PROJECT SUMMARY Acute exposure to ultrapotent synthetic opioids (UPSO), such as fentanyl, represents a significant public health concern. In the ongoing opioid epidemic, it is estimated that UPSO exposure contributes to over 80% of overdose-related deaths. The current defense strategy against UPSO exposure has been the development of reversal agents, such as naloxone, that aim to effectively reverse opioid-induced respiratory depression and related secondary complications with breathing. However, the high potency of UPSO decreases the window of time in which a counteragent can be administered before UPSO-dependent cardiorespiratory collapse (CRC) occurs and immediate resuscitative action is required. Optimal strategies for resuscitation following UPSO- dependent CRC are unknown. Resuscitation increases the risk for hypoxic-ischemic reperfusion injury (HIRI), which can lead to additional morbidity and death despite reversal of the opioid-mediated effects on breathing. Critical knowledge gaps exist in understanding how UPSO exposure impacts post-resuscitative outcomes in vital organ systems such as the heart, lungs, and brain. These gaps are a significant roadblock to successfully minimizing the morbidities and mortalities associated with UPSO exposure. Proposal Objective: Establish a foundational understanding of the cellular and systemic outcomes after reversing fentanyl-induced CRC (fiCRC). We have developed a novel model of fiCRC where we observe pulmonary edema following naloxone administration and reversal of respiratory depression, closely modeling documented clinical observations. Central Hypothesis: Factors beyond respiratory depression contribute to the progression of fentanyl overdose, leading to injury following the reversal of fiCRC; these factors are tractable targets in minimizing injury due to fentanyl overdose and its reversal. We propose the following aims to test this. Aim 1: Characterize how fentanyl and the reversal of OIRD impact the relationship between breathing and O2 consumption, mitochondrial activity, and tissue-specific glucose metabolism. Aim 2: Characterize physiological outcomes of fentanyl overdose and reversal of fiCRC in the cardiopulmonary system and brain. Aim 3: Test the efficacy of adjunctive strategies during naloxone-mediated reversal of fiCRC to improve outcomes in the cardiopulmonary system and brain. This proposal and its aims align with RFA-DA-23-056 to support mechanistic investigations into persistent/delayed pathophysiological effects following acute UPSO exposure.