Administering CO <sub>2</sub> Anesthesia to Male Sprague‐Dawley Rats Prior to Euthanasia for Postmortem Brain Slicing Alters Cardiorespiration (In Vivo) and O <sub>2</sub> ‐/CO <sub>2</sub> ‐sensitive Redox Signaling in Dorsocaudal Medullary Neurons in Brain Slices

Inducing CO 2 anesthesia during euthanasia is anticipated to produce CO 2 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 CO 2 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 CO 2 anesthetized rats would exhibit differences in production of reactive O 2 and N 2 species (RONS) during normocapnic hyperoxia and hypercapnic hyperoxia as compared to brain slices harvested without CO 2 anesthesia prior to euthanasia. In vivo : Telemetric measurements of cortical EEG, respiratory muscle EMG, ECG, and body temperature (T B ) were made in adult male rats implanted with a DSI 4‐ET radio‐transmitter. Gradual replacement of air with CO 2 (0.03–100%) over ~3–5min caused sequential changes in breathing pattern that correlated with changes in the cortical EEG. Onset of CO 2 breathing increased ventilation without measureable changes in EEG, decreased heart rate (HR), and induced exploring behavior. At ~20%CO 2 respiratory frequency (f RESP ) decreased while tidal volume (V T ) was unchanged or increased further. HR either remained unchanged or decreased further. The EEG decreased at ~25–30%CO 2 and exploring behavior ceased with no further movements other than occasional blinking. At ~45%CO 2 inspiratory duration increased for several breaths with visible gasping efforts while HR increased transiently followed by gradual slowing. At ~60%CO 2 there was a transient burst in the EEG lasting ~1.4min during which f RESP 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%CO 2 . HR continued on average for another 5.3min. T B decreased on average by 0.3°C as CO 2 increased from 0.03 to 100%. In vitro : Transverse brain slices (400μm, 34–36°C) harvested from CO 2 anesthetized rats exhibited increased superoxide production (DHE) during normocapnic hyperoxia (40% to 95%O 2 ) compared to slices harvested without using CO 2 anesthesia. CO 2 anesthesia also decreased the stimulatory effects of hypercapnic acidosis (5% to 10%CO 2 ) 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 ±CO 2 anesthesia. We postulate that CO 2 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 CO 2 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 CO 2 ‐peroxynitrite reaction. Support or Funding Information ONR Undersea Medicine Program

Topic: Neuroscience of respiration and sleep
Subfield: Endocrine and Autonomic Systems
Field: Neuroscience
Domain: Life Sciences