Ischemic brain injury inflicted by stroke and cardiac arrest ranks among the leading causes of death and long-term disability in the United States. The brain consumes large amounts of metabolic substrates and oxygen to sustain its energy requirements. Consequently, the brain is exquisitely sensitive to interruptions in its blood supply, and suffers irreversible damage after 10–15 minutes of severe ischemia. Effective treatments to protect the brain from stroke and cardiac arrest have proven elusive, due to the complexities of the injury cascades ignited by ischemia and reperfusion. Although recombinant tissue plasminogen activator and therapeutic hypothermia have proven efficacious for stroke and cardiac arrest, respectively, these treatments are constrained by narrow therapeutic windows, potentially detrimental side effects and the limited availability of hypothermia equipment. Mounting evidence demonstrates the cytokine hormone erythropoietin (EPO) to be a powerful neuroprotective agent and a potential adjuvant to established therapies. Classically, EPO originating primarily in the kidneys promotes erythrocyte production by suppressing apoptosis of proerythroid progenitors in bone marrow. However, the brain is capable of producing EPO, and EPO’s membrane receptors and signaling components also are expressed in neurons and astrocytes. EPO activates signaling cascades that increase the brain’s resistance to ischemia-reperfusion stress by stabilizing mitochondrial membranes, limiting formation of reactive oxygen and nitrogen intermediates, and suppressing pro-inflammatory cytokine production and neutrophil infiltration. Collectively, these mechanisms preserve functional brain tissue and, thus, improve neurocognitive recovery from brain ischemia. This article reviews the mechanisms mediating EPO-induced brain protection, critiques the clinical utility of exogenous EPO to preserve brain threatened by ischemic stroke and cardiac arrest, and discusses the prospects for induction of EPO production within the brain by the intermediary metabolite, pyruvate.

Cardiac arrest remains a leading cause of death and permanent disability worldwide. Although many victims are initially resuscitated, they often succumb to the extensive ischemia-reperfusion injury inflicted on the internal organs, especially the brain. Cardiac arrest initiates a complex cellular injury cascade encompassing reactive oxygen and nitrogen species, Ca(2+) overload, ATP depletion, pro- and anti-apoptotic proteins, mitochondrial dysfunction, and neuronal glutamate excitotoxity, which injures and kills cells, compromises function of internal organs and ignites a destructive systemic inflammatory response. The sheer complexity and scope of this cascade challenges the development of experimental models of and effective treatments for cardiac arrest. Many experimental animal preparations have been developed to decipher the mechanisms of damage to vital internal organs following cardiac arrest and cardiopulmonary resuscitation (CPR), and to develop treatments to interrupt the lethal injury cascades. Porcine models of cardiac arrest and resuscitation offer several important advantages over other species, and outcomes in this large animal are readily translated to the clinical setting. This review summarizes porcine cardiac arrest-CPR models reported in the literature, describes clinically relevant phenomena observed during cardiac arrest and resuscitation in pigs, and discusses numerous methodological considerations in modeling cardiac arrest/CPR. Collectively, published reports show the domestic pig to be a suitable large animal model of cardiac arrest which is responsive to CPR, defibrillatory countershocks and medications, and yields extensive information to foster advances in clinical treatment of cardiac arrest.

Recent studies have demonstrated a pivotal role of protein glycation in brain injury. Methylglyoxal, a by-product of glycolysis and a powerful glycating agent in brain, is detoxified by the glutathione-catalyzed glyoxalase (GLO) system, but the impact of cardiac arrest (CA) and cardiocerebral resuscitation (CCR) on the brain's antiglycation defenses is unknown. This study in a swine model of CA and CCR demonstrated for the first time that the intense cerebral ischemia-reperfusion imposed by CA-resuscitation disabled glyoxalase-1 and glutathione reductase (GR), the source of glutathione for methylglyoxal detoxification. Moreover, intravenous administration of pyruvate, a redox-active intermediary metabolite and antioxidant in brain, prevented inactivation of glyoxalase-1 and GR and blunted protein glycation in cerebral cortex. These findings in a large mammal are first evidence of GLO inactivation and the resultant cerebral protein glycation after CA-resuscitation, and identify novel actions of pyruvate to minimize protein glycation in postischemic brain. AbstractCardiac arrest (CA) and cardiocerebral resuscitation (CCR)-induced ischemia-reperfusion imposes oxidative and carbonyl stress that injures the brain. The ischemic shift to anaerobic glycolysis, combined with oxyradical inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), provokes excessive formation of the powerful glycating agent, methylglyoxal. The glyoxalase (GLO) system, comprising the enzymes glyoxalase 1 (GLO1) and GLO2, utilizes reduced glutathione (GSH) supplied by glutathione reductase (GR) to detoxify methylglyoxal resulting in reduced protein glycation. Pyruvate, a natural antioxidant that augments GSH redox status, could sustain the GLO system in the face of ischemia-reperfusion. This study assessed the impact of CA-CCR on the cerebral GLO system and pyruvate's ability to preserve this neuroprotective system following CA. Domestic swine were subjected to 10 min CA, 4 min closed-chest CCR, defibrillation and 4 h recovery, or to a non-CA sham protocol. Sodium pyruvate or NaCl control was infused (0.1 mmol/kg/min, intravenous) throughout CCR and the first 60 min recovery. Protein glycation, GLO1 content, and activities of GLO1, GR, and GAPDH were analyzed in frontal cortex biopsied at 4 h recovery. CA-CCR produced marked protein glycation which was attenuated by pyruvate treatment. GLO1, GR, and GAPDH activities fell by 86, 55, and 30%, respectively, after CA-CCR with NaCl infusion. Pyruvate prevented inactivation of all three enzymes. CA-CCR sharply lowered GLO1 monomer content with commensurate formation of higher molecular weight immunoreactivity; pyruvate preserved GLO1 monomers. Thus, ischemia-reperfusion imposed by CA-CCR disabled the brain's antiglycation defenses. Pyruvate preserved these enzyme systems that protect the brain from glycation stress.

Koneru B, Bathina CS, Cherry BH, Mifflin SW. Mineralocorticoid receptor in the NTS stimulates saline intake during fourth ventricular infusions of aldosterone. Am J Physiol Regul Integr Comp Physiol 306: R61-R66, 2014. First published November 20, 2013 doi:10.1152/ajpregu.00434.2013.-The purpose of this study was to determine whether neurons within the nucleus tractus solitarius (NTS) that express the mineralocorticoid receptor (MR) play a role in aldosterone stimulation of salt intake. Adult Wistar-Kyoto (WKY) rats received microinjections into the NTS of a short-hairpin RNA (shRNA) for the MR, to site specifically reduce levels of the MR by RNA interference (shRNA; n ϭ 9) or scrambled RNA as a control (scRNA; n ϭ 8). After injection of the viral construct, aldosteronefilled osmotic minipumps were implanted subcutaneously and connected to a cannula extending into the fourth ventricle to infuse aldosterone at a rate of 25 ng/h. Before and after surgeries, rats had ad libitum access to normal sodium (0.26%) rat chow and two graduated drinking bottles filled with either distilled water or 0.3 M NaCl. Before the surgeries, basal saline intake was 1.6 Ϯ 0.6 ml in the scRNA group and 1.56 Ϯ 0.6 ml in the shRNA group. Twenty-four days postsurgery, saline intake was elevated to a greater extent in the scRNA group (5.9 Ϯ 1.07 ml) than in the shRNA group (2.41 Ϯ 0.6 ml). Post mortem immunohistochemistry revealed a significant reduction in the number of NTS neurons exhibiting immunoreactivity for MR in shRNA-injected rats (23 Ϯ 1 cells/section) versus scRNAinjected rats (33 Ϯ 2 cells/section; P ϭ 0.008). shRNA did not alter the level of 11-␤-hydroxysteroid dehydrogenase type II (HSD2) protein in the NTS as judged by the number of HSD2 immunoreactive neurons. These results suggest that fourth ventricular infusions of aldosterone stimulate saline intake, and that this stimulation is at least in part mediated by hindbrain NTS neurons that express MR.

Cardiac electromechanical dysfunction may compromise recovery of patients who are initially resuscitated from cardiac arrest, and effective treatments remain elusive. Pyruvate, a natural intermediary metabolite, energy substrate, and antioxidant, has been found to protect the heart from ischemia-reperfusion injury. This study tested the hypothesis that pyruvate-enriched resuscitation restores hemodynamic, metabolic, and electrolyte homeostasis following cardiac arrest. Forty-two Yorkshire swine underwent pacing-induced ventricular fibrillation and, after 6 min pre-intervention arrest, 4 min precordial compressions followed by transthoracic countershocks. After defibrillation and recovery of spontaneous circulation, the pigs were monitored for another 4 h. Sodium pyruvate or NaCl were infused i.v. (0.1 mmolÁkg À1 Ámin À1 ) throughout precordial compressions and the first 60 min recovery. In 8 of the 24 NaCl-infused swine, the first countershock converted ventricular fibrillation to pulseless electrical activity unresponsive to subsequent countershocks, but only 1 of 18 pyruvate-treated swine developed pulseless electrical activity (relative risk 0.17; 95% confidence interval 0.13-0.22). Pyruvate treatment also lowered the dosage of vasoconstrictor phenylephrine required to maintain systemic arterial pressure at 15-60 min recovery, hastened clearance of excess glucose, elevated arterial bicarbonate, and raised arterial pH; these statistically significant effects persisted up to 3 h after sodium pyruvate infusion, while infusion-induced hypernatremia subsided. These results demonstrate that pyruvate-enriched resuscitation achieves electrocardiographic and hemodynamic stability in swine during the initial recovery from cardiac arrest. Such metabolically based treatment may offer an effective strategy to support cardiac electromechanical recovery immediately after cardiac arrest.

Cardiac arrest is a leading cause of death and permanent disability. Most victims succumb to the oxidative and inflammatory damage sustained during cardiac arrest/resuscitation, but even survivors typically battle long-term neurocognitive impairment. Although extensive research has delineated the complex mechanisms that culminate in neuronal damage and death, no effective treatments have been developed to interrupt these mechanisms. Of importance, many of these injury cascades are also active in the aging brain, where neurons and other cells are under persistent oxidative and inflammatory stress which eventually damages or kills the cells. In light of these similarities, it is reasonable to propose that the brain essentially ages the equivalent of several years within the few minutes taken to resuscitate a patient from cardiac arrest. Accordingly, cardiac arrest-resuscitation models may afford an opportunity to study the deleterious mechanisms underlying the aging process, on an accelerated time course. The aging and resuscitation fields both stand to gain pivotal insights from one another regarding the mechanisms of injury sustained during resuscitation from cardiac arrest and during aging. This synergism between the two fields could be harnessed to foster development of treatments to not only save lives but also to enhance the quality of life for the elderly.

The purpose of this study was to determine if neurons within the NTS that possess the mineralocorticoid receptor (MR) play a role in aldosterone stimulation of salt intake. Adult WKY rats received microinjections into the NTS of a small, hairpin RNA for the MR (shRNA; n=9) or a scrambled RNA (scRNA; n=8). After the viral construct injections, aldosterone‐filled osmotic mini‐pumps were implanted subcutaneously and connected to tubing within the 4th ventricle to infuse aldosterone at a rate of 20ng/h. Prior to and after surgeries, rats had ad libitum access to food and two graduated drinking bottles filled with distilled water and 0.3M NaCl and salt intake expressed as 100 × the ratio of 0.3M NaCl intake to total fluid intake. Prior to surgeries, basal salt intake was 4.7% ± 2.9% in the scRNA group and 3.91% ± 2.6% in the shRNA group. 24 days post‐surgery salt intake was 20.6% ± 2.9% in the scRNA group and 4.3% ± 2.8% in the shRNA group. Post‐mortem immunohistochemistry revealed a significant reduction in the number of NTS neurons exhibiting immunoreactivity for the MR (scRNA 33 ± 2 cells/section; shRNA 23 ± 1 cells/section; p=.008). These results indicate 4th ventricular infusions of aldosterone stimulate salt intake and that at least part of the stimulation of salt intake during 4th ventricular infusions of aldosterone is mediated by hindbrain NTS neurons that possess the MR.

Cardiac arrest, a leading cause of death in the U.S., kills >90% of its victims, and survivors often are disabled by permanent brain injury inflicted by ischemia‐reperfusion. Purkinje cells of the cerebellum and CA1 neurons of the hippocampus are especially vulnerable to post‐ischemic neuronal death. We tested the hypothesis that cardiac arrest in a swine model caused delayed neuronal death. Yorkshire swine (25‐35 kg) were subjected to cardiac arrest‐resuscitation (n = 9) or non‐arrest sham (n = 5) protocols. Ventricular fibrillation was induced by electrical pacing. Precordial compressions (100/min) were given at 6‐10 min arrest, and then sinus rhythm was restored with transthoracic countershocks. NaCl was infused iv at 0.1 mmol/kg/min during CPR and the first 60 min after return of spontaneous circulation (ROSC). At 7 d ROSC, brain regions were fixed in 4% paraformaldehyde and H&E stained. More than 70% of the Purkinje cells were shrunken, lacked dendrites and displayed condensed cytoplasm at 7 d ROSC; in contrast, in shams the majority of Purkinje cells retained the characteristic thick dendrites and well‐defined nuclei. Thus, cardiac arrest‐resuscitation produced marked changes in cerebellar neurons evident 7d after acute insult. Grant Funding Source: NINDS 076975