Cannabinoid exposure in utero exacerbates hyperalgesia in offspring of sickle mice Free

Approximately 5% of pregnant women in the United States and 20% of pregnant women aged 18-24 years in California reported using cannabis (Young-Wolff et al JAMA 2017). Cannabinoid (CB) use in persons with sickle cell disease (SCD) has been reported more than other conditions. Hydroxyurea (HU) improves survival in SCD, however, HU is a teratogen that is withdrawn during pregnancy, thus increasing the probability of seeking CBs to control pain. This may have devastating consequences; a single in utero dose of synthetic- (CP55,940) or phyto-CBs (Δ9-tetrahydrocannabinol) produced developmental changes in the offspring of pregnant C57BL/6 mice (Fish et al Sci. Rep. 2019). We examined if CB use during pregnancy in SCD will have an effect on the development and long-term generational behavioral outcomes related to pain in the offspring. We used humanized transgenic Berkeley (BERK) sickle (SS), hemizygous (AS), and control mice expressing normal human hemoglobin A (AA), to analyze the effect of maternal exposure to non-selective Cannbinoid 1 and 2 receptors on the offspring.

AS and AA females were bred with SS and AA males, respectively (SS females breed poorly). Females were treated with HU (i.p., 50 mg/kg/day), CP55,940 (CP; i.p., 0.3 mg/kg/day), or vehicle (Veh; 2% DMSO in sterile saline) for 2-weeks prior to breeding. During gestation, mice receiving HU were switched to Veh or CP (to recapitulate HU cessation and CB replacement, respectively); mice receiving Veh or CP continued their exposure. Placenta were evaluated at embryonic day (ED) 18. Offspring were assessed for teratogenicity (physical malformations) at postnatal day 1 (PD1) using high-resolution digital images; a subset of offspring were harvested at PD21 to evaluate brain weights; lastly, offspring were evaluated for hyperalgesia at 2- and 3-months (mos) of age.

Replacing HU with CP55,940 (HU+CP) during pregnancy significantly reduced head circumference on PD1 in male and female sickle and control offspring compared to Veh (P < 0.05); HU+CP significantly reduced eye size in male and female sickle offspring compared to Veh (P < 0.05). CP caused limb lengthening in female sickle offspring compared to Veh and HU+CP (P < 0.01). Brains from male PD21 sickle offspring exposed to CP showed significant size reduction compared to Veh and HU+CP (P < 0.05). Further, placenta weights at ED18 were significantly reduced by HU+CP compared to Veh or HU (P < 0.05), which was further exacerbated by CP compared to HU+CP (P < 0.05). Placental histology revealed that CP led to a marked reduction in the junctional zone (JZ), with decreased cellularity, disrupted labyrinth architecture, diminished maternal blood spaces, and focal areas suggestive of hemorrhage and infarction, consistent with ischemic injury. This contrasts with Veh, which showed mildly dilated vascular channels and sinusoidal congestion, likely reflecting SCD-related stress. HU preserved placental structure, maintaining organized vasculature and intact JZ. HU+CP displayed intermediate features, suggesting partial protection. The observed JZ thinning in CP, may reflect CB-induced vasoconstriction or hypoxic remodeling. Placental dysfunction gives rise to poor outcomes for cognition and development, which may contribute to an overt, early onset hyperalgesia phenotype.

SCD presents with chronic hyperalgesia, indicated by greater sensitivity to mechanical and cold stimuli, and reduced grip strength, which typically arises at 3 mos in sickle mice. CP and HU+CP significantly reduced grip strength, indicating greater musculoskeletal hyperalgesia, in male and female sickle offspring starting at 2 mos. CP or HU+CP exacerbated features of chronic hyperalgesia compared to Veh or HU in 3 mos male and female sickle mice, indicated by greater paw withdrawal frequency to mechanical and cold stimuli (P < 0.05). No differences in hyperalgesia were observed in control offspring. Thus, prenatal CB exposure, with or without HU, may have a profound generational effect on pain in offspring, specific to SCD. The offspring with in utero CB exposure are at risk of multi-organ developmental defects, and early onset of hyperalgesia. Our data highlight the risks associated with CB use during pregnancy in SCD, which requires investigation to understand the mechanisms conferring risk to offspring health.