Bilateral carotid sinus nerve transection exacerbates morphine-induced respiratory depression

Abstract

Opioid-induced respiratory depression (OIRD) involves decreased sensitivity of ventilatory control systems to decreased blood levels of oxygen (hypoxia) and elevated levels of carbon dioxide (hypercapnia). Understanding the sites and mechanisms by which opioids elicit respiratory depression is pivotal for finding novel therapeutics to prevent and/or reverse OIRD. To examine the contribution of carotid body chemoreceptors OIRD, we used whole-body plethysmography to evaluate hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses including changes in frequency of breathing, tidal volume, minute ventilation and inspiratory drive, after intravenous injection of morphine (10 mg/kg) in sham-operated (SHAM) and in bilateral carotid sinus nerve transected (CSNX) Sprague-Dawley rats. In SHAM rats, morphine produced sustained respiratory depression (e.g., decreases in tidal volume, minute ventilation and inspiratory drive) and reduced the HVR and HCVR responses. Unexpectedly, morphine-induced suppression of HVR and HCVR were substantially greater in CSNX rats than in SHAM rats. This suggests that morphine did not compromise the function of the carotid body-chemoafferent complex and indeed, that the carotid body acts to defend against morphine-induced respiratory depression. These data are the first in vivo evidence that carotid body chemoreceptor afferents defend against rather than participate in OIRD in conscious rats. As such, drugs that stimulate ventilation by targeting primary glomus cells and/or chemoafferent terminals in the carotid bodies may help to alleviate OIRD.

Keywords: Carotid body; Chemoafferents; Conscious rats; Morphine; Respiratory depression.

Fig. 1.

Schematic of Whole-body plethysmograph setup. Sham-operated (SHAM) and bilateral carotid sinus nerve transected (CNSX) rats were gradually acclimatized to the whole-body plethysmography chamber with a bias chamber air flow. A bias chamber air flow of at least 2 L/min was generated by connecting the chambers to a constant flow vacuum source. However, during normoxia, hypoxia and hypercapnia protocols, a bias flow of room air (PO₂ of 21%), hypoxic (PO₂ of 10%) and hypercapnic (PCO₂ of 5%) gases were generated using a gas mixture. Gas mixture receives inflow from 100% N₂, 100% O₂ and 10% CO₂ cylinders. Gas flow to each plethysmograph chamber (2 L/min) was controlled using flow meters. A respiratory waveform was generated from the expansion and contraction of the air that was exchanged between the rat and the chamber. The cyclic change in air volume during the respiratory cycle elicited oscillating airflow across a calibrated pneumotach in the wall of the plethysmograph chamber. Each pneumotach was calibrated (5.0 mL volume delivered in triplicate) on each study day prior to placing the rats in the chambers. At least 1 hour was permitted for rats to acclimate to the chamber before data collection began. Respiratory waveforms were amplified with Buxco-Amplifiers and captured continuously and stored using FinePointe software later data analysis. Barometric pressure, chamber temperature, chamber partial pressure of water, and body temperature were used to calculate a corrected tidal volume. The first three variables were assumed to be constant throughout the study at 740 mmHg, 25 °C, and 23.7 mmHg, respectively. Minute ventilation was calculated as the product of tidal volume and respiratory frequency.

Fig. 2.

Changes in frequency of breathing in conscious sham-operated rats (SHAM) and in rats with bilateral carotid sinus nerve transection (CSNX) following bolus injection of morphine (10 mg/kg. i.v.), and then exposure to either hypoxic challenge (10% O₂, 90% N₂) for 20 min (top panel) or hypercapnic challenge (5% O₂, 21% O₂, 74% N₂) for 20 min (middle panel) and then re-exposure to room-air. The maximal and total changes in Frequency of breathing (Freq) are shown in the bottom left and right panels, respectively. The data are presented as mean ± SEM. There were 6 rats in each group.

Fig. 3.

Changes in inspiratory time in conscious sham-operated rats (SHAM) and in rats with bilateral carotid sinus nerve transection (CSNX) following bolus injection of morphine (10 mg/kg. i.v.), and then exposure to hypoxic challenge (10% O₂, 90% N₂) for 20 min (top panel) or hypercapnic challenge (5% O₂, 21% O₂, 74% N₂) for 20 min (middle panel) and then re-exposure to room-air. The maximal and total changes in inspiratory time (Ti) are shown in the bottom left and right panels, respectively. The data are presented as mean ± SEM. There were 6 rats in each group.

Fig. 4.

Changes in expiratory time in conscious sham-operated rats (SHAM) and in rats with bilateral carotid sinus nerve transection (CSNX) following bolus injection of morphine (10 mg/kg. i.v.), and then exposure to hypoxic challenge (10% O₂, 90% N₂) for 20 min (top panel) or hypercapnic challenge (5% O₂, 21% O₂, 74% N₂) for 20 min (middle panel) and then re-exposure to room-air. The maximal and total changes in expiratory time (Te) are shown in the bottom left and right panels, respectively. The data are presented as mean ± SEM. There were 6 rats in each group.

Fig. 5.

Changes in tidal volume in conscious sham-operated rats (SHAM) and in rats with bilateral carotid sinus nerve transection (CSNX) following bolus injection of morphine (10 mg/kg. i.v.), and then exposure to hypoxic challenge (10% O₂, 90% N₂) for 20 min (top panel) or hypercapnic challenge (5% O₂, 21% O₂, 74% N₂) for 20 min (middle panel) and then re-exposure to room-air. The maximal and total changes in tidal volume (Vt) are shown in the bottom left and right panels, respectively. The data are presented as mean ± SEM. There were 6 rats in each group.

Fig. 6.

Changes in minute ventilation in conscious sham-operated rats (SHAM) and in rats with bilateral carotid sinus nerve transection (CSNX) following bolus injection of morphine (10 mg/kg. i.v.), and then exposure to hypoxic challenge (10% O₂, 90% N₂) for 20 min (top panel) or hypercapnic challenge (5% O₂, 21% O₂, 74% N₂) for 20 min (bottom panel) and then re-exposure to room-air. Maximal and total changes in minute ventilation (Ve) are shown in the bottom left and right panels, respectively. The data are presented as mean ± SEM. There were 6 rats in each group.

Fig. 7.

Changes in inspiratory drive (tidal volume/inspiratory time, Vt/Ti) in conscious sham-operated rats (SHAM) and in rats with bilateral carotid sinus nerve transection (CSNX) following bolus injection of morphine (10 mg/kg. i.v.), and then exposure to hypoxic challenge (10% O₂, 90% N₂) for 20 min (top panel) or hypercapnic challenge (5% O₂, 21% O₂, 74% N₂) for 20 min (middle panel) and then re-exposure to room-air. Maximal and total changes in tidal volume/inspiratory time (Vt/Ti) are shown in the bottom left and right panels, respectively. The data are presented as mean ± SEM. There were 6 rats in each group.

Fig. 8.

Total changes in frequency of breathing (fR), tidal volume (Vt), minute ventilation (Ve), inspiratory time (TI), expiratory time (Te) and tidal volume/inspiratory time (Vt/ Ti) upon return to room-air following exposure to either hypoxic (10% O₂, 90% N₂) challenge (top panel) or hypercapnic (5% O₂, 21% O₂, 74% N₂) challenge (bottom panel) in morphine-treated sham-operated rats (SHAM) and in morphine-treated rats with bilateral carotid sinus nerve transection (CSNX) * P < 0.05, significant change from pre-hypoxia levels. ^†P< 0.05, CSNX versus SHAM.