Animals

Timed-pregnant Sprague Dawley rats (Envigo Indianapolis, IN, USA) were delivered on gestational day (GD) 13. Animals were housed in our animal facility with temperature maintained at 22–24 °C, a 12-h light–dark photocycle with light onset at 0700 hours and ad libitum access to food and water. This study was conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee at The Medical University of South Carolina (MUSC).

Opioid exposure

Prenatal opioid exposure was achieved using methadone hydrochloride. Methadone was chosen over morphine due to its longer half-life, which eliminates the needs to replace morphine pellets or re-dose morphine postnatally. Although buprenorphine has been used in animal studies, they are less extensive and have limited pharmacokinetics.26 On GD 15, we prepared a 28-day Model 2ML4 osmotic minipump (Alzet®, Palo Alto, CA, USA) to deliver methadone hydrochloride (9 mg/kg/day) for a steady-state blood level13,27,28 or saline (equivalent volume). It was primed overnight in sterile 0.9% saline at 37 °C, according to the manufacturer’s recommendations. Methadone was generously supplied by the National Institute on Drug Abuse (NIDA, Rockville, MD). After a 72-h acclimation period, the minipump was implanted subcutaneously (s.c.) in GD 16 dams, under isoflurane anesthesia (4–5% for induction; 1–3% for maintenance) and carprofen (5 mg/kg, s.c.) for analgesia. After recovery, the dams were placed back into the colony room where they were assessed daily until delivery on GD 21–23. Within 24 h of parturition, litters were culled to a maximum of 10 offspring and pups were weighed and randomly assigned a study ID and treatment group. Methadone exposure continued via ad libitum breast milk13,27,28 until postnatal days (PNDs) 6–7 when methadone- and saline-exposed pups underwent acute or sham withdrawal, behavioral testing, and MRS.26,29,30,31

Acute precipitated withdrawal

In Experiment 1, to establish the model and validate the behavioral scoring, pups were randomized within litter to receive either naloxone hydrochloride (1 mg/kg, intraperitoneal (i.p.)), a dose that has been shown to precipitate acute withdrawal in rats,26,29,30,31 or an equal volume of saline. Treatment groups in Experiment 1 were Sham (pups with neither methadone exposure nor naloxone), SAL OD (pups with methadone exposure, opioid dependent, and saline but no naloxone), and NAL OD (methadone exposure, opioid dependent, and naloxone).

In Experiment 2, to determine the effects of NAC on behavioral scores and CNS metabolites after acute withdrawal, all methadone-exposed pups received naloxone HCl (1 mg/kg, i.p.) and either NAC (50 mg/kg, n = 2, or 100 mg/kg, n = 7) or an equal volume of saline i.p. Pups were randomized within litter based on maternal exposure to either methadone or saline. Treatment groups in Experiment 2 were SAL/NAL OD (methadone exposed, opioid dependent, naloxone and saline) and NAC/NAL OD (methadone exposed, opioid dependent, naloxone, and NAC). Sham (neither methadone exposed nor naloxone) were used as behavioral controls.

Behavioral testing

Behavioral testing was performed on PND 7. Two pups were injected with saline or naloxone and then moved to separate plastic observation chambers (11 cm × 30 cm × 11 cm) heated with small animal heating pads to maintain body temperature while away from the litter and dam. Chamber temperatures were continuously monitored and regulated during the observation period to keep body temperatures at 36 °C (Supplementary Table S1) at the start and end of the behavior testing. Behaviors were continuously recorded from 0 up to 90 min after naloxone/saline administration. Withdrawal behaviors, adapted from Barr et al.,28,32,33 included paw movement, rolling, stretching, body curls, and mild and severe tremors (Table 1), were observed for 30-s intervals every 5 min, and recorded on a checklist. All of the behaviors (except for mild and severe tremors) were given one point for every one occurrence (i.e., 5 body curls in a 30-s period = score of 5) over the 30-s interval. Mild and severe tremors were tallied for every one occurrence and then summed. A scaled scoring system was then used where 0–2 tallies = score of 1; 3–5 tallies = score of 2; 6–8 tallies = score of 3; and ≥9 tallies = score of 4. For each time period, scores for each behavior were summed to get a total score for that given time period and then summed for the duration of the observation period to get the total summed withdrawal behavior score (withdrawal score). In Experiment 1, behaviors were scored from 0 to 60 min. At 60 min, however, behavior scores were still increasing, indicating peak withdrawal was occurring beyond the 60 min of observation. Therefore, in Experiment 2, behaviors were observed for 90 min, and we determined the peak withdrawal period from 35 to 75 min. Withdrawal scores were summed in Experiment 2 from 35 to 75 min.30,33 Two pups were tested at a time (videotaped individually), and videotapes were scored by a blinded observer.

Table 1 Behaviors of opioid withdrawal. Full size table

Magnetic resonance spectroscopy

In Experiment 2 to establish the effects of opioid withdrawal and NAC on CNS metabolism, we performed MRS scans on PND 6 and 7 prior to precipitated withdrawal (Pre-scans) and on PND 7 following the acute precipitated withdrawal, at 30 and 120 min (Post-scans). Owing to time constraints induced by scheduling of MRS before and after the behavioral observation period, not all pups in a litter could be scanned at both 30 and 120 min. Pups were anesthetized with isoflurane 1–2% mixed with warm compressed air/oxygen for the MRS scans, then placed on a MR compatible, heated animal bed (temperature maintained at 38.0 ± 0.2 °C) connected to an animal monitoring unit (SA Instruments, Inc., Stony Brook, NY), which recorded respirations, pulse oximetry, and core temperature. We used a 7 Tesla (T) BioSpec 70/30 horizontal magnetic resonance imaging (MRI) scanner (Bruker© BioSpin, Ettlingen, Germany). The system is equipped with a 12-cm gradient and shim coil set, capable of generating maximum gradient amplitude of 400 mT/m and 4-channel receiver for multi-coil operation. High-resolution T2-weighted anatomy scans were performed for anatomical positioning of the voxel (Fig. 1a). After reconstruction and voxel placement (3 × 3 × 3 mm3), an MRS water reference was acquired for each animal, followed by STEAM sequence (TR 1500 ms, TE = 3 ms, TI = 10 ms, spatial-water-fat shift = 0.467 mm, spectroscopy number of points = 2048, spectral width = 4006.41 Hz, water suppression scheme = VAPOR, bandwidth 200 Hz, outer volume suppression = ON, OVS slice thickness = 10 mm, gap to voxel = 0 mm, number of averages = 512, scanning time = 12.8 min) in the right hemisphere. At the conclusions of all experiments, animals were euthanized according to the euthanasia protocol at MUSC.

Fig. 1: MRS data processing. a T2 image with anatomical placement of the voxel in the right hemisphere; b phantom solution standard curve; c representative spectra output from LCModel. Full size image

MRS data processing

Phantom solutions (0.5–5 mM GSH, 5 mM DTT, 10 mM Choline, 25 mM Creatine, phosphate-buffered saline, pH 7.1) were used to validate [GSH], [Cr], and [Cho] quantification by MRS, using the water peak as a standard.34 Quantification of GSH and Cr in phantoms by LCModel processing with our specialized basis set showed excellent correlation with known concentrations of these metabolites.34 The phantom solution standard curves for [GSH], [Cho], and [Cr] are shown in Fig. 1b.

Rat pup’s MR spectra were included based on spectral quality, using the following criteria as reported by LCModel (full width at half maximum ≤0.1 ppm, signal-to-noise ratio ≥6).35 Scans with obvious artifacts due to gross motion and poor water suppression were excluded. Using explicit formulas for LCModel, a single water attenuation coefficient was calculated and implemented when processing all spectra. We did not make partial volume corrections in these immature animals with small cerebrospinal fluid spaces and largely uniform brain matter. The following metabolites were analyzed for absolute concentrations in the right hemisphere using a validated basis set for LCModel34,36 that included GSH (Glutathione), GLX (Glutamate+Glutamine), Cr (Phosphocreatine+Creatine), NAA (NAA+N-acetylaspartylglutamate), and Cho (Choline+Glycerophosphocholine). Major peaks for these metabolites are noted on a representative spectrum (Fig. 1c). Inclusion of metabolite concentrations into analysis was based on Cramer–Rao <15% for all metabolites. The range of Cramer–Rao bounds for lactate were outside of inclusion criteria. LCModel fitting of the spectra, evaluation of spectral quality, and quantification of metabolites were performed by a researcher blinded to the treatment group.

Statistical analysis

Statistical analysis was performed using SPSS© Statistics, version 24 (IBM©, Armonk, NY). A two-way analysis of variance (ANOVA) with Tukey post hoc analysis was performed on normally distributed data (Experiment 1). In Experiment 2, non-parametric analyses, Wilcoxon signed-rank test and Kruskal–Wallis H Test, with pairwise post hoc analysis, and Spearman’s correlation were performed for behavioral scores and MRS metabolite concentrations, as data were not normally distributed and of unequal sample size. Significance was set at p < 0.05.