Dexmedetomidine alleviates the spinal cord ischemia-reperfusion injury through blocking mast cell degranulation

Abstract

Objective: To investigate the neuro-protective effects of dexmedetomidine (dex) on I/R-induced spinal injury and potential mechanisms.

Methods: sprague-Dawley rats in the treatment group received intraperitoneal injections of 25 mg/kg dexmedetomidine, MC stabilizer cromolyn (100 mg/kg), MCs stimuliser compound 48/80 (80 mg/kg), PBS at 24 h befor IR. Underwent 5 minutes of aortic occlusion via median sternotomy, functional scores were recorded at 12, 24, 36 and 48 hours after reperfusion. Additionally, 3 mice underwent sham surgery with sternotomy and dissection of the aorta and subclavian artery with no occlusion. Spinal cords were examined for protein kinase B (AKT), CREB, and brain-derived neurotrophic factor (BDNF) following treatment alone or ischemia-reperfusion surgery. Collected the serum to observe the expression of pro-inflammation cytokines (TNF-α, INF-γ and IL-1β) and anti-inflammation cytokines (TGF-β, IL-10 and IL-6). Then the MCs were harvested to test the expression surface molecular of FcεR and MCs' degranulation.

Results: Pretreated the rats with dexmedetomidine has higher neurologic function at all time points after I/R injury. We collected the serum of rats then detected the pro-inflammation cytokines TNF-α, INF-γ and IL-1β levels and anti-inflammation cytokinses TGF-β, IL-10 and IL-6 levels, found that the pro-inflammation cytokines of dexmedetomidine group was decreased whereas the anti-inflammation cytokinses was increased. At the same time the protect protein of AKT, CREB and mRNA BDNF were increased. They had the same results with cromolyn group, and opposite with the compound 48/80 group. We pretreated MCs with dexmedetomidine in vitro, and found that the activity surface molecular of MCs was down-regulation, and MCs degranulation was decreased.

Conclusion: We thus demonstrate a possible mechanism by which dexmedetomidine alleviates spinal cord I/R injury through blocking the MCs degranulation.

Keywords: Dexmedetomidine; degranulation; ischemia-reperfusion injury; mast cell.

Figures

Figure 1

Time (h) following the ischemia. Following ischemia-reperfusion (IR) surgery with 5 minutes of aortic occlusion a significant functional deficit in hind-limb function was observed in all rats. 24 hours of pretreatment with dexmedetomidine (IR+ Dex) and cromolyn (IR+ cromolyn) resulted in significantly higher neurologic function at all time-point postoperatively. The rats pretreated with compound 48/80 had opposite result. Error bars represent mean ± SEM. *P<0.05.

Figure 2

The expression of pro-inflammation cytokines. Dexmedetomidine suppressed I/R induced upregulation of TNF-α, INF-γ and IL-1β levels expressions in spinal. ELISA was performed to detect the serum expression of TNF-α, INF-γ and IL-1β levels 24 h following I/R insult (n = 5; *P<0.05).

Figure 3

The expression of anti-inflammation cytokines. Dexmedetomidine induced upregulation of TGF-β, IL-10 and IL-6 levels expressions in spinal to alleviate the spinal I/R injury. ELISA was performed to detect the serum expression of TGF-β, IL-10 and IL-6 levels 24 h following I/R insult (n = 5; *P<0.05).

Figure 4

The protein of AKT expression. Dexmedetomidine induced enhancement in spinal AKT expression. A. Western blots assay was performed to evaluate AKT protein expression in the spinal 24 h following I/R insult. B. Bands corresponding to AKT and β-actin protein were quantified densitometrically. Results are expressed as the ratio of full-length AKT to β-actin. Data are means ± SD of three independent experiments performed in triplicate or a representative result. *P<0.05.

Figure 5

The protein of CREB expression. Dexmedetomidine induced enhancement in spinal CREB expression. A. Western blots was performed to evaluate CREB protein expression in the spinal 24 h following I/R insult. B. Bands corresponding to CREB and β-actin protein were quantified densitometrically. Results are expressed as the ratio of full-length CREBto β-actin. Data are means ± SD of three independent experiments performed in triplicate or a representative result. *P<0.05.

Figure 6

The mRNA of BDNF expression. A. Quantitative polymerase chain reaction for production of brain-derived neurotrophic factor (BDNF) transcripts was performed. Pretreatment with dexmedetomidine resulted in a significant up-regulation BNDF messenger RNA expression. B. Bands corresponding to BDNF and GAPDH were quantified densitometrically. Data are means ± SD of three independent experiments performed in triplicate. *P<0.05.

Figure 7

Expression of FcεR on the surface of MCs. MCs pretreated with PBS DEX cromolyn compound 48/80. A. FcεR expression on MCs decreased with DEX (b) and cromolyn (d). PBS (a) and compound 48/80 (c) had the opposite results. B. Expression of the FcεR on the MCs surface shown as mean fluorescence intensity (MFI) measured by flow cytometry after incubation for 24 h (with isotope control subtracted). Data are presented as the percentage MFI of MCs alone which was set as 100%. Data are means ± SEM of three independent experiments. Statistical significance was calculated using the Student’s t-test. *P

Figure 8

MCs degranulation induced by anti –DNPIgE mAb and DNP-HAS was evaluated by 4-Nitrophenyl N-acetyl-β-D-glucosaminide after pretreatment. Compared with the control group, pretreatment with PBS, compound 48/80 obviously elicited MCs degranulation, whereas the DEX and cromolyn groups decreased the MCs degranulation, there were not significantly different. Data are shown as the mean ± SD of three independent experiments performed in triplicate. *P