Hypoxic Air Inhalation and Ischemia Interventions Both Elicit Preconditioning Which Attenuate Subsequent Cellular Stress In vivo Following Blood Flow Occlusion and Reperfusion

schemic preconditioning (IPC) is valid technique which elicits reductions in femoral blood flow occlusion mediated reperfusion stress (oxidative stress, Hsp gene transcripts) within the systemic blood circulation and/or skeletal muscle. It is unknown whether systemic hypoxia, evoked by hypoxic preconditioning (HPC) has efficacy in priming the heat shock protein (Hsp) system thus reducing reperfusion stress following blood flow occlusion, in the same manner as IPC. The comparison between IPC and HPC being relevant as a preconditioning strategy prior to orthopedic surgery. In an independent group design, 18 healthy men were exposed to 40 min of (1) passive whole-body HPC (FiO2 = 0.143; no ischemia. N = 6), (2) IPC (FiO2 = 0.209; four bouts of 5 min ischemia and 5 min reperfusion. n = 6), or (3) rest (FiO2 = 0.209; no ischemia. n = 6). The interventions were administered 1 h prior to 30 min of tourniquet derived femoral blood flow occlusion and were followed by 2 h subsequent reperfusion. Systemic blood samples were taken pre- and post-intervention. Systemic blood and gastrocnemius skeletal muscle samples were obtained pre-, 15 min post- (15PoT) and 120 min (120PoT) post-tourniquet deflation. To determine the cellular stress response gastrocnemius and leukocyte Hsp72 mRNA and Hsp32 mRNA gene transcripts were determined by RT-qPCR. The plasma oxidative stress response (protein carbonyl, reduced glutathione/oxidized glutathione ratio) was measured utilizing commercially available kits. In comparison to control, at 15PoT a significant difference in gastrocnemius Hsp72 mRNA was seen in HPC (−1.93-fold; p = 0.007) and IPC (−1.97-fold; p = 0.006). No significant differences were observed in gastrocnemius Hsp32 and Hsp72 mRNA, leukocyte Hsp72 and Hsp32 mRNA, or oxidative stress markers (p > 0.05) between HPC and IPC. HPC provided near identical amelioration of blood flow occlusion mediated gastrocnemius stress response (Hsp72 mRNA), compared to an established IPC protocol. This was seen independent of changes in systemic oxidative stress, which likely explains the absence of change in Hsp32 mRNA transcripts within leukocytes and the gastrocnemius. Both the established IPC and novel HPC interventions facilitate a priming of the skeletal muscle, but not leukocyte, Hsp system prior to femoral blood flow occlusion. This response demonstrates a localized tissue specific adaptation which may ameliorate reperfusion stress

Lung recruitment and endotracheal suction in ventilated preterm infants measured with electrical impedance tomography

Aims Although suctioning is a standard airway maintenance procedure, there are significant associated risks, such as loss of lung volume due to high negative suction pressures. This study aims to assess the extent and duration of change in end-expiratory level (EEL) resulting from endotracheal tube (ETT) suction and to examine the relationship between EEL and regional lung ventilation in ventilated preterm infants with respiratory distress syndrome. Methods A prospective observational clinical study of the effect of ETT suction on 20 non-muscle-relaxed preterm infants with respiratory distress syndrome (RDS) on conventional mechanical ventilation was conducted in a neonatal intensive care unit. Ventilation distribution was measured with regional impedance amplitudes and EEL using electrical impedance tomography. Results ETT suction resulted in a significant increase in EEL post-suction (P < 0.01). Regionally, anterior EEL decreased and posterior EEL increased post-suction, suggesting heterogeneity. Tidal volume was significantly lower in volume-guarantee ventilation compared with pressure-controlled ventilation (P = 0.04). Conclusions ETT suction in non-muscle-relaxed and ventilated preterm infants with RDS results in significant lung volume increase that is maintained for at least 90 min. Regional differences in distribution of ventilation with ETT suction suggest that the behaviour of the lung is heterogeneous in nature

The impact of remote ischaemic preconditioning on the human plasma proteome

© 2014 Dr. Joanne Michele HepponstallPublications included in thesis:Hepponstall, M., Ignjatovic, V., Binos, S., Monagle, P., Jones, B., Cheung, M. H. H., d’Udekem, Y. & Konstantinov, E. (2012). Remote ischemic preconditioning (RIPC) modifies plasma proteome in humans. PLoS ONE, 7(11), e48284. DOI: 10.1371/journal.pone.0048284Pepe, S., Liaw, N. Y., Hepponstall, M., Sheeran, F. L., Yong, M. S., d'Udekem, Y., Cheung, M. M. & Konstantinov, I. E. (2013). Effect of remote ischemic preconditioning on phosphorylated protein signalling in children undergoing tetralogy of Fallot repair: a randomised controlled trial. Journal of the American Heart Association, 2(3), e000095. DOI: 10.1161/JAHA.113.000095Remote Ischemic Preconditioning (RIPC) is an intervention involving intermittent periods of ischaemia and reperfusion of peripheral tissue to provide multi-organ protection, including the heart, from ischaemia-reperfusion (IR) injury. RIPC has been shown to improve clinical outcomes in a variety of settings. It appears that humoral factors that are released into the blood in response to the RIPC stimulus. Previous studies have demonstrated a global genomic response to RIPC in healthy adult volunteers. The focus of the studies described in this thesis was to examine the proteomic changes in plasma in response to RIPC in healthy adult volunteers and children undergoing repair of Tetralogy of Fallot (ToF) with cardiopulmonary bypass (CPB). The latter was in the setting of a randomised controlled trial (RCT). RIPC was induced by cyclic inflations of a standard blood pressure cuff applied to a limb, to a pressure exceeding systolic in order to completely interrupt blood flow. Complete interruption of blood flow was confirmed by pulse oximetry. The protocol involved four cycles of five minutes of ischaemia alternating with five minutes of reperfusion. In the preclinical study, the proteomic change in plasma originating from the limb subjected to the RIPC and the proteomic response to RIPC at both 15 min and 24 h post RIPC was studied. Forty eight proteins were found to be differentially expressed with up-regulation dominating during the cycles of reperfusion and down-regulation evident at 15 min and 24 h post RIPC stimulus. These results suggested that in response to brief episodes of ischaemia and reperfusion in an upper limb, proteomic changes in the plasma are induced. This may result in a protective state systemically, that significantly modifies both the early and late proteomic response to IR injury. We then extended this idea into the clinical setting to determine if a similar proteomic response could be detected in children with ToF undergoing repair with CPB. A double blind RCT was performed to investigate whether RIPC modifies clinical markers of IR injury and cardiopulmonary function after CPB in children undergoing ToF repair. Although there was no clinical evidence that RIPC promotes recovery of cardiac function, reduces post-operative complications or inotrope requirement in the first 24 h post CPB, the proteomic response to the RIPC was noticeable. We presented the results characterising the proteomic changes in response to CPB that are most apparent at 6 h and 12 h post CPB and return to baseline within 24 h. CPB induced significant changes to the global proteomic response in plasma of children undergoing cardiac surgery for repair of ToF. We further investigated the impact of the RIPC on modifying the global plasma proteomic response during the peri-operative period in plasma from children undergoing ToF repair with CPB. There were no differences between the control group and the RIPC group at baseline or at the end of CPB. At 6h post CPB, there were 48 peptides that were found to be differentially expressed which related to six proteins. There was a return to the baseline within 24 h of CPB. In conclusion, the findings of this thesis provided evidence for the first time that there were proteomic changes in human plasma in response to RIPC, supporting the hypothesis that humoral factors are released into the blood to render a protective state against IR injury. Together, findings from the preclinical study and the RCT indicated that the observed proteomic changes were consistent in healthy adults and children undergoing heart surgery

Exploring the human plasma proteome for humoral mediators of remote ischemic preconditioning - A word of caution

Despite major advances in early revascularization techniques, cardiovascular diseases are still the leading cause of death worldwide, and myocardial infarctions contribute heavily to this. Over the past decades, it has become apparent that reperfusion of blood to a previously ischemic area of the heart causes damage in and of itself, and that this ischemia reperfusion induced injury can be reduced by up to 50% by mechanical manipulation of the blood flow to the heart. The recent discovery of remote ischemic preconditioning (RIPC) provides a non-invasive approach of inducing this cardioprotection at a distance. Finding its endogenous mediators and their operative mode is an important step toward increasing the ischemic tolerance. The release of humoral factor(s) upon RIPC was recently demonstrated and several candidate proteins were published as possible mediators of the cardioprotection. Before clinical applicability, these potential biomarkers and their efficiency must be validated, a task made challenging by the large heterogeneity in reported data and results. Here, in an attempt to reproduce and provide more experimental data on these mediators, we conducted an unbiased in-depth analysis of the human plasma proteome before and after RIPC. From the 68 protein markers reported in the literature, only 28 could be mapped to manually reviewed (Swiss-Prot) protein sequences. 23 of them were monitored in our untargeted experiment. However, their significant regulation could not be reproducibly estimated. In fact, among the 394 plasma proteins we accurately quantified, no significant regulation could be confidently and reproducibly assessed. This indicates that it is difficult to both monitor and reproduce published data from experiments exploring for RIPC induced plasma proteomic regulations, and suggests that further work should be directed towards small humoral factors. To simplify this task, we made our proteomic dataset available via ProteomeXchange, where scientists can mine for novel potential targets

Remote Ischemic Preconditioning (RIPC) Modifies Plasma Proteome in Humans

Remote Ischemic Preconditioning (RIPC) induced by brief episodes of ischemia of the limb protects against multi-organ damage by ischemia-reperfusion (IR). Although it has been demonstrated that RIPC affects gene expression, the proteomic response to RIPC has not been determined. This study aimed to examine RIPC induced changes in the plasma proteome. Five healthy adult volunteers had 4 cycles of 5 min ischemia alternating with 5 min reperfusion of the forearm. Blood samples were taken from the ipsilateral arm prior to first ischaemia, immediately after each episode of ischemia as well as, at 15 min and 24 h after the last episode of ischemia. Plasma samples from five individuals were analysed using two complementary techniques. Individual samples were analysed using 2Dimensional Difference in gel electrophoresis (2D DIGE) and mass spectrometry (MS). Pooled samples for each of the time-points underwent trypsin digestion and peptides generated were analysed in triplicate using Liquid Chromatography and MS (LC-MS). Six proteins changed in response to RIPC using 2D DIGE analysis, while 48 proteins were found to be differentially regulated using LC-MS. The proteins of interest were involved in acute phase response signalling, and physiological molecular and cellular functions. The RIPC stimulus modifies the plasma protein content in blood taken from the ischemic arm in a cumulative fashion and evokes a proteomic response in peripheral blood

Effect of Remote Ischemic Preconditioning on Phosphorylated Protein Signaling in Children Undergoing Tetralogy of Fallot Repair: A Randomized Controlled Trial

BACKGROUND: Our previous randomized controlled trial demonstrated cardiorespiratory protection by remote ischemic preconditioning (RIPC) in children before cardiac surgery. However, the impact of RIPC on myocardial prosurvival intracellular signaling remains unknown in cyanosis. RIPC may augment phosphorylated protein signaling in myocardium and circulating leukocytes during tetralogy of Fallot (ToF) repair. METHODS AND RESULTS: Children (n=40) undergoing ToF repair were double-blind randomized to RIPC (n=11 boys, 9 girls) or control (sham RIPC: n=9 boys, 11 girls). Blood samples were taken before, immediately after, and 24 hours after cardiopulmonary bypass. Resected right ventricular outflow tract muscle and leukocytes were processed for protein expression and mitochondrial respiration. There was no difference in age (7.1 ± 3.4 versus 7.1 ± 3.4 months), weight (7.7 ± 1.8 versus 7.5 ± 1.9 kg), or bypass or aortic cross-clamp times between the groups (control versus RIPC, mean±SD). No differences were seen between the groups for an increase in the ratio of phosphorylated to total protein for protein kinase B, p38 mitogen activated protein kinase, signal transducer and activator of transcription 3, glycogen synthase kinase 3β, heat shock protein 27, Connexin43, or markers associated with promotion of necrosis (serum cardiac troponin I), apoptosis (Bax, Bcl-2), and autophagy (Parkin, Beclin-1, LC3B). A high proportion of total proteins were in phosphorylated form in control and RIPC myocardium. In leukocytes, mitochondrial respiration and assessed protein levels did not differ between groups. CONCLUSIONS: In patients with cyanotic heart disease, a high proportion of proteins are in phosphorylated form. RIPC does not further enhance phosphorylated protein signaling in myocardium or circulating leukocytes in children undergoing ToF repair. CLINICAL TRIAL REGISTRATION: URL: (http://www.anzctr.org.au/trial_view.aspx?id=335613. Unique identifier: Australian New Zealand Clinical Trials Registry number ACTRN12610000496011