Synergistically Induced Hypothermia and Enhanced Neuroprotection by Pharmacological and Physical Approaches in Stroke
Zhang Jun1, Liu Kaiyin2, Elmadhoun Omar2, Ji Xunming1,*, Duan Yunxia1, Shi Jingfei1, He Xiaoduo1, Liu Xiangrong1, Wu Di1, Che Ruiwen1, Geng Xiaokun2,3, Ding Yuchuan1,2,3
1China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing, China 2Department of Neurosurgery, Wayne State University School of Medicine, Detroit, MI 48201, USA 3China-America Institute of Neuroscience, Luhe Hospital, Capital Medical University, Beijing, China
Hypothermia is considered as a promising neuroprotective treatment for ischemic stroke but with many limitations. To expand its clinical relevance, this study evaluated the combination of physical (ice pad) and pharmacological [transient receptor potential vanilloid channel 1 (TRPV1) receptor agonist, dihydrocapsaicin (DHC)] approaches for faster cooling and stronger neuroprotection. A total of 144 male Sprague Dawley rats were randomized to 7 groups: sham (n=16), stroke only (n=24), stroke with physical hypothermia at 31ºC for 3 h after the onset of reperfusion (n=24), high-dose DHC (H-DHC)(1.5 mg/kg, n=24), low-dose DHC (L-DHC)(0.5 mg/kg, n=32) with (n=8) or without (n=24) external body temperature control at ~38 ºC (L-DHC, 38 ºC), and combination therapy (L-DHC+ ice pad, n=24). Rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h. Infarct volume, neurological deficits and apoptotic cell death were determined at 24 h after reperfusion. Expression of pro- and anti-apoptotic proteins was evaluated by Western blot. ATP and reactive oxygen species (ROS) were detected by biochemical assays at 6 and 24 h after reperfusion. Combination therapy of L-DHC and ice pad significantly improved every measured outcome compared to monotherapies. Combination therapy achieved hypothermia faster by 28.6% than ice pad, 350% than L-DHC and 200% than H-DHC alone. Combination therapy reduced (p<0.05) neurological deficits by 63% vs. 26% with L-DHC. No effect was observed when using ice pad or H-DHC alone. L-DHC and ice pad combination improved brain oxidative metabolism by reducing (p<0.05) ROS at 6 and 24 h after reperfusion and increasing ATP levels by 42.9% compared to 25% elevation with L-DHC alone. Finally, combination therapy decreased apoptotic cell death by 48.5% vs. 24.9% with L-DHC, associated with increased anti-apoptotic protein and reduced pro-apoptotic protein levels (p<0.001). Our study has demonstrated that combining physical and pharmacological hypothermia is a promising therapeutic approach in ischemic stroke, and warrants further translational investigations.
Zhang Jun,Liu Kaiyin,Elmadhoun Omar, et al. Synergistically Induced Hypothermia and Enhanced Neuroprotection by Pharmacological and Physical Approaches in Stroke[J]. Aging and disease,
2018, 9(4): 578-589.
Figure 1 Changes of body temperature during hypothermia procedure in rat after MCAO
The figure shows changes in rectal temperature after reperfusion (0-180 min) for different therapies in MCAO groups, including L-DHC (0.5 mg/kg), H-DHC (1.5 mg/kg), ice pad, L-DHC combined with ice pad, and L-DHC with normal temperature group. The baseline temperatures were measured immediately after reperfusion, and all therapies were initiated after recording the baseline temperatures. No statistical differences were observed in baseline temperatures, target temperatures or endpoint temperatures in all these groups. In the combination therapy group, rectal temperature was reduced from 37.7 ± 0.07 °C to 31 ± 0.15 °C in less than 40 minutes. Ice pad rapidly reduced temperature from 37.6 ± 0.10 °C to 31 ± 0.09 °C after 50 minutes. The time for low and high DHC monotherapies to reach 31? were 180 and 120 min, respectively.
Figure 2 Neurological deficits and brain infarct volumes at 24 h of reperfusion
Higher scores represent greater deficits in neurological function in the 5-point (A) and 12-point scale (B) systems after stroke. At 24 h after reperfusion, slightly lower scores (3.3 ± 0.7, 6.8 ± 0.5) were observed in L-DHC group than no treatment group (4.3 ± 0.3, 10.6 ± 0.4) (##p<0.01, ###p<0.001). In the L-DHC with normal temperature (4.3±0.4, 10.6 ± 0.3) (##p<0.01, ###p<0.001), these reductions were statistically reversed. The combination therapy largely reduced deficit score (2.4 ± 0.5, 4.3 ± 0.4) (###p<0.001, ###p<0.001) as compared with L-DHC and ice pad each alone. Neither ice pad (4.3 ± 0.3, 9.4 ± 0.3) nor H-DHC group (3.9±0.4, 9.1±0.5) showed significant therapeutic effects. (C) Representatives of brain slices with TTC staining after ischemia/reperfusion in each group are shown. (D) At 24 h of reperfusion, the stroke group without treatment and L-DHC with normal temperature (37°C) showed infarct volume of 49.5 ± 12.4% and 50.0 ± 11.2%, respectively. L-DHC significantly (##p<0.01) reduced infarct volume (36.7 ± 9.9%). The combination of L-DHC with ice pad further reduced infarct volume to 18.2 ± 5.8% (###p<0.001). Neither ice pad (47.9 ± 10.3%) nor H-DHC (1.5mg/kg) (43.3 ± 9.4%) alone showed a therapeutic effect.
Figure 3 ROS production, ATP levels and cell death
At 6 (A) and 24 h (B) after reperfusion, reactive oxygen species (ROS) levels were significantly elevated (***p<0.001, ***p<0.001) in no treatment group as compared to sham operation control group (reference as 1). Ice pad group significantly reduced ROS levels at 6 (##p<0.01) and 24 h (###p<0.001). L-DHC group significantly (###p<0.001) reduced ROS levels at only 24 h. Again, combination therapy largely enhanced the reduction in ROS levels at both time points (###p<0.001, #p<0.05). (C) ATP production was compared among treatment groups at 24 h reperfusion. MCAO reduced ATP levels significantly (#p<0.05) compared with sham operation group (artificially set to 1). L-DHC group significantly (**p<0.01) elevated ATP levels, and this was further enhanced by combination therapy (*p<0.05). Other therapies did not show significant changes in ATP. (D) Apoptotic cell death profile was compared among groups at 24 h reperfusion. The degree of apoptotic cell death was significantly (***p<0.001) increased in no treatment stroke group than sham operation group. A moderate reduction (#p<0.05) in cell death was observed in L-DHC group, and this reduction was enhanced by the combination therapy (#p<0.05). Other treatment groups did not show significant reduction in cell death.
Figure 4 Apoptotic protein expression evaluated by Western blotting
(A) Bax protein expression was significantly (***p<0.001, *p<0.05) increased in no treatment group at 6 (***p<0.001) and 24 h (*p<0.05) compared with sham control (artificially set at 1). At 6 h, the combination therapy significantly (###p<0.001) reduced protein expression. At 24 h, L-DHC group significantly (##p<0.01) reduced protein expression. This reduction was enhanced by combination therapy (##p<0.01). (B) Cleaved caspase-3 protein expression was significantly increased after stroke at 6 (*p<0.05) and 24 h (**p<0.01) of reperfusion. The combination therapy significantly (###p<0.001) reduced protein expression at 6 h of reperfusion. At 24 h, the reduced effect of L-DHC on cleaved caspase-3 protein expression (###p<0.001), was significantly (###p<0.001) enhanced by combination therapy. (C) Bcl-2 protein expression was significantly reduced after stroke at both 6 (###p<0.001) and 24 (###p<0.001) h of reperfusion. L-DHC and ice pad significantly increased the protein expression at 6 (*p<0.05, ***p<0.001) and 24 h (***p<0.001, **p<0.01) of reperfusion. The combination therapy augmented the increase seen in L-DHC (***p<0.001, ***p<0.001) and ice pad groups (***p<0.001, ***p<0.001) at both time-points. (D) Bcl-xl protein expression was significantly reduced after stroke at the two-time points after reperfusion (#p<0.05, #p<0.05). While L-DHC significant increased the protein expression at both time-points (***p<0.001, **p<0.01), the combination therapy enhanced the increase at 6 (*p<0.05) and 24 (*p<0.05) of reperfusion.
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