Characterization of cannabinoid plasma concentration, maternal health, and cytokine levels in a rat model of prenatal Cannabis smoke exposure

Abstract

Cannabis sativa has gained popularity as a "natural substance", leading many to falsely assume that it is not harmful. This assumption has been documented amongst pregnant mothers, many of whom consider Cannabis use during pregnancy as benign. The purpose of this study was to validate a Cannabis smoke exposure model in pregnant rats by determining the plasma levels of cannabinoids and associated metabolites in the dams after exposure to either Cannabis smoke or injected cannabinoids. Maternal and fetal cytokine and chemokine profiles were also assessed after exposure. Pregnant Sprague-Dawley rats were treated daily from gestational day 6-20 with either room air, i.p. vehicle, inhaled high-Δ⁹-tetrahydrocannabinol (THC) (18% THC, 0.1% cannabidiol [CBD]) smoke, inhaled high-CBD (0.7% THC, 13% CBD) smoke, 3 mg/kg i.p. THC, or 10 mg/kg i.p. CBD. Our data reveal that THC and CBD, but not their metabolites, accumulate in maternal plasma after repeated exposures. Injection of THC or CBD was associated with fewer offspring and increased uterine reabsorption events. For cytokines and chemokines, injection of THC or CBD up-regulated several pro-inflammatory cytokines compared to control or high-THC smoke or high-CBD smoke in placental and fetal brain tissue, whereas smoke exposure was generally associated with reduced cytokine and chemokine concentrations in placental and fetal brain tissue compared to controls. These results support existing, but limited, knowledge on how different routes of administration contribute to inconsistent manifestations of cannabinoid-mediated effects on pregnancy. Smoked Cannabis is still the most common means of human consumption, and more preclinical investigation is needed to determine the effects of smoke inhalation on developmental and behavioural trajectories.

Figure 1

Experimental timeline for gestational Cannabis exposure. Rats were bred and treated once daily between gestational day (GD) 6 and 20 for 15 consecutive days of treatment. Maternal and fetal tissues were harvested 30 min following the first (GD6) and last (GD20) treatment. THC, CBD, and their metabolites were quantified in maternal plasma using high performance liquid chromatography tandem mass spectroscopy (HPLC–MS/MS). Protein samples from placenta and whole fetal brain were prepared for cytokine and chemokine quantification. Created with Biorender.com.

Figure 2

Comparison of THC, CBD, and metabolite levels in maternal plasma according to treatment between first exposure on GD6 and last exposure on GD20. Plasma levels of phytocannabinoids were quantified on GD6 and GD20 30 min after cannabinoid exposure in the following treatment groups: (A) 3 mg/kg/day THC i.p., (B) 10 mg/kg/day CBD i.p., (C) High-THC smoke, and (D) High-CBD smoke. Note the different scaling of the y-axes of the panels. Data are mean ± S.E.M. n = 5–8 dams/per treatment. Values falling below LLOQ were deemed not detectable (n.d.).

Figure 3

Comparison of THC, CBD, and metabolite levels among treatments in maternal plasma on GD20 after repeated exposure to either 3 mg/kg/day THC i.p., high THC smoke, 10 mg/kg/day CBD i.p., or high CBD smoke. Mean maternal plasma concentrations of (A) THC, (B) 11-OH-THC, (C) 11-COOH-THC, (D) CBD, and (E) 7-OH-CBD. All samples were collected on GD20, 30 min following final treatment. Note the different scaling of the y-axes of the panels. Data are mean ± S.E.M. n = 5–8 dams/treatment; *p < 0.05, ** p < 0.01 as determined by Kruskal–Wallis (KW) one-way ANOVA or Kolmogorov–Smirnov (KS) test.

Figure 4

Markers of maternal and litter health during gestation. (A) Rates of maternal weight gain, (B) uterine resorption discovered, (C) litter size (D), mean fetal body weight per litter, (E) mean fetal brain to body weight ratio per litter, and (F) mean fetal bodyweight to placenta ratio per litter, and (G) Ratio of male to female offspring per litter. Data are mean ± S.E.M, n = 4–8 dams/per treatment. Measurements in panels B-G were taken on GD20. *p < 0.05, as determined by one-way ANOVA followed by Holm-Šídák post-hoc analysis.

Figure 5

Placental cytokine and chemokine levels 30 min after final treatment on GD20. Cytokines and chemokines are organized by type: species within the red squircle are broadly considered pro-inflammatory, species within the blue squircle are broadly considered anti-inflammatory. Because the role of IL-4, IL-6 and IL-10 can be pro- or anti-inflammatory depending on context, these species are within both squircles. Data are displayed as mean ± S.E.M, n = 6–8 dams/group. An average of two replicates was used when possible. Note the y-axis varies in each panel depending on the cytokine measured. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as indicated between groups.

Figure 6

Fetal brain cytokine and chemokine concentrations 30 min after final treatment on GD20. Cytokines and chemokines are organized by type: species within the red squircle are broadly considered pro-inflammatory, species within the blue squircle are broadly considered anti-inflammatory. Because the role of IL-2, IL-4, IL-6, IL-10 and VEGF can be pro- or anti-inflammatory depending on context, these species are within both squircles. Data are displayed as mean ± S.E.M, n = 12–15 samples/group. Closed circles are data points from males. Open circles are data points from females. An average of two replicates was used when possible. Note the y-axis varies in each panel depending on the cytokine measured. *p < 0.05, **p < 0.01 as indicated between groups.