ABSTRACT: Effect of methylene blue on the genomic response to reperfusion injury induced by cardiac arrest and cardiopulmonary resuscitation in porcine brain
Project description:Background: Cerebral ischemia/reperfusion injury is a common secondary effect of cardiac arrest which is largely responsible for postresuscitative mortality. Therefore development of therapies which restore and protect the brain function after cardiac arrest is essential. Methylene blue (MB) has been experimentally proven neuroprotective in a porcine model of global ischemia-reperfusion in experimental cardiac arrest. However, no comprehensive analyses have been conducted at gene expression level. Methods: Pigs underwent either untreated cardiac arrest (CA) or CA with subsequent cardiopulmonary resuscitation (CPR) accompanied with an infusion of saline or an infusion of saline with MB. Genome-wide transcriptional profiling using the Affymetrix porcine microarray was performed to 1) gain understanding of delayed neuronal death initiation in porcine brain during ischemia and after 30, 60 and 180 min following reperfusion, and 2) identify the mechanisms behind the neuroprotective effect of MB after ischemic injury (at 30, 60 and 180 min). Results: Our results show that restoration of spontaneous circulation (ROSC) induces major transcriptional changes related to stress response, inflammation, apoptosis and even cytoprotection. In contrast, the untreated ischemic and anoxic insult affected only few genes mainly involved in intra-/extracellular ionic balance. Furthermore, our data show that the neuroprotective role of MB is diverse and fulfilled by regulation of the expression of soluble guanylate cyclase and biological processes accountable for inhibition of apoptosis, modulation of stress response, neurogenesis and neuroprotection. Conclusions: Our results support that MB could be a valuable intervention and should be investigated as a therapeutic agent against neural damage associated with I/R injury induced by cardiac arrest. One group of pigs underwent cardiac arrest (CA) without subsequent cardiopulmonary resuscitation (CPR) and the brain of the animals was removed after 5, 20 or 30 min post CA for genome-wide expression study. Two groups of pigs underwent first CA for 12 min with a subsequent 8 min-long CPR and received either an infusion of saline or an infusion of saline with MB from one minute after the start of CPR until 50 min after return of spontaneous circulation (ROSC). In both latter groups the brains were removed for microarray analysis at the following time points: 30, 60, 180 min.
Project description:Background: Cerebral ischemia/reperfusion injury is a common secondary effect of cardiac arrest which is largely responsible for postresuscitative mortality. Therefore development of therapies which restore and protect the brain function after cardiac arrest is essential. Methylene blue (MB) has been experimentally proven neuroprotective in a porcine model of global ischemia-reperfusion in experimental cardiac arrest. However, no comprehensive analyses have been conducted at gene expression level. Methods: Pigs underwent either untreated cardiac arrest (CA) or CA with subsequent cardiopulmonary resuscitation (CPR) accompanied with an infusion of saline or an infusion of saline with MB. Genome-wide transcriptional profiling using the Affymetrix porcine microarray was performed to 1) gain understanding of delayed neuronal death initiation in porcine brain during ischemia and after 30, 60 and 180 min following reperfusion, and 2) identify the mechanisms behind the neuroprotective effect of MB after ischemic injury (at 30, 60 and 180 min). Results: Our results show that restoration of spontaneous circulation (ROSC) induces major transcriptional changes related to stress response, inflammation, apoptosis and even cytoprotection. In contrast, the untreated ischemic and anoxic insult affected only few genes mainly involved in intra-/extracellular ionic balance. Furthermore, our data show that the neuroprotective role of MB is diverse and fulfilled by regulation of the expression of soluble guanylate cyclase and biological processes accountable for inhibition of apoptosis, modulation of stress response, neurogenesis and neuroprotection. Conclusions: Our results support that MB could be a valuable intervention and should be investigated as a therapeutic agent against neural damage associated with I/R injury induced by cardiac arrest.
Project description:The consequences of cardiac arrest are often fatal, including brain injury after resuscitation. It has been reported that few people patients can recover to the neurological state before cardiac arrest. MiRNAs are short non-protein-coding RNA molecules that are evolutionarily conserved and ubiquitously expressed. Numerous pieces of research have reviewed the role of miRNAs in regulating neuronal apoptosis, regeneration, and plasticity of neurons, and inflammatory after cardiac arrest . As the stability of miRNAs in the bloodstream and the function in the regulation of neurological impairment after ischemia-reperfusion injury, microRNAs have been the most potential new biomarkers and therapeutic targets after cardiac arrest to alleviate neurological impairment . In this work, we found that the level of miR-483-5p is correlated to the prognosis of neurological function. To investigate the function of miR-483-5p on neurons after ischemia-reperfusion injury,we established highly differentiated PC12 cell lines in which miR-483-5p was overexpressed by transfection with miR-483-5p mimcis.We then performed gene expression profiling analysis using data obtained from RNA-seq of PC12 cells in different groups.
Project description:Acute cardiorenal syndrome (CRS-1) is a morbid complication of acute cardiovascular disease. Mechanistic investigations have focused on intrarenal cellular signaling induced by ischemia/reperfusion. Additional cardiorenal connector signals have been postulated, but investigation in CRS-1 has been limited by a paucity of animal models and technical limitations precluding discovery studies of glomerular filtrate. To address these limitations, we developed a translational model of CRS-1, cardiac arrest and cardiopulmonary resuscitation (CA/CPR) in mice. Quantitative proteomics using isobaric tagging (10-plex TMT reagents, Thermo Fisher) was performed on 24h urine collections from mice with deficient tubular endocytosis and from littermate controls, before and after CA/CPR. Data acquisition used the SPS MS3 method on a Thermo Fusion Tribrid mass spectrometer for accurate reporter ion signals. The findings confirmed CA/CPR-specific cardiac proteins in urine and identified a novel CA/CPR-specific filtrate component: Cardiac LIM protein (CSRP3).
Project description:To investigate changes in cardiac transcription profiles caused by on-pump cardiac surgery, we collected myocardial samples, prior and after grafting, from patients undergoing on-pump coronary artery bypass grafting with cardiopulmonary bypass and cardiac arrest. The transcriptional profile of the mRNA in these samples was measured with gene array technology. Changes in transcriptional profiles can be correlated with the stress response of heart to surgery, cardiopulmonary bypass and cardiac arrest. Keywords: human, cardiac, CABG coronary surgery, gene expression, cardiopulmonary bypass. Myocardial samples were collected, prior and after grafting, from patients undergoing on-pump coronary artery bypass grafting with cardiopulmonary bypass and cardiac arrest.
Project description:Acute cardiorenal syndrome (CRS-1) is a morbid complication of acute cardiovascular disease. Mechanistic investigations have focused on intrarenal cellular signaling induced by ischemia/reperfusion. Additional signals, “cardiorenal connectors”, have been postulated, but investigation in CRS-1 has been limited by a paucity of animal models and technical limitations precluding discovery studies of glomerular filtrate. To address these limitations we developed a translational model of CRS-1, cardiac arrest and cardiopulmonary resuscitation (CA/CPR) and now report findings from a nanoscale mass spectrometry assay allowing proteomic exploration of Bowman’s space aspirate 2h after CA/CPR or sham procedure. Imaging, molecular weight and charge distribution, and minimal contribution of proteins from surrounding cell types confirmed the acquisition of filtrate. We detected filtration of low-molecular weight proteins specific to the heart following CA/CPR. Additional mass spectrometry performed on 24h urine collections from mice with deficient tubular endocytosis confirmed CA/CPR-specific cardiac protein filtration, and identified a novel, CA/CPR-specific, filtrate component: Cardiac LIM protein. Cardiac arrest-induced plasma release of Cardiac LIM protein occurred in mice and in critically-ill human cardiac arrest survivors and administration of recombinant cardiac LIM protein to mice altered renal function. Our findings demonstrate that glomerular filtrate is accessible to nanoscale proteomics and elucidate the population of proteins filtered 2h after acute cardiovascular crisis. The presence and identification of cardiac-specific proteins in renal filtrate suggest a potential novel signaling mechanism in CRS-1. We expect these findings to advance understanding of cardiorenal syndrome.
Project description:Pigs underwent 5 min of untreated cardiac arrest followed by 8 min of basic life support and 22 min of advanced life support (ALS). Following this conventional cardiopulmonary resuscitation, all pigs were treated 120 min with extracorporeal circulation (eCPR). Blood samples were drawn at baseline, after ALS and after eCPR and were processed to serum samples, which were depleted for Albumin and IgG before analysing them via explorative mass spectrometry.
Project description:Complete global brain ischemia (CGBI) and reperfusion occur following resuscitation from cardiac arrest. Different brain neurons are selectively vulnerable to CGBI: pyramidal neurons of hippocampal CA3 survive 10 min CGBI but those of CA1 die at 3 days following 10 min CGBI. CA3 neurons are expected to have more robust stress responses and repair responses than CA1 neurons. We used microarrays to compared total and polysome-bound mRNAs in CA1 and CA3 at 8 hr reperfusion after 10 min CGBI in Long Evans male rats to ascertain differences in total vs polysome-bound gene expression.