Synergistic benefits of melatonin and therapeutic exercise as a unified strategy for managing stroke and doxorubicin-induced cardiotoxicity

Figures

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  Figure 1. (A) Experimental design and animal groups in present study. This in vivo study followed the scheme shown here, which is summarized in “materials and methods.” Transient MCAo: Transient middle cerebral artery occlusion. Veh and control were considered same in TTC staining to reduce the number of animals used in this study. (B) Schematic diagram of experimental design. Dox-induced cardiotoxic animals were randomly divided into four groups, vehicle control, melatonin, exercise, and exercise + melatonin. Heart weight, heart weight/tibia length ratio, and histological analysis were performed after 5 weeks of treatment.

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  Figure 2. Sequential tracing of body weight and neurological deficits after cerebral ischemic injury. (A) Alterations of body weight were measured in experimental animals for 4 weeks after MCAo. **p < 0.01 compared with Veh. Data shown are means ± SD. (B) Effect of melatonin and exercise on recovery of neurological behaviors. The degree of neurological deficiency was assessed by the mNSS criteria. (C) 2% TTC-defined ischemic infarct volume after MCAo. (A) TTC-stained images of experimental animal. The brain of the control model was intact; the white color on the right shows the infarction in the MCAo brains. (D) Quantification of infarct volume using Image J software. **p < 0.01 and *p < 0.05 compared with Veh; ##p < 0.01 compared with MT + Ex; data are presented as mean ± SD.

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  Figure 3. Downregulation of neuronal injury by melatonin plus exercise showing neuroprotective effects. (A) The expressions of 46- and 48-kDa of NeuN isoforms in the infarct region (B) GFAP (C) nNOS and (D) mBDNF expression was analyzed in the infarct region. *p < 0.05 compared with Con; #p < 0.05 compared with Veh; ap < 0.05 compared with MT; bp < 0.05 compared with Ex; data are presented as mean ± SD. The full images of western blots are given in Supplementary material 1.

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  Figure 4. Melatonin and exercise promote neuronal migration and differentiation after ischemic insult. DCX expression was determined in infarct region of MCAo rats. *p < 0.05 compared with Con; #p < 0.05 compared with Veh; ap < 0.05 compared with MT; bp < 0.05 compared with Ex; data are presented as mean ± SD. The full images of western blots are given in Supplementary material 1.

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  Figure 5. Golgi-cox-stained sections of the MCAo brain. (A) Region of analyzed neurons in the ipsilateral cortex. (B) Quantification of neurite outgrowth. Magnification = 400×. Scale bars = 500 μm. **p < 0.01 compared with Veh, ##p < 0.01 compared with MT + Ex, data are presented as mean ± SD.

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  Figure 6. Pre-treatment effect of MT on the DIC. (A) Experimental scheme. (B) Morphology. (C) Cell viability, ap < 0.05 vs. Veh; bp < 0.05 vs. Resv. (D) Changes of beating frequency, ap < 0.05 vs. Con; bp < 0.05 vs. Veh; cp < 0.05 vs. Resv. Post-treatment effect of MT on the DIC. (E). Experimental scheme. (F). Morphology. (G). Cell viability, ap < 0.05 vs. Veh; bp < 0.05 vs. Resv. (H). Changes of beating frequency, ap < 0.05 vs. Con; bp < 0.05 vs. Veh; cp < 0.05 vs. Resv. Statistical significance was assessed by Kruskal–Wallis ANOVA and the Mann–Whitney U-test. Data shown are means ± S.E.M. Dox, doxorubicin; DIC, dox-induced cardiotoxicity; Ex, exercise; H.w., heart weight; MT, melatonin; T.l., tibia length; Veh, vehicle.

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  Figure 7. (A) Changes in body weight following the DIC and various intervention such as Veh, MT, Ex, and MT + Ex. Statistical significance was assessed by Kruskal–Wallis ANOVA and the Mann–Whitney U-test. No significant difference was found between the groups. (B,C) Changes in heart weight H.w. and H.w./T.l. following the DIC and various intervention such as Veh, MT, Ex, and MT + Ex. Statistical significance was assessed by Kruskal–Wallis ANOVA and the Mann–Whitney U-test. No significant difference was found between the groups. Data shown are means ± S.E.M. Dox, doxorubicin; DIC, dox-induced cardiotoxicity; Ex, exercise; H.w., heart weight; MT, melatonin; T.l., tibia length; Veh, vehicle.

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  Figure 8. Histopathological changes induced by Dox and protective effect of MT, Ex, and MT + Ex in rat heart (H&E staining, magnification = 40×). (A) Cardiac section from Veh treated rats showing degenerative changes. (B) Cardiac sections from Dox-treated rats followed by Ex training. (C) Represents cardiac section from Dox-treated rats followed by MT administration. (D) Represents cardiac section from Dox-treated rats followed by MT + Ex treatment. Dox, doxorubicin; Ex, exercise; MT, melatonin; Veh, vehicle.