Steve T. Yeh

Oxygen requirement during cardiopulmonary resuscitation (CPR) to effect return of spontaneous circulation

BACKGROUND Recent scientific evidence has demonstrated the importance of good quality chest compressions without interruption to improve cardiac arrest resuscitation rates, and suggested that a de-emphasis on minute ventilation is needed. However, independent of ventilation, the role of oxygen and the optimal oxygen concentration during CPR is not known. Previous studies have shown that ventilation with high oxygen concentration after CPR is associated with worse neurologic outcome. We tested the hypothesis that initial ventilation during CPR without oxygen improves resuscitation success. METHODS Sprague-Dawley rats were anesthetized with ketamine/xylazine (IP), intubated and ventilated with room air. A KCl bolus (0.04 mg/g) was given (IV) to induce asystolic cardiac arrest and ventilation was stopped. At 6 min, CPR was started with an automated chest compressor at a rate of 200-240/min and epinephrine (0.01 mg/kg) was given 1 min later. During CPR, the ventilation rate was 50% of baseline with one of three oxygen concentrations: (1) 0% O2 (100% N2), (2) 21% O2, or (3) 100% O2. The prescribed oxygen concentration was continued for 2 min after return of spontaneous circulation (ROSC) and then all animals were switched to 100% oxygen for 1h prior to extubation. Blood gases were measured at baseline, 2 min and 1h after ROSC. Group comparisons were done using Fishers exact test and ANOVA. RESULTS ROSC was achieved in 1/10 (0% O2), 9/11 (21% O2) and 10/12 (100% O2, p<0.001). ROSC times after starting CPR were 80s in the 0% O2, 115+/-87 s in the 21% O2 group and 95+/-33 s in the 100% O2 group (mean+/-SD, p=0.5). Aortic end-diastolic pressure before ROSC was not different among groups. 100% oxygen ventilation in the first 2 min resulted in higher PaO2 at ROSC 2 min (109+/-44 mm Hg vs. 33+/-8 mm Hg, p<0.001). Survival to 72 h was 0/1 (0% O2), 7/9 (21% O2) and 8/10 (100% O2) with a low neurologic deficit score in both O2 groups (NDS range 5-25). CONCLUSIONS In a mild cardiac arrest model with generally good neurologic recovery, initial CPR ventilation with no O2 did not allow for ROSC. In contrast, CPR coupled with room air or higher oxygen levels result in a high rate of ROSC with good neurologic recovery. During CPR, the level of oxygenation must be considered, which if too low may preclude initial ROSC.

Biphasic modulation of the mitochondrial electron transport chain in myocardial ischemia and reperfusion

Mitochondrial electron transport chain (ETC) is the major source of reactive oxygen species during myocardial ischemia-reperfusion (I/R) injury. Ischemic defect and reperfusion-induced injury to ETC are critical in the disease pathogenesis of postischemic heart. The properties of ETC were investigated in an isolated heart model of global I/R. Rat hearts were subjected to ischemia for 30 min followed by reperfusion for 1 h. Studies of mitochondrial function indicated a biphasic modulation of electron transfer activity (ETA) and ETC protein expression during I/R. Analysis of ETAs in the isolated mitochondria indicated that complexes I, II, III, and IV activities were diminished after 30 min of ischemia but increased upon restoration of flow. Immunoblotting analysis and ultrastructural analysis with transmission electron microscopy further revealed marked downregulation of ETC in the ischemic heart and then upregulation of ETC upon reperfusion. No significant difference in the mRNA expression level of ETC was detected between ischemic and postischemic hearts. However, reperfusion-induced ETC biosynthesis in myocardium can be inhibited by cycloheximide, indicating the involvement of translational control. Immunoblotting analysis of tissue homogenates revealed a similar profile in peroxisome proliferator-activated receptor-γ coactivator-1α expression, suggesting its essential role as an upstream regulator in controlling ETC biosynthesis during I/R. Significant impairment caused by ischemic and postischemic injury was observed in the complexes I- III. Analysis of NADH ferricyanide reductase activity indicated that injury of flavoprotein subcomplex accounts for 50% decline of intact complex I activity from ischemic heart. Taken together, our findings provide a new insight into the molecular mechanism of I/R-induced mitochondrial dysfunction.

Preservation of mitochondrial function with cardiopulmonary resuscitation in prolonged cardiac arrest in rats.

During cardiac arrest (CA), myocardial perfusion is solely dependent on cardiopulmonary resuscitation (CPR) although closed-chest compressions only provide about 10-20% of normal myocardial perfusion. The study was conducted in a whole animal CPR model to determine whether CPR-generated oxygen delivery preserves or worsens mitochondrial function. Male Sprague-Dawley rats (400-450 g) were randomly divided into four groups: (1) BL (instrumentation only, no cardiac arrest), (2) CA(15) (15 min cardiac arrest without CPR), (3) CA(25) (25 min cardiac arrest without CPR) and (4) CPR (15 min cardiac arrest, followed by 10 min CPR). The differences between groups were evaluated by measuring mitochondrial respiration, electron transport chain (ETC) complex activities and mitochondrial ultrastructure by transmission electron microscopy (TEM). The CA(25) group had the greatest impairment of mitochondrial respiration and ETC complex activities (I-III). In contrast, the CPR group was not different from the CA(15) group regarding all measures of mitochondrial function. Complex I was more susceptible to ischemic injury than the other complexes and was the major determinant of mitochondrial dysfunction. Observations of mitochondrial ultrastructure by TEM were compatible with the biochemical results. The findings suggest that, despite low blood flow and oxygen delivery, CPR is able to preserve heart mitochondrial function and viability during ongoing global ischemia. Preservation of complex I activity and mitochondrial function during cardiac arrest may be an important mechanism underlying the beneficial effects of CPR which have been shown in clinical studies.

Post-cardiac arrest hyperoxia and mitochondrial function

INTRODUCTION Rapid post-ischemic re-oxygenation is necessary to minimize ischemic injury, but itself can induce further reperfusion injury through the induction of reactive oxygen species. Utilization of oxygen within the cell primarily occurs in the mitochondria. The objective of this study was to determine heart mitochondrial function after 1 h of controlled arterial oxygenation following cardiac arrest and restoration of spontaneous circulation (ROSC). We hypothesized that arterial hyper-oxygenation following ROSC would result in greater impairment of heart mitochondrial function. METHODS KCl cardiac arrest was induced in anesthetized rats. Following 6.5 min of cardiac arrest, animals were resuscitated with standard thumper CPR, ventilation and epinephrine. Following ROSC, all animals were ventilated for 60 min with either 100% O(2) or 40% O(2) titrated to achieve normoxia utilizing pulse oximetry. At the end of 1 h, heart mitochondria were isolated and mitochondrial respiratory function was measured. RESULTS Post-ROSC arterial PaO2 was 280 ± 40 in the 100% O2 group and 105 ± 10 in the 40% O2 group. One hour after ROSC, heart mitochondrial state 3 respirations and respiration control ratio (state 3/4 respiration) were significantly reduced from baseline in animals ventilated with 100% O(2), but not with 40% O(2). CONCLUSION Post-ROSC arterial hyperoxia after a short cardiac arrest exacerbates impaired mitochondrial function. The overall clinical significance of these findings is unclear and requires additional work to better understand the role of post-arrest hyperoxia on cardiac and mitochondrial function.

Hyperoxemic reperfusion after prolonged cardiac arrest in a rat cardiopulmonary bypass resuscitation model

BACKGROUND The effect of hyperoxygenation at reperfusion, particularly in the setting of cardiac arrest, remains unclear. This issue was studied in a prolonged cardiac arrest model consisting of 25 min cardiac arrest in a rat resuscitated with cardiopulmonary bypass (CPB). The objective of this study was to determine the effect of hyperoxygenation following prolonged cardiac arrest resuscitation on mitochondrial and cardiac function. METHODS Male Sprague-Dawley rats (400-450 g) were anesthetized with ketamine and xylazine and instrumented for closed chest cardiopulmonary bypass (CPB). Following a 25-min KCl-induced cardiac arrest, the animals were resuscitated by CPB with 100% oxygen. Three minutes after successful return of spontaneous circulation (ROSC), the animals received either normoxemic reperfusion (CPB with 40-50% oxygen) or hyperoxemic reperfusion (CPB with 100% oxygen) for 1 h. Post-resuscitation hemodynamics, cardiac function, mitochondrial function and immunostaining of 3-nitrotyrosine were compared between the two different treatment groups. RESULTS At 1 h after ROSC, the hyperoxemic reperfusion group had a significant higher mean arterial pressure, less metabolic acidosis and better diastolic function than the normoxemic reperfusion group. Cardiac mitochondria from the hyperoxemic reperfusion group had a higher respiratory control ratio (RCR) and cardiac tissue showed less nitroxidative stress compared to the normoxemic reperfusion group. CONCLUSIONS One hour of hyperoxemic reperfusion after 25 min of cardiac arrest in an in vivo CPB model resulted in significant short-term improvement in myocardial and mitochondrial function compared with 1h of normoxemic reperfusion. This myocardial response may differ from previously reported post-arrest hyperoxia mediated effects following shorter arrest times.

Measurement of hydrogen peroxide and oxidant stress in a recirculating whole blood-perfused rat heart model ☆

AIM OF STUDY Isolated hearts used in the study of ischemia-reperfusion induced myocardial reactive oxygen species (ROS) have typically been perfused with crystalloid buffer. Limitations of crystalloid buffer which may exaggerate the production of ROS, include a requirement for higher oxygen tension and the absence of the intrinsic erythrocyte antioxidant defenses. Using a novel recirculating blood-perfused rat heart model, we measured H(2)O(2) concentration in the blood (as an indicator of ROS formation) and tissue glutathione concentration (an overall measure of oxidant stress) following ischemia and reperfusion. METHODS Autologous blood was obtained and the heart isolated from pentobarbital-anesthetized male Sprague-Dawley rats and placed on a recirculating perfusion circuit with an in-line peristaltic pump and oxygenator. Blood temperature was maintained at 37°C. Hearts underwent normal perfusion for 120min (Sham Group, n=7) or 35min of normal perfusion, 25min of global ischemia, followed by 60min of reperfusion with baseline coronary blood flow levels (IR group, n=6). Oxygen delivery was compared with a group of buffer-perfused hearts perfused at 85mmHg. RESULTS LV function in the sham group remained stable for 2h under normal physiologic oxygen conditions. The oxygen tension and coronary flow were significantly decreased but the myocardial oxygen delivery was significantly increased with blood perfusion compared with buffer perfusion. In the blood IR group, a significant increase in H(2)O(2) was seen early in reperfusion and a reduction in tissue GSH was noted at the end of reperfusion. CONCLUSION This model offers significant physiologic advantages in the study of ischemia and reperfusion, particularly in terms of oxygen delivery, compared with the more commonly used acellular buffer-perfused isolated heart systems.

Blood Pressure Lowering and Safety Improvements With Liver Angiotensinogen Inhibition in Models of Hypertension and Kidney Injury

Uncontrolled hypertension is an important contributor to cardiovascular disease. Despite the armamentarium of antihypertensive treatments, there remains a need for novel agents effective in individuals who cannot reach acceptable blood pressure levels. Inhibitors targeting the renin–angiotensin–aldosterone system (RAAS) are widely used but may not optimally inhibit RAAS and demonstrate an acceptable safety profile. Experiments were conducted to characterize a series of AGT (angiotensinogen) antisense oligonucleotides (ASOs) and compare their efficacy and tolerability to traditional RAAS blockade. AGT ASOs which target multiple systemic sites of AGT versus an N-acetylgalactosamine-conjugated AGT ASO that targets the liver were compared with captopril and losartan. Spontaneously hypertensive rats fed an 8% NaCl diet, a model of malignant hypertension resistant to standard RAAS inhibitors, demonstrated robust and durable blood pressure reductions with AGT ASO treatments, which was not observed with standard RAAS blockade. Studies in rat models of acute kidney injury produced by salt deprivation revealed kidney injury with ASO treatment that reduced kidney-expressed AGT, but not in animals treated with the N-acetylgalactosamine AGT ASO despite comparable plasma AGT reductions. Administration of either captopril or losartan also produced acute kidney injury during salt deprivation. Thus, intrarenal RAAS derived from kidney AGT, and inhibited by the standard of care, contributes to the maintenance of renal function during severe RAAS challenge. Such improvements in efficacy and tolerability by a liver-selective AGT inhibitor could be desirable in individuals not at their blood pressure goal with existing RAAS blockade.

Sivelestat attenuates myocardial reperfusion injury during brief low flow postischemic infusion.

The neutrophil elastase inhibitor sivelestat (ONO-5046) possesses unknown mechanisms of cardioprotection when infused following global ischemia, even in the absence of neutrophils. Since myocardial ischemia-reperfusion injury is strongly associated with endothelial dysfunction and reactive oxygen species (ROS) generation during reperfusion, we have tested the hypothesis that infusion of sivelestat during postischemic low flow would preserve endothelial and contractile function and reduce infarct size through an ROS-mediated mechanism. Isolated male rat hearts, subjected to global ischemia of 25 minutes, were reperfused with low flow with or without sivelestat followed by a full flow reperfusion. Hearts treated with sivelestat showed a significant improvement of LV contractile function and a reduction in infarct size. Infusion of L-NAME (nonspecific blocker of endothelial nitric oxide synthase (eNOS)) along with sivelestat during reperfusion reversed the preservation of contractile function and infarct size. In vitro EPR spin trapping experiments showed that sivelestat treatment decreased superoxide adduct formation in bovine aortic endothelial cells (BAECs) subjected to hypoxia-reoxygenation. Similarly, dihydroethidine (DHE) staining showed decreased superoxide production in LV sections from sivelestat-treated hearts. Taken together, these results indicate that sivelestat infusion during postischemic low flow reduces infarct size and preserves vasoreactivity in association with decreased ROS formation and the preservation of nitric oxide.

Simplified method for concentration of mitochondrial membrane protein complexes

Extracting and concentrating mitochondrial protein complexes from gel strips after blue native PAGE (BN‐PAGE) can be daunting tasks using the traditional methods, such as electroelution, passive diffusion and centrifugal concentration. We present a simplified gel electrophoresis method to concentrate mitochondrial protein complexes with excellent recovery rate. Mitochondrial complex I present in a long gel strip from BN‐PAGE can be easily concentrated into a 0.8 cm gel strip when a second BN‐PAGE is performed with a Y‐shaped gel and the addition of 0.01% n‐dodecyl β‐D‐maltoside and 0.001% SDS in the cathode buffer. Once completed, the concentrated protein complex in the gel strip is ready for SDS‐PAGE or proteomic studies.