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Walsh et al. APNM 2015-0410 Accepted Neurotrophic Growth Factor Responses to Lower Body Resistance Training in Older Adults
Jeremy J. Walsh1, Trisha D. Scribbans1, Robert F. Bentley1, J. Mikhail Kellawan2, Brendon Gurd1, and Michael E. Tschakovsky1.
1
School of Kinesiology and Health Studies, Queen’s University, Kingston, ON, Canada 2
Department of Kinesiology, University of Wisconsin, Madison, WI, USA
Corresponding Author: Michael Tschakovsky Mailing Address: 28 Division St., Kingston, ON, Canada, K7L 3N6 Telephone: 613-533-6000 ext. 74697 Fax: 613-533-2009 Email: [email protected]
1
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Walsh et al. APNM 2015-0410 Accepted ABSTRACT Resistance exercise is an efficacious stimulus for improving cognitive function in older adults, which may be mediated by the upregulation of blood-borne neurotrophic growth factors (NTFs) like brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1). However, the NTF response to resistance exercise and training in older adults is poorly understood. Therefore, the purpose of this study was to characterize the timing and magnitude of the NTF response following an acute bout of resistance exercise before and after 8 weeks of resistance training. Methods: 10 cognitively normal, older adults (ages 60 – 77, 5 men) were examined. The acute NTF response to resistance exercise was assessed via serum samples drawn at designated time points following exercise. This procedure was then repeated following 8 weeks of resistance training. Results: BDNF increased immediately post-exercise (∆9% pre-training, ∆11% posttraining) then returned to resting levels while IGF-1 remained stable following resistance exercise before and after 8 weeks of resistance training. Basal levels of both NTFs were unaffected by the 8-week training period. Conclusion: We report a transient increase in serum BDNF following a bout of resistance exercise in older adults, which could have implications for the design of interventions seeking to maximize cognitive function in older adults. Key Words: BDNF, IGF-1, neuroplasticity, cognitive function, aging
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Walsh et al. APNM 2015-0410 Accepted INTRODUCTION Aging is associated with marked declines in muscle mass (Liu-Ambrose and Donaldson, 2009), bone density (Parkhouse et al. 2000), brain tissue (Fjell et al. 2009), and a concomitant decrease in cognitive function (Liu-Ambrose and Donaldson 2009). Interventions that protect and improve muscle strength and cognitive function in older adults have a critical role to play in healthy aging. In this regard, the American College of Sports Medicine advocates for the participation of older adults in regular resistance training as a means of combatting frailty, improving body composition, and more recently improving brain function (Nelson and Rejeski 2007). Both short- and longduration resistance training programs have been shown to improve response inhibition, selective attention, processing speed, and other executive functions in older adults (Chang et al. 2012), with some benefits persisting for up to 1 year post-training (PerrigChiello et al. 1998). More recently, long-term resistance training has been shown to stimulate brain plasticity, with alterations in cortical activation being functionally coupled with enhanced flanker task performance (Liu-Ambrose et al. 2012).
Cognitive training represents another strategy for combatting age-related cognitive decline and enhancing select cognitive functions among older adults. While a lack of transferable improvements has been a critique of cognitive training paradigms (Ball et al. 2002), these do lead to significant improvements in cognitive domains that are directly trained by the program, such as processing speed, attention, and memory (Gates and Valenzuela 2010). In addition, emerging evidence suggests that combining exercise and
3
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Walsh et al. APNM 2015-0410 Accepted cognitive training (multi-modal training) may exact greater benefit to cognitive function than either performed alone (Fabre et al. 2002; Oswald et al. 2006).
Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) are key orchestrators of the positive neurobiological adaptations garnered through exercise and cognitive training (Cotman et al. 2007; Valenzuela et al.2007). BDNF is essential for learning and memory (Cotman et al. 2007) and plays a key role in regulating the growth, maintenance, and survival of neurons (Marosi and Mattson 2014). Cognitive activity evokes localized BDNF production in the brain, which plays a key role in neuroplasticity (Valenzuela et al. 2007). Furthermore, neuroplasticity also appears sensitive to bloodborne BDNF levels as evidenced by increased neuroplasticity responses with central infusion of BDNF in rats (Hoshaw et al. 2005) and mice (D’Amore et al. 2013). Bloodborne IGF-1 appears to be essential for exercise-induced neuroplasticity, as blocking entry of circulating IGF-1 into the brain completely abrogates the neurogenic and neuroprotective effects of exercise (Carro et al. 2000, 2001; Trejo et al. 2007).
Animal models demonstrate divergent pathways of exercise-induced neural plasticity for resistance vs. aerobic training, wherein resistance training may evoke its effects through the actions IGF-1 and aerobic through BDNF (Cassilhas et al. 2012). Indeed, Cassilhas et al. (2012) observed a 43% increase in systemic and 95% increase in hippocampal IGF1 following a period of resistance training in rats. Further, these changes in IGF-1 were coupled with improved hippocampal dependent memory, suggesting a direct impact of IGF-1 on improved cognitive function in animals. This IGF-1 mediated improvement 4
Page 5 of 40
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Walsh et al. APNM 2015-0410 Accepted may be also be indirect, as the expression of BDNF in the hippocampus is dependent on the presence of IGF-1 (Ding et al. 2006).
Based on this, our current working hypothesis is that combining exercise and cognitive training to maximize NTF delivery to the microcirculation of areas of the brain involved in cognitive processing may improve the benefits of these interventions. So far, no “multi-modal” training studies based their timing of exercise and cognitive training bouts on enhancing NTF dose (Fabre et al. 2002; Gates et al. 2011; Oswald et al. 2006).
Maximizing delivery to cerebral microcirculation is a function of local perfusion and blood [NTF’s]. We know that neural activity evokes rapid and substantial increases in local cerebral perfusion (neurovascular coupling response) (Attwell et al. 2010). What remains poorly understood is the timing of resistance exercise-evoked increases in blood borne NTF’s in older adults; the very group that would benefit considerably from interventions that improve strength and cognition. Specifically, the blood-borne BDNF response to an acute bout of resistance exercise or how this might be altered by resistance training in this population has yet to be determined. While the blood-borne IGF-1 response to resistance exercise in older adults has been investigated somewhat, findings are conflicting with some reports of no change (Frystyk 2010; Kraemer and Ratamess 2005) and others of immediate and sustained (6 hours) increases in IGF-1 in older adults following a single bout of resistance exercise (Bermon et al. 1999).
5
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Walsh et al. APNM 2015-0410 Accepted Therefore, the objective of this study was to identify timing and magnitude of changes in blood-borne BDNF and IGF-1 in response to a bout of resistance exercise, and whether 8 weeks of resistance training affected this response. Based on previous work and evidence from animal models, we hypothesize that an acute bout of resistance exercise will result in a significant increase in IGF-1 levels, whereas BDNF will remain unchanged. Further, we hypothesize that basal IGF-1 will increase following 8 week of resistance training, while basal BDNF levels will remain unaffected. METHODS Participants Ten cognitively normal older adults (5 males, mean age 66 ±5.3 years; Table 1) from the Kingston, Ontario community were recruited for this study. The study procedures were approved by the Health Sciences Human Research Ethics Board at Queen’s University. All participants read and signed a consent form, which explicitly detailed all of the experimental procedures and the associated risks. Inclusion criteria were older (≥ 60 years old), cognitively normal adults as assessed by the Montreal Cognitive Assessment (MoCA), free of major adverse neurological events or mental illness including depression, free from known cardiovascular disease, no history of substance abuse, not taking any psychiatric medication, were non-smokers/non-nicotine users, no regular resistance training within the last 6 months, and free from contraindications to resistance training. All female participants were post-menopausal and were not on hormonereplacement therapy. The current study was part of a larger, multi-modal intervention where participants performed resistance training followed immediately by 40 minutes of
6
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Walsh et al. APNM 2015-0410 Accepted cognitive training, performed 3x/week for 8 weeks. However, participants did not perform cognitive training during on the NTF testing days. Strength Training Program Participants performed supervised resistance training 3 times per week (Monday / Wednesday / Friday) for an 8-week period. The program followed the principle of progressive overload with active recovery sessions implemented to facilitate recovery. Each training session began with a 7 minute warm-up on a leg ergometer followed by a 5 minute routine of dynamic movements, including walking knee-raises, walking heelkicks, and lateral shuffling. The training program consisted of 3 lower-body exercises: 1) Split Squat (performed with body weight or dumbbells), 2) Double Leg Press, and 3) Single Leg Press (Atlantis C-204 Dual Pivot Press, Quebec, Canada). Participants completed 4 consecutive sets of each exercise with 90-second rest periods between sets. Following completion of these exercises, participants cooled down on a leg-ergometer for 7 minutes. Each set consisted of 8 -10 repetitions at 60% 1-repetition maximum (1RM) during the first week, 70% during the second and fourth weeks, and 80% 1RM for the remainder of the study. The active recovery weeks consisted of the same exercises performed for 12 – 15 reps at 50% 1RM and were performed during the third and sixth weeks of training. Exercise supervisors tracked the performance of participants on a daily basis and weights were individually adjusted on a weekly basis to match the desired intensity. Orientation Prior to strength testing participants were oriented with the resistance training program, where proper weight lifting technique, breathing, posture, and lifting cadence were
7
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Walsh et al. APNM 2015-0410 Accepted taught. On this day participants performed the regular resistance training routine. The selected weight was relatively low and could be completed for >15 repetitions. Weight was modestly increased between sets; however, all exercises were of submaximal intensity. Participants were unaccustomed to resistance training, therefore, an additional purpose of this visit was to stimulate delayed onset of muscle soreness (DOMS) and allow adequate recovery to ensure that strength was fully restored for the strength assessment session. Strength Assessment Five days after the training orientation visit, participants performed submaximal strength testing using a predicted 1-repitition maximum (1RM) protocol as described by Reynolds et al. (2006). Strength was re-assessed on the second last day of training. Participants performed the previously described resistance training routine with a lifting cadence of 2:2 (concentric:eccentric phases) and rested for 3 minutes between sets to allow for proper recovery and avoid fatigue effects. NTF Testing Blood collection was performed prior to the initiation of regular training (Pre-Training) and on the final training session (Post-Training). Participants arrived at the lab at 7 a.m. following an overnight fast and were fed a standardized breakfast (toasted bagel [~190 kcal; 1 g fat, 36 g CHO, 7 g protein] with 15 g of cream cheese [~45 kcal; 4 g fat, 1 g CHO, 1 g protein] and a 200 mL juice pack [~100 kcal, 0 g fat, 26 g CHO, 0 g protein]). Following breakfast, a vein of the forearm was catheterized using a 20-gauge Teflon catheter (BD Insyte Autoguard, Oakville, Canada) and the resting (baseline) blood
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Walsh et al. APNM 2015-0410 Accepted sample was drawn following 30 minutes of rest. Following the rest period and the regular warm-up routine, participants performed the 4 sets of each resistance exercise at 80% of their 1RM for 8-10 reps, with 90 seconds of rest between sets. Weight was adjusted to ensure 80% relative intensity during the Post-training NTF testing session to account for strengths gains due to training. Serial blood samples were drawn at Rest (prior to exercise) and at 0, 10, 20, 30, 40, 60, and 120 minutes of recovery following a bout of resistance exercise. A schematic of the NTF Testing protocol and blood-draw time points can be viewed in Figure 1. Consumption of water was allowed ad libitum throughout the training and bed rest periods. Blood Analysis Serum BDNF and IGF-1 samples were collected in serum separator tubes and left to clot for 30 minutes at room temperature. Samples were centrifuged for 15 minutes at 1000 x g at 4oC, aliquoted, and immediately stored at -80oC. Serum BDNF was analyzed using the DBD000 enzyme-linked immunoassay (ELISA) kit from Quanitkine (R&D Systems, Minneapolis, MN, USA). The kit detection range was 62.5 pg/mL - 4,000 pg/mL, and the intra- and interassay coefficients of variation were 5% and 9%, respectively. While the assay was performed in accordance with the manufacturer’s instructions, we optimized the kit specifically for our samples. Briefly, serum samples were diluted 20fold with the supplied calibrator diluent. Plates were read at 450 and 540 nm with a micro plate reader (Synergy 2, BioTek Instruments, Winooski, VT, USA). Absorbance readings at 450 nm were subtracted from 540 nm to correct for background noise associated with the plate. A standard curve was created using a four-parameter logistic curve-fit approach. IGF-1 samples were analyzed using the DG100 ELISA kit from
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Walsh et al. APNM 2015-0410 Accepted Quantikine (R&D Systems, Minneapolis, MN, USA). The kit detection range was .094 ng/mL – 6 ng/mL, and the intra- and interassay coefficients of variation were 4% and 7.9%, respectively. The assay was performed in accordance with the manufacturer’s instructions. Briefly, in order to release IGF-1 from binding proteins, serum samples were processed in accordance with the pretreatment protocol. Plates were read at 450 and 540 nm with a micro plate reader and absorbance readings at 450 nm were subtracted from 540 nm to correct for background noise associated with the plate. Samples were multiplied by a dilution factor of 100 and a standard curve was created using a linear regression curve-fit approach. Samples were analyzed within 5 months of collection. Statistical Analysis and Sample Size Determination A two-way repeated measured ANOVA was used to determine differences in NTF levels across the 8 blood sampling time points in the Pre-training and Post-training testing sessions. A Bonferroni correction for multiple comparisons was performed where appropriate. To assess the effect of 8 weeks of training on measures of strength, twotailed paired t-tests were used to compare strength from pre- to post-training. All analysis was performed using SigmaPlot Statistical Analysis and Scientific Graphing Software version 12.0. All data is expressed as mean ± standard deviation (SD) with statistical significance set at p ≤ 0.05. Sample size was calculated using a two-tailed paired t-test with α set at 0.05 and desired power of 0.90. Using the baseline serum BDNF values reported by Yarrow et al. 2010 (23,304 ± 1835 pg/mL) with an desired minimum increase of 10%, we determined the required sample size for this study was n = 9 with an effect size (d) = 1.27 (Faul et al. 2009). RESULTS
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Walsh et al. APNM 2015-0410 Accepted The Effect of 8-Weeks of Resistance Training on Strength Post-training strength measures significantly improved in all exercises compared to Pretraining measures (p < 0.001; Table 2). Split squat results are not reported as a majority of participants began with bodyweight only and in some cases, used supports for balance. As such, Pre-training strength measures would not accurately reflect an individual’s1RM for that exercise. There were no reported injuries or adverse events throughout the study.
The Effect of 8-Weeks of Resistance Training on Basal BDNF and IGF-1 Basal blood samples were drawn at 8:30 a.m. while participants rested following their standardized breakfast. There was no difference in serum BDNF between Pre and Posttraining (22652.6 ±4757.3 pg/mL vs. 22961.1 ±5202.4 pg/mL respectively, F(1,9) = 0.866 p = 0.376). Basal levels of serum IGF-1 levels also remained unchanged between Pre and Post-training (89.98 ±32.52 ng/mL vs. 81.64 ±34.41 ng/mL respectively, p = 0.16). BDNF and IGF-1 Responses to Acute Resistance Exercise Serial blood samples were drawn at Rest prior to exercise and 0, 10, 20, 30, 40, 60, and 120 minutes of recovery following a bout of resistance exercise. Serum levels of IGF-1 remained unchanged across the 7 measured time points following resistance exercise in both the Pre and Post-training sessions (Figure 2A). Serum levels of BDNF significantly increased immediately after exercise (0 min) in both Pre- (+ 2411.5 pg/mL) and Posttraining (+2849.0 pg/mL; F(7, 63) = 8.12, p =
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Walsh et al. APNM 2015-0410 Accepted Neurotrophic Growth Factor Responses to Lower Body Resistance Training in Older Adults
Jeremy J. Walsh1, Trisha D. Scribbans1, Robert F. Bentley1, J. Mikhail Kellawan2, Brendon Gurd1, and Michael E. Tschakovsky1.
1
School of Kinesiology and Health Studies, Queen’s University, Kingston, ON, Canada 2
Department of Kinesiology, University of Wisconsin, Madison, WI, USA
Corresponding Author: Michael Tschakovsky Mailing Address: 28 Division St., Kingston, ON, Canada, K7L 3N6 Telephone: 613-533-6000 ext. 74697 Fax: 613-533-2009 Email: [email protected]
1
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Walsh et al. APNM 2015-0410 Accepted ABSTRACT Resistance exercise is an efficacious stimulus for improving cognitive function in older adults, which may be mediated by the upregulation of blood-borne neurotrophic growth factors (NTFs) like brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1). However, the NTF response to resistance exercise and training in older adults is poorly understood. Therefore, the purpose of this study was to characterize the timing and magnitude of the NTF response following an acute bout of resistance exercise before and after 8 weeks of resistance training. Methods: 10 cognitively normal, older adults (ages 60 – 77, 5 men) were examined. The acute NTF response to resistance exercise was assessed via serum samples drawn at designated time points following exercise. This procedure was then repeated following 8 weeks of resistance training. Results: BDNF increased immediately post-exercise (∆9% pre-training, ∆11% posttraining) then returned to resting levels while IGF-1 remained stable following resistance exercise before and after 8 weeks of resistance training. Basal levels of both NTFs were unaffected by the 8-week training period. Conclusion: We report a transient increase in serum BDNF following a bout of resistance exercise in older adults, which could have implications for the design of interventions seeking to maximize cognitive function in older adults. Key Words: BDNF, IGF-1, neuroplasticity, cognitive function, aging
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Walsh et al. APNM 2015-0410 Accepted INTRODUCTION Aging is associated with marked declines in muscle mass (Liu-Ambrose and Donaldson, 2009), bone density (Parkhouse et al. 2000), brain tissue (Fjell et al. 2009), and a concomitant decrease in cognitive function (Liu-Ambrose and Donaldson 2009). Interventions that protect and improve muscle strength and cognitive function in older adults have a critical role to play in healthy aging. In this regard, the American College of Sports Medicine advocates for the participation of older adults in regular resistance training as a means of combatting frailty, improving body composition, and more recently improving brain function (Nelson and Rejeski 2007). Both short- and longduration resistance training programs have been shown to improve response inhibition, selective attention, processing speed, and other executive functions in older adults (Chang et al. 2012), with some benefits persisting for up to 1 year post-training (PerrigChiello et al. 1998). More recently, long-term resistance training has been shown to stimulate brain plasticity, with alterations in cortical activation being functionally coupled with enhanced flanker task performance (Liu-Ambrose et al. 2012).
Cognitive training represents another strategy for combatting age-related cognitive decline and enhancing select cognitive functions among older adults. While a lack of transferable improvements has been a critique of cognitive training paradigms (Ball et al. 2002), these do lead to significant improvements in cognitive domains that are directly trained by the program, such as processing speed, attention, and memory (Gates and Valenzuela 2010). In addition, emerging evidence suggests that combining exercise and
3
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Walsh et al. APNM 2015-0410 Accepted cognitive training (multi-modal training) may exact greater benefit to cognitive function than either performed alone (Fabre et al. 2002; Oswald et al. 2006).
Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) are key orchestrators of the positive neurobiological adaptations garnered through exercise and cognitive training (Cotman et al. 2007; Valenzuela et al.2007). BDNF is essential for learning and memory (Cotman et al. 2007) and plays a key role in regulating the growth, maintenance, and survival of neurons (Marosi and Mattson 2014). Cognitive activity evokes localized BDNF production in the brain, which plays a key role in neuroplasticity (Valenzuela et al. 2007). Furthermore, neuroplasticity also appears sensitive to bloodborne BDNF levels as evidenced by increased neuroplasticity responses with central infusion of BDNF in rats (Hoshaw et al. 2005) and mice (D’Amore et al. 2013). Bloodborne IGF-1 appears to be essential for exercise-induced neuroplasticity, as blocking entry of circulating IGF-1 into the brain completely abrogates the neurogenic and neuroprotective effects of exercise (Carro et al. 2000, 2001; Trejo et al. 2007).
Animal models demonstrate divergent pathways of exercise-induced neural plasticity for resistance vs. aerobic training, wherein resistance training may evoke its effects through the actions IGF-1 and aerobic through BDNF (Cassilhas et al. 2012). Indeed, Cassilhas et al. (2012) observed a 43% increase in systemic and 95% increase in hippocampal IGF1 following a period of resistance training in rats. Further, these changes in IGF-1 were coupled with improved hippocampal dependent memory, suggesting a direct impact of IGF-1 on improved cognitive function in animals. This IGF-1 mediated improvement 4
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Walsh et al. APNM 2015-0410 Accepted may be also be indirect, as the expression of BDNF in the hippocampus is dependent on the presence of IGF-1 (Ding et al. 2006).
Based on this, our current working hypothesis is that combining exercise and cognitive training to maximize NTF delivery to the microcirculation of areas of the brain involved in cognitive processing may improve the benefits of these interventions. So far, no “multi-modal” training studies based their timing of exercise and cognitive training bouts on enhancing NTF dose (Fabre et al. 2002; Gates et al. 2011; Oswald et al. 2006).
Maximizing delivery to cerebral microcirculation is a function of local perfusion and blood [NTF’s]. We know that neural activity evokes rapid and substantial increases in local cerebral perfusion (neurovascular coupling response) (Attwell et al. 2010). What remains poorly understood is the timing of resistance exercise-evoked increases in blood borne NTF’s in older adults; the very group that would benefit considerably from interventions that improve strength and cognition. Specifically, the blood-borne BDNF response to an acute bout of resistance exercise or how this might be altered by resistance training in this population has yet to be determined. While the blood-borne IGF-1 response to resistance exercise in older adults has been investigated somewhat, findings are conflicting with some reports of no change (Frystyk 2010; Kraemer and Ratamess 2005) and others of immediate and sustained (6 hours) increases in IGF-1 in older adults following a single bout of resistance exercise (Bermon et al. 1999).
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Walsh et al. APNM 2015-0410 Accepted Therefore, the objective of this study was to identify timing and magnitude of changes in blood-borne BDNF and IGF-1 in response to a bout of resistance exercise, and whether 8 weeks of resistance training affected this response. Based on previous work and evidence from animal models, we hypothesize that an acute bout of resistance exercise will result in a significant increase in IGF-1 levels, whereas BDNF will remain unchanged. Further, we hypothesize that basal IGF-1 will increase following 8 week of resistance training, while basal BDNF levels will remain unaffected. METHODS Participants Ten cognitively normal older adults (5 males, mean age 66 ±5.3 years; Table 1) from the Kingston, Ontario community were recruited for this study. The study procedures were approved by the Health Sciences Human Research Ethics Board at Queen’s University. All participants read and signed a consent form, which explicitly detailed all of the experimental procedures and the associated risks. Inclusion criteria were older (≥ 60 years old), cognitively normal adults as assessed by the Montreal Cognitive Assessment (MoCA), free of major adverse neurological events or mental illness including depression, free from known cardiovascular disease, no history of substance abuse, not taking any psychiatric medication, were non-smokers/non-nicotine users, no regular resistance training within the last 6 months, and free from contraindications to resistance training. All female participants were post-menopausal and were not on hormonereplacement therapy. The current study was part of a larger, multi-modal intervention where participants performed resistance training followed immediately by 40 minutes of
6
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Walsh et al. APNM 2015-0410 Accepted cognitive training, performed 3x/week for 8 weeks. However, participants did not perform cognitive training during on the NTF testing days. Strength Training Program Participants performed supervised resistance training 3 times per week (Monday / Wednesday / Friday) for an 8-week period. The program followed the principle of progressive overload with active recovery sessions implemented to facilitate recovery. Each training session began with a 7 minute warm-up on a leg ergometer followed by a 5 minute routine of dynamic movements, including walking knee-raises, walking heelkicks, and lateral shuffling. The training program consisted of 3 lower-body exercises: 1) Split Squat (performed with body weight or dumbbells), 2) Double Leg Press, and 3) Single Leg Press (Atlantis C-204 Dual Pivot Press, Quebec, Canada). Participants completed 4 consecutive sets of each exercise with 90-second rest periods between sets. Following completion of these exercises, participants cooled down on a leg-ergometer for 7 minutes. Each set consisted of 8 -10 repetitions at 60% 1-repetition maximum (1RM) during the first week, 70% during the second and fourth weeks, and 80% 1RM for the remainder of the study. The active recovery weeks consisted of the same exercises performed for 12 – 15 reps at 50% 1RM and were performed during the third and sixth weeks of training. Exercise supervisors tracked the performance of participants on a daily basis and weights were individually adjusted on a weekly basis to match the desired intensity. Orientation Prior to strength testing participants were oriented with the resistance training program, where proper weight lifting technique, breathing, posture, and lifting cadence were
7
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Walsh et al. APNM 2015-0410 Accepted taught. On this day participants performed the regular resistance training routine. The selected weight was relatively low and could be completed for >15 repetitions. Weight was modestly increased between sets; however, all exercises were of submaximal intensity. Participants were unaccustomed to resistance training, therefore, an additional purpose of this visit was to stimulate delayed onset of muscle soreness (DOMS) and allow adequate recovery to ensure that strength was fully restored for the strength assessment session. Strength Assessment Five days after the training orientation visit, participants performed submaximal strength testing using a predicted 1-repitition maximum (1RM) protocol as described by Reynolds et al. (2006). Strength was re-assessed on the second last day of training. Participants performed the previously described resistance training routine with a lifting cadence of 2:2 (concentric:eccentric phases) and rested for 3 minutes between sets to allow for proper recovery and avoid fatigue effects. NTF Testing Blood collection was performed prior to the initiation of regular training (Pre-Training) and on the final training session (Post-Training). Participants arrived at the lab at 7 a.m. following an overnight fast and were fed a standardized breakfast (toasted bagel [~190 kcal; 1 g fat, 36 g CHO, 7 g protein] with 15 g of cream cheese [~45 kcal; 4 g fat, 1 g CHO, 1 g protein] and a 200 mL juice pack [~100 kcal, 0 g fat, 26 g CHO, 0 g protein]). Following breakfast, a vein of the forearm was catheterized using a 20-gauge Teflon catheter (BD Insyte Autoguard, Oakville, Canada) and the resting (baseline) blood
8
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Walsh et al. APNM 2015-0410 Accepted sample was drawn following 30 minutes of rest. Following the rest period and the regular warm-up routine, participants performed the 4 sets of each resistance exercise at 80% of their 1RM for 8-10 reps, with 90 seconds of rest between sets. Weight was adjusted to ensure 80% relative intensity during the Post-training NTF testing session to account for strengths gains due to training. Serial blood samples were drawn at Rest (prior to exercise) and at 0, 10, 20, 30, 40, 60, and 120 minutes of recovery following a bout of resistance exercise. A schematic of the NTF Testing protocol and blood-draw time points can be viewed in Figure 1. Consumption of water was allowed ad libitum throughout the training and bed rest periods. Blood Analysis Serum BDNF and IGF-1 samples were collected in serum separator tubes and left to clot for 30 minutes at room temperature. Samples were centrifuged for 15 minutes at 1000 x g at 4oC, aliquoted, and immediately stored at -80oC. Serum BDNF was analyzed using the DBD000 enzyme-linked immunoassay (ELISA) kit from Quanitkine (R&D Systems, Minneapolis, MN, USA). The kit detection range was 62.5 pg/mL - 4,000 pg/mL, and the intra- and interassay coefficients of variation were 5% and 9%, respectively. While the assay was performed in accordance with the manufacturer’s instructions, we optimized the kit specifically for our samples. Briefly, serum samples were diluted 20fold with the supplied calibrator diluent. Plates were read at 450 and 540 nm with a micro plate reader (Synergy 2, BioTek Instruments, Winooski, VT, USA). Absorbance readings at 450 nm were subtracted from 540 nm to correct for background noise associated with the plate. A standard curve was created using a four-parameter logistic curve-fit approach. IGF-1 samples were analyzed using the DG100 ELISA kit from
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Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 11/23/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.
Walsh et al. APNM 2015-0410 Accepted Quantikine (R&D Systems, Minneapolis, MN, USA). The kit detection range was .094 ng/mL – 6 ng/mL, and the intra- and interassay coefficients of variation were 4% and 7.9%, respectively. The assay was performed in accordance with the manufacturer’s instructions. Briefly, in order to release IGF-1 from binding proteins, serum samples were processed in accordance with the pretreatment protocol. Plates were read at 450 and 540 nm with a micro plate reader and absorbance readings at 450 nm were subtracted from 540 nm to correct for background noise associated with the plate. Samples were multiplied by a dilution factor of 100 and a standard curve was created using a linear regression curve-fit approach. Samples were analyzed within 5 months of collection. Statistical Analysis and Sample Size Determination A two-way repeated measured ANOVA was used to determine differences in NTF levels across the 8 blood sampling time points in the Pre-training and Post-training testing sessions. A Bonferroni correction for multiple comparisons was performed where appropriate. To assess the effect of 8 weeks of training on measures of strength, twotailed paired t-tests were used to compare strength from pre- to post-training. All analysis was performed using SigmaPlot Statistical Analysis and Scientific Graphing Software version 12.0. All data is expressed as mean ± standard deviation (SD) with statistical significance set at p ≤ 0.05. Sample size was calculated using a two-tailed paired t-test with α set at 0.05 and desired power of 0.90. Using the baseline serum BDNF values reported by Yarrow et al. 2010 (23,304 ± 1835 pg/mL) with an desired minimum increase of 10%, we determined the required sample size for this study was n = 9 with an effect size (d) = 1.27 (Faul et al. 2009). RESULTS
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Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 11/23/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.
Walsh et al. APNM 2015-0410 Accepted The Effect of 8-Weeks of Resistance Training on Strength Post-training strength measures significantly improved in all exercises compared to Pretraining measures (p < 0.001; Table 2). Split squat results are not reported as a majority of participants began with bodyweight only and in some cases, used supports for balance. As such, Pre-training strength measures would not accurately reflect an individual’s1RM for that exercise. There were no reported injuries or adverse events throughout the study.
The Effect of 8-Weeks of Resistance Training on Basal BDNF and IGF-1 Basal blood samples were drawn at 8:30 a.m. while participants rested following their standardized breakfast. There was no difference in serum BDNF between Pre and Posttraining (22652.6 ±4757.3 pg/mL vs. 22961.1 ±5202.4 pg/mL respectively, F(1,9) = 0.866 p = 0.376). Basal levels of serum IGF-1 levels also remained unchanged between Pre and Post-training (89.98 ±32.52 ng/mL vs. 81.64 ±34.41 ng/mL respectively, p = 0.16). BDNF and IGF-1 Responses to Acute Resistance Exercise Serial blood samples were drawn at Rest prior to exercise and 0, 10, 20, 30, 40, 60, and 120 minutes of recovery following a bout of resistance exercise. Serum levels of IGF-1 remained unchanged across the 7 measured time points following resistance exercise in both the Pre and Post-training sessions (Figure 2A). Serum levels of BDNF significantly increased immediately after exercise (0 min) in both Pre- (+ 2411.5 pg/mL) and Posttraining (+2849.0 pg/mL; F(7, 63) = 8.12, p =
