Acute resistance exercise induces antinociception by activation of the endocannabinoid system in rats
Giovane Galdino, Thiago Romero, José Felippe Pinho da Silva, Daniele Aguiar, Ana Maria de Paula, Jader Cruz, Cosimo Parrella, Fabiana Piscitelli, Igor Duarte, Vincenzo Di Marzo, Andrea Perez, Giovane Galdino, Thiago Romero, José Felippe Pinho da Silva, Daniele Aguiar, Ana Maria de Paula, Jader Cruz, Cosimo Parrella, Fabiana Piscitelli, Igor Duarte, Vincenzo Di Marzo, Andrea Perez
Abstract
Background: Resistance exercise (RE) is also known as strength training, and it is performed to increase the strength and mass of muscles, bone strength, and metabolism. RE has been increasingly prescribed for pain relief. However, the endogenous mechanisms underlying this antinociceptive effect are still largely unexplored. Thus, we investigated the involvement of the endocannabinoid system in RE-induced antinociception.
Methods: Male Wistar rats were submitted to acute RE in a weight-lifting model. The nociceptive threshold was measured by a mechanical nociceptive test (paw pressure) before and after exercise. To investigate the involvement of cannabinoid receptors and endocannabinoids in RE-induced antinociception, cannabinoid receptor inverse agonists, endocannabinoid metabolizing enzyme inhibitors, and an anandamide reuptake inhibitor were injected before RE. After RE, CB1 cannabinoid receptors were quantified in rat brain tissue by Western blot and immunofluorescence. In addition, endocannabinoid plasma levels were measured by isotope dilution-liquid chromatography mass spectrometry.
Results: RE-induced antinociception was prevented by preinjection with CB1 and CB2 cannabinoid receptor inverse agonists. By contrast, preadministration of metabolizing enzyme inhibitors and the anandamide reuptake inhibitor prolonged and enhanced this effect. RE also produced an increase in the expression and activation of CB1 cannabinoid receptors in rat brain tissue and in the dorsolateral and ventrolateral periaqueductal regions and an increase in endocannabinoid plasma levels.
Conclusions: The present study suggests that a single session of RE activates the endocannabinoid system to induce antinociception.
Figures
The systemic involvement of cannabinoid receptors and endocannabinoids in the antinociception induced by acute resistance exercise (RE). (A) Rats were pretreated with subcutaneous injections of AM251 (4 mg/kg) or AM630 (4 mg/kg) 10 min before to onset of exercise. (B) Rats were pretreated with subcutaneous injections of VDM11 (2.5 mg/kg), methylester phosphonofluoridic acid (MAFP) (2 mg/kg) or JZL184 (4 mg/kg) 10 min before onset of exercise. Mechanical nociceptive thresholds were measured before and after 1, 15 and 30 min of RE. Data are expressed as the mean + S.E.M. of 6 animals per group. ***P < .01, indicates statistical significance compared to the control group (Co), immediately after RE (post hoc pairwise comparisons with Bonferroni correction). ###P < .01, indicates statistical significance of RE+AM251 and RE+AM630 groups compared to RE group, immediately after exercise. †††P < .01, indicates statistical significance of RE+VDM11, RE+MAFP and RE+JZL184 groups when compared to RE group, immediately, after 15 and 30 min of exercise. One-way ANOVA followed Student's t-test with equal variances. BL: pre-exercise baseline latency; Vec: vehicle.
The systemic involvement of cannabinoid receptors and endocannabinoids in the antinociception induced by acute resistance exercise (RE). (A) Rats were pretreated with subcutaneous injections of AM251 (4 mg/kg) or AM630 (4 mg/kg) 10 min before to onset of exercise. (B) Rats were pretreated with subcutaneous injections of VDM11 (2.5 mg/kg), methylester phosphonofluoridic acid (MAFP) (2 mg/kg) or JZL184 (4 mg/kg) 10 min before onset of exercise. Mechanical nociceptive thresholds were measured before and after 1, 15 and 30 min of RE. Data are expressed as the mean + S.E.M. of 6 animals per group. ***P < .01, indicates statistical significance compared to the control group (Co), immediately after RE (post hoc pairwise comparisons with Bonferroni correction). ###P < .01, indicates statistical significance of RE+AM251 and RE+AM630 groups compared to RE group, immediately after exercise. †††P < .01, indicates statistical significance of RE+VDM11, RE+MAFP and RE+JZL184 groups when compared to RE group, immediately, after 15 and 30 min of exercise. One-way ANOVA followed Student's t-test with equal variances. BL: pre-exercise baseline latency; Vec: vehicle.
The spinal involvement of cannabinoid receptors and endocannabinoids in the antinociception induced by acute resistance exercise (RE). (A) Rats were pretreated with intrathecal injections of AM251 (30 μg/10 μL) and AM630 (10 μg/10 μL) immediately before the onset of exercise. (B) Rats were pretreated with intrathecal injections of VDM11 (10 μg/10 μL), methylester phosphonofluoridic acid (MAFP) (10 μg/10 μL) or JZL184 (7 μg/10 μL) immediately before the onset of exercise. Mechanical nociceptive thresholds were measured before and after 1, 15 and 30 min of RE. Data are expressed as the mean + S.E.M. of 6 animals per group. ***P < .01, indicates statistical significance compared to the control group (Co), immediately after RE. ###P < .01, indicates statistical significance of RE+AM251 and RE+AM630 groups compared to RE group, immediately after exercise.†††P < .01, indicates statistical significance of RE+VDM11, RE+MAFP and RE+JZL184 groups compared to the RE group, immediately, 15 and 30 min after exercise. One-way ANOVA followed Student's t-test with equal variances. BL: pre-exercise baseline latency; Vec: vehicle.
The spinal involvement of cannabinoid receptors and endocannabinoids in the antinociception induced by acute resistance exercise (RE). (A) Rats were pretreated with intrathecal injections of AM251 (30 μg/10 μL) and AM630 (10 μg/10 μL) immediately before the onset of exercise. (B) Rats were pretreated with intrathecal injections of VDM11 (10 μg/10 μL), methylester phosphonofluoridic acid (MAFP) (10 μg/10 μL) or JZL184 (7 μg/10 μL) immediately before the onset of exercise. Mechanical nociceptive thresholds were measured before and after 1, 15 and 30 min of RE. Data are expressed as the mean + S.E.M. of 6 animals per group. ***P < .01, indicates statistical significance compared to the control group (Co), immediately after RE. ###P < .01, indicates statistical significance of RE+AM251 and RE+AM630 groups compared to RE group, immediately after exercise.†††P < .01, indicates statistical significance of RE+VDM11, RE+MAFP and RE+JZL184 groups compared to the RE group, immediately, 15 and 30 min after exercise. One-way ANOVA followed Student's t-test with equal variances. BL: pre-exercise baseline latency; Vec: vehicle.
Supraspinal involvement of cannabinoid receptors and endocannabinoids in the antinociception induced by acute resistance exercise (RE). (A) Rats were pretreated with intracerebroventricular injections of AM251 (6 μg/5 μL) or AM630 (10 μg/5 μL) immediately before the onset of exercise. (B) Rats were pretreated with intracerebroventricular injections of VDM11 (10 μg/5 μL), methylester phosphonofluoridic acid (MAFP) (0.4 μg/5 μL) or JZL184 (7 μg/5 μL) immediately before the onset of exercise. Mechanical nociceptive thresholds were measured before and after 1, 15 and 30 min of RE. Data are expressed as the mean + S.E.M. of 6 animals per group. *** P < .01, indicates statistical significance compared to the control group (Co), immediately after RE. ### P < .01, indicates statistical significance of RE+AM251 and RE+AM630 groups compared to RE group, immediately after exercise † Indicates statistical significance of RE+VDM11, RE+MAFP and RE+JZL184 groups compared to RE group P < .01, immediately after, 15 and 30 min of exercise. One-way ANOVA followed Student's t-test with equal variances. BL: pre-exercise baseline latency; Vec: vehicle.
Supraspinal involvement of cannabinoid receptors and endocannabinoids in the antinociception induced by acute resistance exercise (RE). (A) Rats were pretreated with intracerebroventricular injections of AM251 (6 μg/5 μL) or AM630 (10 μg/5 μL) immediately before the onset of exercise. (B) Rats were pretreated with intracerebroventricular injections of VDM11 (10 μg/5 μL), methylester phosphonofluoridic acid (MAFP) (0.4 μg/5 μL) or JZL184 (7 μg/5 μL) immediately before the onset of exercise. Mechanical nociceptive thresholds were measured before and after 1, 15 and 30 min of RE. Data are expressed as the mean + S.E.M. of 6 animals per group. *** P < .01, indicates statistical significance compared to the control group (Co), immediately after RE. ### P < .01, indicates statistical significance of RE+AM251 and RE+AM630 groups compared to RE group, immediately after exercise † Indicates statistical significance of RE+VDM11, RE+MAFP and RE+JZL184 groups compared to RE group P < .01, immediately after, 15 and 30 min of exercise. One-way ANOVA followed Student's t-test with equal variances. BL: pre-exercise baseline latency; Vec: vehicle.
Resistance exercise (RE) increases CB1 cannabinoid receptor expression in the rat brain. Immediately after exercise or control conditions, brains samples were stained with antibodies against CB1, transblotted and analyzed by ImageJ (A). Representative immunoblots for CB1 cannabinoid receptor expression with corresponding α-Tubulin controls are shown (B). Data expressed as the mean + SE.M. of 5 animals per group.*Indicates statistical significance compared to control group (Co, rats nonexercised). P < .01, t test.
Resistance exercise produces an equivalent increase of AEA, anandamide (A); 2-AG, 2-arachidonoylglycerol (B); PEA, palmitoylethanolamide (C) and OEA, oleoylethanolamide (D) levels in rat plasma. Data are expressed as the mean + SE.M. of 5 animals per group. *Indicates statistical significance compared to control group (Co, rats non-exercised). P < .01, t test.
Resistance exercise produces an equivalent increase of AEA, anandamide (A); 2-AG, 2-arachidonoylglycerol (B); PEA, palmitoylethanolamide (C) and OEA, oleoylethanolamide (D) levels in rat plasma. Data are expressed as the mean + SE.M. of 5 animals per group. *Indicates statistical significance compared to control group (Co, rats non-exercised). P < .01, t test.
Resistance exercise produces an equivalent increase of AEA, anandamide (A); 2-AG, 2-arachidonoylglycerol (B); PEA, palmitoylethanolamide (C) and OEA, oleoylethanolamide (D) levels in rat plasma. Data are expressed as the mean + SE.M. of 5 animals per group. *Indicates statistical significance compared to control group (Co, rats non-exercised). P < .01, t test.
Resistance exercise produces an equivalent increase of AEA, anandamide (A); 2-AG, 2-arachidonoylglycerol (B); PEA, palmitoylethanolamide (C) and OEA, oleoylethanolamide (D) levels in rat plasma. Data are expressed as the mean + SE.M. of 5 animals per group. *Indicates statistical significance compared to control group (Co, rats non-exercised). P < .01, t test.
CB1 cannabinoid receptor and c-FOS activation and co-expression in neurons of the dorsolateral periaqueductal gray region (dlPAG) after resistance exercise (RE). (A) Representative double immunofluorescence staining CB1 cannabinoid receptor (green) and c-FOS (red) in dlPAG. Scale bars, 50 μm. (B) Number of neurons in dlPAG after RE. Data are expressed as the mean + S.E.M. of 5 animals per group. *Indicates statistical significance of CB1 cannabinoid receptor activation and co-expression after RE (CB1/RE) compared to the control group (CB1/Co). #Indicates statistical significance of c-FOS activation and co-expression after RE (c-FOS/RE) compared to control group (c-FOS/Co). †Indicates statistical significance of double immunofluorescence for CB1 cannabinoid receptor and c-FOS activation and co-expression after RE (CB1+c-FOS/RE) compared to control group (CB1+c-FOS/Co). P < .01, t test. (C) Schematic representation of coronal section dlPAG (arrowheads on dark circle) of the rat brain (based on Paxinos and Watson atlas).
CB1 cannabinoid receptors and c-FOS activation and co-expression in neurons of the ventrolateral periaqueductal gray region (vlPAG) after resistance exercise (RE). (A) Representative double immunofluorescence staining CB1 cannabinoid receptor (green) and c-FOS (red) in vlPAG. Scale bars, 50 μm. (B) Number of neurons in vlPAG after RE. Data are expressed as the mean + S.E.M. of 5 animals per group. *Indicates statistical significance of CB1 cannabinoid receptor activation and co-expression after RE (CB1/RE) compared to the control group (CB1/Co). #Indicates statistical significance of c-FOS activation and co-expression after RE (c-FOS/RE) compared to control group (c-FOS/Co). †Indicates statistical significance of double immunofluorescence for CB1 cannabinoid receptor and c-FOS activation and co-expression after RE (CB1+c-FOS/RE) compared to control group (CB1+c-FOS/Co). P < .01, t test. (C) Schematic representation of coronal section vlPAG (arrowheads on dark circle) of the rat brain (based on Paxinos and Watson atlas).
Effects of the CB1 receptor inverse agonist AM251 (100 pmol) in the activation and co-expression of CB1 cannabinoid receptors (CB1/RE+AM 251) and c-FOS (c-FOS /RE+AM 251) in neurons of the dorsolateral periaqueductal gray region (dlPAG) after resistance exercise (RE). (A) Representative double immunofluorescence staining CB1 cannabinoid receptor (green) and c-FOS (red) in dlPAG. Scale bars, 50 μm. (B) Number of neurons in dlPAG after RE. Data are expressed as the mean + S.E.M. of 5 animals per group. *Indicates statistical significance of CB1 cannabinoid receptor activation and co-expression after RE followed by pretreatment with AM 251 (CB1/RE+AM251) compared to the control group (CB1/Co). P < .01, t test. (C) Schematic representation of coronal section dlPAG (arrowheads on dark circle) of the rat brain (based on Paxinos and Watson atlas).
Effects of the CB1 receptor inverse agonist AM251 (100 pmol) in the activation and co-expression of CB1 cannabinoid receptors (CB1/RE+AM 251) and c-FOS (c-FOS /RE+AM 251) in neurons of the ventrolateral periaqueductal gray region (vlPAG) after resistance exercise (RE). (A) Representative double immunofluorescence staining CB1 cannabinoid receptor (green) and c-FOS (red) in vlPAG. Scale bars, 50 μm. (B) Number of neurons in vlPAG after RE. Data are expressed as the mean + S.E.M. of 5 animals per group. *Indicates statistical significance of CB1 cannabinoid receptor activation and co-expression after RE followed by pretreatment with AM251 (CB1/RE+AM 251) compared to the control group (CB1/Co). P < .01, t test. (C) Schematic representation of coronal section vlPAG (arrowheads on dark circle) of the rat brain (based on Paxinos and Watson atlas).
Source: PubMed