Administering CO2 Anesthesia to Male Sprague‐Dawley Rats Prior to Euthanasia for Postmortem Brain Slicing Alters Cardiorespiration (In Vivo) and O2‐/CO2‐sensitive Redox Signaling in Dorsocaudal Medullary Neurons in Brain Slices
Authors: Ciarlone Geoffrey E. , Landon Carol S. , Dean Jay B. 
Published: November 2023

Inducing CO2 anesthesia during euthanasia is anticipated to produce CO2 narcosis in parallel with escalating levels of cellular hypercapnia, acidosis, and hypoxia during the 3–5min preceding euthanasia. The goal of this study was to identify cardiorespiratory responses activated in CO2 anesthetized rats used to harvest brain slices for studies of redox and nitrosative signaling in neurons in a cardiorespiratory control center (caudal solitary complex, cSC). We hypothesized that medullary tissue slices harvested from CO2 anesthetized rats would exhibit differences in production of reactive O2 and N2 species (RONS) during normocapnic hyperoxia and hypercapnic hyperoxia as compared to brain slices harvested without CO2 anesthesia prior to euthanasia. In vivo: Telemetric measurements of cortical EEG, respiratory muscle EMG, ECG, and body temperature (TB) were made in adult male rats implanted with a DSI 4‐ET radio‐transmitter. Gradual replacement of air with CO2 (0.03–100%) over ~3–5min caused sequential changes in breathing pattern that correlated with changes in the cortical EEG. Onset of CO2 breathing increased ventilation without measureable changes in EEG, decreased heart rate (HR), and induced exploring behavior. At ~20%CO2 respiratory frequency (fRESP) decreased while tidal volume (VT) was unchanged or increased further. HR either remained unchanged or decreased further. The EEG decreased at ~25–30%CO2 and exploring behavior ceased with no further movements other than occasional blinking. At ~45%CO2 inspiratory duration increased for several breaths with visible gasping efforts while HR increased transiently followed by gradual slowing. At ~60%CO2 there was a transient burst in the EEG lasting ~1.4min during which fRESP increased but the rat remained prone and inactive. Cortical activity completely ceased following the EEG burst and breathing slowly decreased until the last breath ~1min later at ~95%CO2. HR continued on average for another 5.3min. TB decreased on average by 0.3°C as CO2 increased from 0.03 to 100%. In vitro: Transverse brain slices (400μm, 34–36°C) harvested from CO2 anesthetized rats exhibited increased superoxide production (DHE) during normocapnic hyperoxia (40% to 95%O2) compared to slices harvested without using CO2 anesthesia. CO2 anesthesia also decreased the stimulatory effects of hypercapnic acidosis (5% to 10%CO2) on aggregate RONS production (DHR123) during hyperoxia in brain slices. By contrast, nitric oxide production (DAF‐FM DA) during normocapnic hyperoxia was unchanged in brain slices harvested from rats ±CO2 anesthesia. We postulate that CO2 anesthesia activates multiple respiratory patterns and affects HR leading to long‐lasting changes in redox signaling mechanisms in cSC neurons studied under in vitro conditions. Specifically, we hypothesize that CO2 anesthesia decreases superoxide dismutase activity in brain slices causing increased superoxide accumulation while also shifting downstream RONS production during hypercapnic hyperoxia from the H+‐Fenton reaction to the CO2‐peroxynitrite reaction.Support or Funding InformationONR Undersea Medicine Program