Role Of Cardiac-Splenic Axis In Acutemyocardial Ischemia And Reperfusion Injury

Part Ⅰ Role of the spleen in acute myocardial ischemia and reperfusion injuryOBJECTIVE:Inflammatory responses play critical roles in myocardial ischemia/reperfusion (I/R) injury. Recently, spleen has been reported to regulate the process of ventricular remodeling after myocardial infarction. However, the role of spleen during acute phase of post-ischemic reperfusion injury remains unknown. The present study was designed to investigate if the spleen participates the myocardial post-ischemic reperfusion injury.METHODS:C57BL6 mice underwent various durations (10,20,30,40 and 50 minutes) of LAD occlusion followed by 60 minutes reperfusion with or without acute splenectomy (SPLX) prior to I/R injury. Acute splenocyte adoptive transfer (SPAT) was performed by Ⅳ injection of 5×106 live splenocytes. Viability (>95%) of the splenic leukocytes was determined with Trypan Blue staining. TTC & Blue staining was used to analyze the myocardial infarct size after reperfusion.RESULTS:SPLX significantly decreased myocardial infarct size (IS) in 40’/60’ and 50’/60’ groups; however, had no effect in 10’/60’,20’/60’and 30’/60’ groups. SPAT significantly increased IS of 40’/60’ splenectomized mice but not the splenectomized mice from 20’/60’ groups.CONCLUSION:These data indicates that spleen play important roles in acute myocardial ischemia and reperfusion injury after prolonged ischemic insults (≥ 40 min in mice).Part Ⅱ The Cardiac (HMGB1)-Splenic (RAGE) axis participates myocardial I/R injury process and exacerbates the myocardial infarct size after reperfusion.OBJECTIVE:It has been reported that injured tissue will release endogenous ’danger’ signals referred to as danger associated molecular patterns (DAMPs), which triggers innate immune responses. Among the DAMPs, HMGB1 has been reported to play roles in myocardial ischemia and reperfusion injury by acting on RAGE receptors from immune cells. Thus, we hypothesized that prolonged ischemic insults may lead myocardial release more HMGB1, which, in turn, activate splenic leukocytes by RAGE receptors and exacerbate the myocardial reperfusion injury.METHODS:C57BL6 and RAGE knockout mice were subjected to myocardial ischemia and reperfusion injury model. Based on the results from part Ⅰ, we chose 20 minutes ischemia and 60 minutes reperfusion as a minor injury control group. Splenectomy and SPAT were performed as described in part Ⅰ. Ischemic myocardial homogenate (IHH) were obtained after 10 minutes and 40 minutes myocardial ischemia without reperfusion, respectively. The protein concentration of the IHH supernatant was determined by BCA protein assay kit. IHH was iv injected 5 minutes before reperfusion at a dose of 10μg/g. Myocardial infarct size was determined by TTC & Blue staining. Immunoprecipitation was used to separate the HMGB1 from the IHH. HMGB1 levels were detected by western blot.RESULTS:In the 20’/60’ group, infusion of 40-minute ischemic heart homogenate (40-IHH) upon reperfusion increased IS by>3-fold versus infusion of 10-IHH (p<0.05). Splenectomy abolished the infarct exacerbating effect of 40-IHH, which was restored by splenic leukocyte adoptive transfer (SPAT). Furthermore, depletion of HMGB1 in the 40-IHH abolished its infarct exacerbation effects (p<0.05), and 40-IHH failed to increase IS in both RAGEKO mice and splenectomized wild-type mice with SPAT from RAGEKO mice.CONCLUSION:A cardio (HMGB1)-splenic (RAGE receptor) axis exists and contributes to myocardial infarct exaggeration during reperfusion after prolonged ischemic insults.Part Ⅲ Cardio-splenic axis mediates neutrophils activation, infiltration and exacerbate the myocardial infarctionOBJECTIVE:Neutrophils infiltration is critical in the acute phase of myocardial reperfusion injury. A recent study demonstrated that activation of HMGB1-RAGE pathway triggers recruitment of neutrophils after liver ischemic necrosis, which mediates subsequent amplification of liver injury. Our present study was designed to investigate whether cardio-splenic axis could mediate the neutrophil activation and exacerbate the reperfusion injury.METHODS:C57BL6 mice underwent 20 minutes or 40 minutes ischemia followed by 60 minutes reperfusion. Splenectomy 10-IHH or 40-IHH treated groups were performed as described in Part I and II. In N-formyl peptide receptor 1 (FPR1) antagonist treated mice, cFLFLF was administered 5 minutes before reperfusion. Peripheral neutrophils were detected by HemaVet system. Immunofluorescence staining was used to detect neutrophils in spleen and myocardium. Myocardial infarct size was determined by TTC & Blue staining.RESULTS:40’/60’ I/R injury caused significant neutrophilia and more myocardial neutrophil infiltration than 20’/60’I/R or sham surgery, which was attenuated by splenectomy.40-IHH induced significant neutrophilia on 20’/60’ mice, which was also attenuated by splenectomy.40-IHH caused a significantly higher myocardial neutrophil infiltration in 20’/60’ mice than 10-IHH. Immunofluorescence staining revealed that 40-IHH activated splenic leukocytes in the marginal zone by elevating FPR1 expression, which is a key receptor that regulates neutrophil chemoattraction. Confocal imaging further showed that the elevated FPR1 expressions were mainly localized with Ly6G+ cells, which represent neutrophils. cFLFLF significantly reduced circulating neutrophils and myocardial neutrophil infiltration in 40-IHH-treated 20’/60’ I/R mice. The myocardial infarct sizes of 40-IHH-treated 20’/60’I/R mice were significantly reduced by cFLFLF pretreatment.CONCLUSION:Cardio-splenic axis mediates neutrophils activation during reperfusion. FPR1 is a potential therapeutic target for inhibiting the neutrophils that augments infarct size after I/R.Part IV Splenic leukocytes mediate the hyperglycemic exacerbation of myocardial infarct size in miceOBJECTIVE:Acute hyperglycemia (HG) potentiates systemic inflammatory responses and exacerbates myocardial infarct (MI) size in both pre-clinical and clinical studies. Accumulating evidence now shows that the spleen plays critical roles during myocardial infarction and subsequent healing. The current study was designed to determine whether splenocytes mediate the detrimental myocardial infarct exacerbation of ischemia/reperfusion injury caused by HG.METHODS:C57BL6 (B6) mice were assigned to 10 groups and underwent 30 min of LAD occlusion followed by 60 min of reperfusion. Acute hyperglycemia was induced by intraperitoneal injection of 20% dextrose 20 minutes prior to LAD occlusion at a dose of 2μl/g body weight. Acute splenectomy (SPLX) was performed 10 min before dextrose or vehicle injection with or without acute splenocyte adoptive transfer after splenectomy. Isolation of splenocytes was performed using gentle MACS single splenocyte isolation system according to manufacturer’s protocol. Viability (>95%) of the splenocytes was determined with Trypan Blue staining. Acute splenocyte adoptive transfer (SPAT) was performed by injecting 5* 106 single live splenocytes (≈100μl PBS) into splenectomized mice 5 minutes after removing the spleen. Infarct size (IS) and risk region (RR) were determined by TTC & Phthalo blue staining, respectively. Peripheral white blood cell counting was performed with a HemaVet hematology system. Spleens and hearts were harvested at 60 minutes after saline or Dextrose injection for ROS detection. Spleens were weighed after removing excess moisture and immunofluorescence staining was used to detect monocytes, neutrophils and FPR1 expression in spleens or hearts.RESULTS:HG significantly increased IS by 43% in mice (p<0.05). Splenectomy (SPLX) performed 30 min before ischemia abolished the infarct exacerbating effect of HG. Acute adoptive transfer of splenocytes (SPAT) from B6 mice performed 25 min before ischemia effectively restored the effects of HG on IS in SPLX mice. Acute hyperglycemia activated splenic leukocytes by increasing FPR1 expression and reactive oxygen species production before ischemia, and enhanced splenic neutrophil release with resultant peripheral neutrophilia and increased myocardial neutrophil infiltration during reperfusion.CONCLUSION:Acute hyperglycemia directly activates splenic leukocytes, especially neutrophils, by increasing FPR1 expression and ROS production. Acute hyperglycemia also facilitates mobilization of activated splenic neutrophils and induces neutrophilia early during reperfusion, which may directly exacerbate myocardial infarct size.