Abstract
Neuroinflammation initiated by damage-associated molecular patterns, including high mobility group box 1 protein (HMGB1), has been implicated in adverse neurological outcomes following lethal hemorrhagic shock and polytrauma. Emergency preservation and resuscitation (EPR) is a novel method of resuscitation for victims of exsanguinating cardiac arrest, shown in preclinical studies to improve survival with acceptable neurological recovery. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has emerged as a key regulator of metabolic and energy stress response pathways in the brain and a pharmacological target to induce a neuronal pro-survival phenotype. This study aims to examine whether systemic administration of an Annexin-A1 bioactive peptide (ANXA1sp) could resolve neuroinflammation and induce sirtuin-3 regulated cytoprotective pathways in a novel rat model of exsanguinating cardiac arrest and EPR. Adult male rats underwent hemorrhagic shock and ventricular fibrillation, induction of profound hypothermia, followed by resuscitation and rewarming using cardiopulmonary bypass (EPR). Animals randomly received ANXA1sp (3 mg/kg, in divided doses) or vehicle. Neuroinflammation (HMGB1, TNFα, IL-6, and IL-10 levels), cerebral cell death (TUNEL, caspase-3, pro and antiapoptotic protein levels), and neurologic scores were assessed to evaluate the inflammation resolving effects of ANXA1sp following EPR. Furthermore, western blot analysis and immunohistochemistry were used to interrogate the mechanisms involved. Compared to vehicle controls, ANXA1sp effectively reduced expression of cerebral HMGB1, IL-6, and TNFα and increased IL-10 expression, which were associated with improved neurological scores. ANXA1sp reversed EPR-induced increases in expression of proapoptotic protein Bax and reduction in antiapoptotic protein Bcl-2, with a corresponding decrease in cerebral levels of cleaved caspase-3. Furthermore, ANXA1sp induced autophagic flux (increased LC3II and reduced p62 expression) in the brain. Mechanistically, these findings were accompanied by upregulation of the mitochondrial protein deacetylase Sirtuin-3, and its downstream targets FOXO3a and MnSOD in ANXA1sp-treated animals. Our data provide new evidence that engaging pro-resolving pharmacological strategies such as Annexin-A1 biomimetic peptides can effectively attenuate neuroinflammation and enhance the neuroprotective effects of EPR after exsanguinating cardiac arrest.
Highlights
Exsanguinating hemorrhage, leading to cardiac arrest and multiple organ failure, remains the most common cause of death among trauma patients without traumatic brain injury, and neurologic outcomes in survivors are poor
Acute neuroinflammation is characterized by increased cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), HMGB1 and Abbreviations: ANXA1sp, Annexin A1 short peptide; CPB, cardiopulmonary bypass; emergent preservation and resuscitation (EPR), emergency preservation and resuscitation; HMGB1, high mobility group box 1; HS, hemorrhagic shock; LC3-II, microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate; MnSOD, manganese superoxide dismutase; p62/SQSTM1, sequestosome-1; ROS, reactive oxygen species; SIRT3, sirtuin-3
Cells was further confirmed by confocal microscopy, with SIRT3 immunoreactivity colocalizing with the mitochondrial protein COXIV (Figure 6E)
Summary
Exsanguinating hemorrhage, leading to cardiac arrest and multiple organ failure, remains the most common cause of death among trauma patients without traumatic brain injury, and neurologic outcomes in survivors are poor. An new therapeutic paradigm – termed EPR – involves rapidly cooling victims of exsanguinating cardiac arrest to deep or profound hypothermia levels (≤20◦C), in an effort to extend ischemic time, maintain organ viability during severe shock, allow operative repair of injuries and resuscitation, and improve survival and preserve neurological function (Tisherman et al, 2017). Neuroinflammation is a complex immune response commonly observed following acute brain insults such as HS and associated warm ischemia, ischemia-reperfusion injury (I/RI), therapeutic hypothermia, exposure to CPB, and post-cardiac arrest syndrome. Acute neuroinflammation is characterized by increased cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), HMGB1 and Abbreviations: ANXA1sp, Annexin A1 short peptide; CPB, cardiopulmonary bypass; EPR, emergency preservation and resuscitation; HMGB1, high mobility group box 1; HS, hemorrhagic shock; LC3-II, microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate; MnSOD, manganese superoxide dismutase; p62/SQSTM1, sequestosome-1; ROS, reactive oxygen species; SIRT3, sirtuin-3
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