Oxytocin and Vasopressin Gene Expression in the Brain as Potential Biomarkers for Cannabidiol Exposure and Anti-inflammatory Efficacy

Over the past few years there has been an increased interest in cannabidiol (CBD) for general use as well as alternative therapeutic for various diseases. Although CBD has only been FDA approved for seizures under the oral formulation, Epidiolex®, the public has used various unregulated forms of CBD to treat a variety of ailments from pain to anxiety and insomnia. Moreover, an increasing subset of CBD advocates take CBD daily to prevent inflammation. This expanded use of CBD strongly suggests that more research is necessary to elucidate the safety and efficacy of CBD and determine the mechanisms by which it acts. Thus, we conducted two separate studies. In the first, RNA sequencing (RNA-Seq) analysis of brains of female mice undergoing experimental autoimmune encephalomyelitis (EAE) in the presence and absence of CBD was conducted in order to identify potential genes that mediated CBD's neuroprotective effects. In the second, we assessed some of the same genes in male and female mice treated with CBD in the absence of an immune stimulus. Together these data showed that CBD modestly increased oxytocin (Oxt) and vasopressin (Avp) gene expression in the brains of mice, regardless of whether there was active inflammation, suggesting they might serve as biomarkers for CBD exposure, and perhaps, anti-inflammatory efficacy. Overall design: Mice were anesthetized using 3% isoflurane and EAE was induced by subcutaneously injecting the MOG35-55 peptide emulsion. The emulsion was prepared with vigorous mixing for 30 min of a solution containing 100 µg MOG35–55 peptide, 500 µg HKMT, and CFA. Each mouse received 100 µL emulsion delivered in 4 sites (2 at the hips, 2 at the shoulders; 25 µL per site). Control mice received 15 µL saline (SAL) at each site. Twenty-four hours after EAE induction, mice were dosed orally with 75 mg/kg CBD in 100µL of corn oil (CO) or 100µL CO for five days. Mice were observed over an 18-day period, and scores were given based on the following scale: 0-Asymptomatic; 0.5-Flaccid tail; 1-Hindlimb paresis/waddling; 1.5-Waddling gait; 2-Unable to prevent being placed in dorsal recumbency; 2.5-Hindlimb dragging; 3-Single hindlimb paralysis; 3.5-Single hindlimb paralysis with other hindlimb dragging; and 4-Complete hindlimb paralysis. Any score above 0 was considered symptomatic. Mice were never allowed to progress past a score of 4 for animal welfare purposes. Scores and samples analyzed were from two, independent cohorts. Mice were divided into three treatment groups: SAL/CO (control), EAE/CO, and EAE/CBD. SAL/CO mice did not show any clinical signs and were not included in any downstream analyses. For all results and discussion of our findings, “EAE” will refer to active immunization with MOG in CFA supplemented with HKMT (and no administration of PTx).