2), 2 (range = 1–3), 2 (range = 1–3), and 2 (range = 1–3) in the MC, ME, MAg, and MEAg groups, respectively. Neurological scores did not differ significantly between groups, and no rats died during the experimental period.
Injection of IGF-I receptor inhibitor
Rats in the MAg and MEAg groups were cannulated over the right motor cortex (AP = +2.0 mm, RL = +2.4 mm, V = −1.8 mm) and right striatum (AP = −0.3 mm, RL = +3.4 mm, V = −4.4 mm from the bregma) as described by Paxinos and Watson (27). The skull was exposed, and a guided cannula was implanted through a boring hole over the right motor cortex and striatum. Animals were given 7 d to recover from surgery before receiving the MCAO procedures as described. During the study period, rats in these two groups received a daily injection of IGF-I receptor inhibitor (3-bromo-5-t-butyl-4-hydroxy-benzylidene-malonitrile, AG1024, 30 μg in 3 μL of phosphate-buffered saline) through a cannula under light ether anesthesia and were allowed to totally recover from anesthesia (approximately 30 min). After recovery, the rats were then exercised on the treadmill (MEAg group) or remained relatively inactive (MAg group).
A motor-driven treadmill (T410E; Singa Co., Taipei, Taiwan) was used for the exercise training. Rats were placed on a moving belt facing away from the electrified grid. Rats ran in the direction opposite to the movement of the belt to avoid light foot shocks (intensity, 1.0 mA with a 1-s “on” phase and 2-s “off” phase; Stimulus Controller Model D48E; DRI Co., Kwei-Shan,Taoyuan, Taiwan). The speed of the treadmill was set at 20 m·min−1 and 30 min·d−1 for 14 consecutive days (38). Rats in the MC and MAg groups remained relatively inactive, whereas those in the ME and MEAg groups exercised on the treadmill. According to our pilot study, rats administered with AG1024 injection may not be able to complete the 30-min treadmill training. If rats stayed on the electrical grid for >5 s, they were removed from the treadmill and rested for 5 min. If the rats failed to run on the treadmill again within 1 min after the rest, the training session for that time was then terminated. The total duration of training per day in each rat was recorded in both exercise groups.
Motor function measurement
The parallel bar crossing test was used to test motor function (12). The two parallel bars (diameter = 1.0 cm, length = 110 cm, interrod distance = 2.5 cm) were connected to platforms (15 × 50 cm) at each end. In each trial, the rats were placed on a platform and encouraged to walk on the parallel bars for 1 min. Errors were recorded when both hind paws were placed on one row, when a hind paw slipped over the rod, or when the animals fell or swung from the rods. We calculated the errors made per meter in 1 min. The motor function test was performed 24 h after MCAO (pretest) and 14 d after MCAO (posttest).
After the final exercise for the training group (or the same period of relative inactivity for the control group), half of the rats in each group were anesthetized with pentobarbital (50 mg·kg−1). Plasma samples were collected from 1 mL of blood with heparin (10 IU) saline by heart puncture and centrifuged at 10,000g for 1 min at 4°C. The supernatant was collected and stored at −20°C. After plasma collection, the rats were sacrificed by decapitation, and the regions of the affected motor cortex and striatum were removed, quickly frozen in liquid nitrogen, and stored at −80°C.
Samples from the affected motor cortex and striatum were weighed and homogenized in the lysis buffer, containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1 μg·mL−1 aprotinin, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.2 mM phenylmethylsulfonyl fluoride. A 1:100 dilution of an inhibitor cocktail was added to the buffer. The lysates were centrifuged at 12,500g for 30 min at 4°C. Total protein concentrations in the supernatants were determined using the Bradford-red protein assay. After adding sample buffer containing 0.1 M Tris–HCl (pH 6.8), 25% glycerol, 2% SDS, 0.02% bromophenol blue, and 5% β-mercaptoethanol, the sample was boiled for 10 min. Proteins were resolved in 8% SDS–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking with 0.1% Tween 20/Tris-buffered saline (TBST) containing 5% nonfat milk at room temperature for 60 min, the membranes were probed with appropriate primary antibodies including rabbit anti–phosphorylated Aktser473 (p-Aktser473) or anti–total Akt (t-Akt; Millipore; 1:1000) antibodies in TBST at 4°C overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase–conjugated goat antirabbit IgG secondary antibodies (Millipore; 1:6000) in TBST at room temperature for 1 h. The signals were visualized with Western blot chemiluminescence reagent (Amersham Biosciences, Buckinghamshire, UK). The levels of protein expression were quantified by densitometry using image analysis software (ImageQuant; Amersham Biosciences). The ratios of p-Aktser473 densitometry data to that of t-Akt from the affected motor cortex and striatum were calculated.
IGF-I concentrations in the affected motor cortex, striatum, and plasma were measured using an IGF-I ELISA kit (R&D Systems, Minneapolis, MN). The plasma and supernatants from tissue sample were diluted 1000-fold and 6-fold with the calibrator diluent RD5-38 buffer. Calibrator diluent buffer (50 μL) was added to each well followed by 50 μL of sample, including standard dilutions. Plates were incubated at room temperature on a horizontal orbital microplate shaker set at 500 ± 50 rpm for 2 h. After aspirating and washing each well five times, 100-μL mouse IGF-I conjugate was added and incubated at room temperature on the shaker for 2 h. After five cycles of aspiration and washing, 100 μL of substrate solution was added and incubated at room temperature in the dark for 30 min. After incubation, 100 μL of stop solution was added, and the plate was gently tapped to ensure mixing. The optical density of each well was determined with a microplate reader at 450 nm. The IGF-I concentration in each sample was calculated from the standard curve and presented as nanograms per gram of tissue weight for affected motor cortex and striatum and presented as nanograms per milliliter for plasma samples.
Infarct volume measurement
The other half of the rats in each group were sacrificed under anesthesia by intracardiac perfusion with 100 mL in 0.1 M phosphate-buffered saline (pH 7.2). The brain was removed and dissected into coronal 2-mm sections using a brain slicer. The fresh brain slices were immersed in a 2% 2,3,5-triphenyltetrazolium chloride saline solution at 37°C for 10 min and then fixed in 10% phosphate-buffered formalin. The unstained areas on each brain slice, defined as infarct areas, were measured by an image analyzer (Image-Pro Plus; Total Smart Technology, Taiwan). The damaged areas were mainly confined to the cerebral cortex and its adjacent caudate nucleus, putamen, and hippocampus (38). The total of measured infarct volume (MV) for each brain was calculated by summation of the infarct areas of all brain slices (area of infarct in square millimeters multiplied by the thickness, 2 mm). Right (RV) and left hemisphere volumes (LV) were measured and calculated to compensate for the effect of brain edema on the MV in ischemic hemisphere. The corrected infarct volume was used and calculated as previously described: LV − (RV − MV) (20,37,38). The corrected infarct volume was then presented as a percentage to the volume of the left hemisphere.
The results from motor function, infarct volume, expression of p-Aktser473, IGF-I concentration, and exercise duration were presented as mean ± SEM. Differences in the infarct volume and expression of p-Aktser473 and IGF-I concentration were examined by two-way ANOVA. Differences in the motor function and exercise duration were examined by repeated-measure two-way ANOVA. A post hoc test was used to determine the differences among groups. The correlation between exercise duration and IGF-I signaling was examined by Pearson correlation. Significance was set at P < 0.05.
Results of the infarct volume are shown in Figure 1. Two-way ANOVA indicated an interaction between exercise and IGF-I inhibition (P < 0.05). The post hoc test showed a significant decrease in infarct volume in the ME group when compared with the MC group (P < 0.01). However, IGF-I receptor inhibitor increased the infarct volume in the MAg group when compared with the MC group (P < 0.01) and in the MEAg group when compared with the ME group (P < 0.01). There was no significant difference between the MAg and MEAg groups.
Results of the parallel bar crossing test are demonstrated in Figure 2. Two-way ANOVA indicated an interaction between exercise and IGF-I inhibition (P < 0.05). The post hoc test indicated no significant difference among groups measured 24 h after MCAO. The motor function in the ME group measured 14 d after MCAO showed significant improvement in comparison to the MC group (P < 0.01). There were significant differences between the MAg and MC groups (P < 0.01), between the MEAg and ME groups (P < 0.01), and between the MAg and MEAg groups (P < 0.01).
Results of the IGF-I concentration in the affected motor cortex and striatum are shown in Figure 3A. Two-way ANOVA indicated an interaction between exercise and IGF-I inhibition (motor cortex, P < 0.01; striatum, P < 0.01). The post hoc test indicated a significant difference between the ME and MC groups (motor cortex, P < 0.01; striatum, P < 0.01). There was no significant difference in brain IGF-I concentration between the MC and MAg groups. However, there were significant differences between the ME and MEAg groups (motor cortex, P < 0.01; striatum, P < 0.01) and between the MAg and MEAg groups (motor cortex, P < 0.01; striatum, P < 0.01).
IGF-I concentrations in the plasma are shown in Figure 3B. Two-way ANOVA indicated an interaction between exercise and IGF-I inhibition (P < 0.01). The post hoc test indicated a significant difference between the ME and MC groups (P < 0.01). IGF-I concentrations in the plasma from the MAg and MEAg groups were not significantly different from that of the MC group. However, there was a significant difference between the ME and MEAg groups (P < 0.05).
The IGF-I signaling was examined by assessing the expression of p-Aktser473. The ratios of p-Aktser473 densitometry data to that of total Akt in the affected motor cortex and striatum are shown in Figure 4. Two-way ANOVA indicated an interaction between exercise and IGF-I inhibition (motor cortex, P < 0.01; striatum, P < 0.01). The post hoc test indicated a significant difference between the ME and MC groups (motor cortex, P < 0.01; striatum, P < 0.01). There were significant differences between the MAg and MC groups (motor cortex, P < 0.05; striatum, P < 0.05), between the MEAg and ME groups (motor cortex, P < 0.01; striatum, P < 0.01), and between the MAg and MEAg groups (motor cortex, P < 0.01; striatum, (P < 0.01).
In this study, rats in the MEAg group did not complete the 30-min exercise training session, and the exercise duration was decreased with increasing IGF-I inhibition (Fig. 5). The average exercise duration was 10.6 ± 0.6 min for rats in the MEAg group; however, all rats in the ME group completed every 30-min exercise training session. There were strong positive correlations between the p-Aktser473 level in the brain and exercise duration (motor cortex, r = 0.85, P < 0.01; striatum, r = 0.85, P < 0.01). There were also strong positive correlations between the exercise duration and IGF-I concentration in the brain (motor cortex, r = 0.95, P < 0.01; striatum, r = 0.94, P < 0.01). Furthermore, there was a strong negative correlation between exercise duration and IGF-I concentration in the plasma (r = −0.81, P < 0.01).
In this study, we found that 2 wk of treadmill training significantly reduced infarct volume and improved motor function with involvement of IGF-I signaling. Treadmill training enhanced IGF-I entrance into the ischemic brain and increased IGF-I signaling expression. Inhibiting IGF-I signaling in the ischemic brain reduced such protective effects. We also noted that IGF-I signaling was involved in regulating the motor activity after brain ischemia.
The IGF-I concentration was decreased in the plasma but increased in the affected motor cortex and striatum in the exercise group. Previous studies suggest that exercise training enhances the uptake of circulating IGF-I by the normal brain, thereby mediating its plasticity and regulating cognitive functions (4,24,36). Our results are in agreement with those of previous studies and suggest that exercise enhances the uptake of circulating IGF-I by the ischemic brain for neuroprotection. IGF-I signaling is thought to play an important role in regulating cell survival, differentiation, and metabolism (3,10). Our results also found that the expression of p-Aktser473 in the ischemic brain increased with respect to decreased infarct volume after exercise training. Moreover, administration of the IGF-I receptor inhibitor inhibited the increase in brain IGF-I concentration in the exercise group. Although the brain IGF-I concentration in exercise animals administered with IGF-I receptor inhibitor was higher than that in the ischemic control, the increase was significantly lower than in animals who only exercised (Fig. 3). The protective effects of exercise were also reduced by IGF-I inhibition. These results indicate that IGF-I signaling is involved in exercise-mediated neuroprotection after brain ischemia. Although a previous study indicated that 1 wk of exercise training does not significantly increase IGF-I concentration in the ischemic hemisphere (30); the differences between this study and ours might be due to the differences in the exercise period and intensity.
We noted that rats administered with IGF-I receptor inhibitor did not complete the 30-min training session throughout the 14-d study period and that the exercise duration was significantly decreased immediately after the first injection of IGF-I receptor inhibitor. The p-Aktser473 level in the affected motor cortex and striatum was strongly correlated with exercise duration. These results indicate that IGF-I signaling might participate in regulating motor activity. IGF-I is suggested to modulate neuronal activity by enhancing the biological activity of brain-derived neurotrophic factor (BDNF) or modify electrophysiological properties of neurons by modulating ion channels and glutamate receptors (22,26,35). It is also suggested to increase cell survival by increasing glucose metabolism and angiogenesis and inhibiting apoptotic mediators (15,21,33,34). These modulations may all contribute to the protective effects of IGF-I. In support, the inhibition of IGF-I signaling resulted in progressive cell death after brain ischemia and decreased ability to exercise. In addition, the motor cortex and striatum are known as the major control areas for motor function; inhibiting the IGF-I signaling in these areas may further contribute to decreases in the ability to exercise. These results indicate that IGF-I signaling is at least in part contributive toward improved motor functions.
We noted that exercise duration correlated positively with IGF-I concentration in the affected motor cortex and striatum. We also noted that rats only receiving IGF-I receptor inhibitor did not show significant changes in the brain IGF-I concentration compared with the ischemic control group (Fig. 3). This result indicates that the IGF-I receptor inhibitor does not influence the basal IGF-I concentration in ischemic brain. Therefore, we suggest that exercise duration is an important factor for brain to uptake circulating IGF-I. In addition, the infarct volume was decreased but not significantly in rats administered with IGF-I receptor inhibitor and undergoing exercise training compared to rats only administered with IGF-I receptor inhibitor. This result indicates that other pathways may also be involved in the protective effects of exercise. It is suggested that exercise training increases BDNF expression in the ischemic brain (7,31). BDNF is suggested to be a survival-promoting factor similar to IGF-I on neurons and is thought to protect neurons from ischemic insult in vitro and in vivo (6,18,31). Other studies also suggest that BDNF participates in neuronal plasticity in both animal and human subjects (1,17,32). These results indicate that BDNF may also participate in exercise-mediated protective effects after brain ischemia. Previous studies show that IGF-I enhances the expression of BDNF receptor (TrkB) and the bioactivity of BDNF (11,22). Therefore, the involvement of IGF-I signaling in the neuroprotective effects of exercise after brain ischemia cannot be excluded on the basis of our results.
In conclusion, 2 wk of treadmill training enhanced IGF-I entrance into the ischemic brain. Moreover, inhibiting IGF-I signaling in the ischemic brain not only attenuated exercise-mediated neuroprotection but also contributed to the decrease in exercise ability and motor function. Taken together, these data suggest that IGF-I is involved in mediating the protective effects of exercise training after brain ischemia.
This study was supported by a grant (NSC95-2314-B-010-041-MY3) from the National Science Council of the Republic of China.
The authors have no conflict of interests to report.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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