Ventilatory parameters were recorded in freely moving rats by whole body plethysmography (PLY3223; Data Sciences International, St. Paul, MN) as described previously23 (link)–27 (link). The rats were placed in individual chambers and given 60 min to acclimatize to allow true resting ventilatory parameters to be established. Study 1—see Supplemental Table S1 : Two groups of rats received a bolus injection of morphine (10 mg/kg, IV) and after 15 min, one group received an injection of vehicle (saline) whereas the other received an injection of d -cystine diEE (500 μmol/kg, IV) and ventilatory parameters were recorded for a further 75 min. Study 2—see Supplemental Table S3 : Two groups of rats received a bolus injection of morphine (10 mg/kg, IV) and after 15 min, one group received an injection of vehicle (saline) whereas the other received an injection of d -cystine (500 μmol/kg, IV) and ventilatory parameters were recorded for a further 75 min. Study 3—see Supplemental Table S4 : Since Trivedi and Deth7 (link) proposed that administration of N-acetyl- l -cysteine (l -NAC) may help reverse the redox-based epigenetic status of opioid addiction, we thought it appropriate to also determine whether the thiolester, l -N-acetylcysteine methyl ester (l -NACme), which is a highly cell penetrable reducing agent21 (link), would reverse the negative effects of morphine on breathing. Two groups of rats received a bolus injection of morphine (10 mg/kg, IV) and after 15 min, one group received an injection of vehicle (saline) whereas the other received an injection l -NACme (500 μmol/kg, IV)21 (link). The rats received another injection of vehicle or l -NACme (500 μmol/kg, IV) 15 min later and ventilatory parameters were recorded for a further 60 min.
Due to the closeness of the body weights of all of the groups of rats, ventilatory data are shown without any corrections for body weight. The provided software (Fine Pointe, BUXCO) constantly corrected digitized values for changes in chamber temperature and humidity. Pressure changes associated with the respiratory waveforms were then converted to volumes (i.e., TV, PIF and PEF) using the algorithm of Epstein and colleagues28 (link)–30 (link). Specifically, factoring in chamber temperature and humidity, the cycle analyzers filtered the acquired signals, and BUXCO algorithms (Fine Pointe) generated an array of box flow data that identified a waveform segment as an acceptable breath. From that data vector, the minimum and maximum values were determined. Flows at this point were considered to be “box flow” signals. From this array, the minimum and maximum box flow values were determined and multiplied by a compensation factor provided by the selected algorithm50 (link),51 (link), thus producing TV, PIF and PEF values that were used to determine accepted and rejected waveforms, with rejected waveforms remaining below 5% throughout all phases of the protocols except for a transient rise in rejection of breaths to 15–20% for 1–2 min after injection of morphine (data not shown).
Due to the closeness of the body weights of all of the groups of rats, ventilatory data are shown without any corrections for body weight. The provided software (Fine Pointe, BUXCO) constantly corrected digitized values for changes in chamber temperature and humidity. Pressure changes associated with the respiratory waveforms were then converted to volumes (i.e., TV, PIF and PEF) using the algorithm of Epstein and colleagues28 (link)–30 (link). Specifically, factoring in chamber temperature and humidity, the cycle analyzers filtered the acquired signals, and BUXCO algorithms (Fine Pointe) generated an array of box flow data that identified a waveform segment as an acceptable breath. From that data vector, the minimum and maximum values were determined. Flows at this point were considered to be “box flow” signals. From this array, the minimum and maximum box flow values were determined and multiplied by a compensation factor provided by the selected algorithm50 (link),51 (link), thus producing TV, PIF and PEF values that were used to determine accepted and rejected waveforms, with rejected waveforms remaining below 5% throughout all phases of the protocols except for a transient rise in rejection of breaths to 15–20% for 1–2 min after injection of morphine (data not shown).
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