Others see this drug for its therapeutic use in the treatment of diseases, such as cancer, glaucoma, nerve pain, seizure disorders, Crohn's disease, and muscle spasms due to multiple sclerosis (Elikkottil et al., 2009). The term cannabis describes the dried components of the hemp plant, such as leaves, seeds and stems, which are often smoked or eaten (Campbell et al., 2017). Both recreational and medical marijuana can have the same harmful effects during pregnancy, such as small birth size, stillbirth, and birth deformities (Gunn et al., 2016).
Many medical marijuana patients in California and other states with legalized medical marijuana often feel stigmatized by others (Satterlund et al., 2015). These mechanisms may explain the ability of CBD to act as a negative allosteric modulator at CB1 receptor sites (Laprairie et al., 2015). In animal models, CBD has been shown to have no adverse psychoactive effects, no abuse potential, and a better defined anticonvulsant profile than THC (Rosenberg et al., 2015).
Endocannabinoid system
Arachidonyl ethanolamine
Arachidonyl dopamine
Past cannabinoid receptor studies yield inconclusive data
The literature has shown that deletion of CB1 results in different phenotypes within species (Lerner et al., 2008). For example, two contrasting studies regarding bone formation were conducted in different strains of mice. In another strain, deletion of CB1 resulted in a higher bone mass phenotype and genetic protection from ovariectomy-induced bone loss (Bab and Zimmer, 2008).
Because of conflicting studies such as these, the exact function of CB1 has not been elucidated. Even less is known about the exact function of CB2 than CB1. CB2 has also been linked to bone formation; the deletion of the cannabinoid receptor 2 gene in mice caused increased bone turnover and subsequent low bone mass.
Adverse outcomes of cannabinoid exposure
11 smoking, can lead to problems with lung function, such as chronic cough and breathing difficulties (Russell et al., 2018). Overexposure to THC can cause adverse effects, including intense nausea and vomiting, increased heart rate, which can increase one's risk of heart attack, and psychological disorders, such as depression, anxiety, schizophrenia, psychosis, and neurodevelopmental deficits in youth (Borgelt et al., 2013; Meier et al., 2012). The developmental effects of transplacental and postnatal exposure to THC have not been well studied.
Studies also show that marijuana use during pregnancy can cause adverse effects on the fetus, such as low birth weight and body deformities (Shiono et al., 1995). However, the association between cannabis use during pregnancy and other outcomes such as stillbirth, developmental deformities, etc. is still unclear (Gunn et al., 2016). The potential developmental effects of CBD exposure at an early age have also not been well studied.
Zebrafish as a model organism
Previous studies of THC/CBD in zebrafish have shown that exposure at an early age leads to physical deformity and behavioral changes (Carty et al., 2018).
Research goals
Methods
- Zebrafish breeding and egg collection
- Exposure
- Larval behavior
- Developmental deformity scoring
- Statistical analysis
The goal was to treat embryos at or near the beginning of the gastrula period at the 50%. Starting at 5 hpf allows enough time to sort and raise the embryos; it also allows for the development of CB1 and CB2 receptors from the four- and one-cell stage, respectively, to the end of the blastula stage (Kimmel et al., 1995; Paria and Dey, 2000). Embryos are all staged to be within 30 minutes of each other to provide precision in this developmental study, which subsequently ensures experimental reproducibility and the ability to add new observations and details (Kimmel et al., 1995).
We routinely plated zebrafish within one hour of collection to confirm that they were fertilized at the same time. Embryos were then assessed one hour prior to exposure to ensure that exposure would begin at the correct stage (50% epiboly). These concentrations of CBD and THC were based on experimental data described previously (Carty et al., 2018).
Prepared embryos were then randomly sorted into scintillation vials, n=5 vials per exposure group; 20 embryos per vial (diluted to 10 at 24 hpf), containing egg water. For each well, the duration of the larvae's journey at a velocity greater than or equal to 5 mm/sec was recorded. These data were then pooled per scintillation vial and statistical analyzes were performed using a One-Way ANOVA test to analyze the dark cycle data.
Physical deformity data comparison between both treatment and strain was assessed using a two-way ANOVA followed by a Tukey's post hoc test. Data for each deformity were also assessed using one-way ANOVA followed by Tukey's post-hoc test (p≤0.05; n=5). During a 30-min period, we continuously collected movement data for each larval zebrafish at a speed of ≥ 5 mm/s for statistical assessment of larval zebrafish locomotor behavior.
Dark behavioral data were averaged for each fish, pooled by concentration iteration, and analyzed using a.
Results
- Lethality of THC and CBD
- CBD morphology
- Behavior of cnr2 -/- exposed to CBD
- THC morphology
- Behavior of cnr2 -/- exposed to THC
The percentage of physical deformities in both fli and cnr2 -/- fish was significant when comparing both loading and treatment by two-way ANOVA (p ≤ 0.05). Indeed, yolk sac edema and pericardial edema showed a significantly higher incidence as a result of loading and treatment (p ≤ 0.05). In fli larvae at 0.6 mg/l, the incidence of PE was significantly greater than the PE incidence in all other fli exposure treatments.
There was a significant 27.8% percent higher incidence with 0.6 mg/L CBD in fli compared to the cnr2-/- fish. Percent incidence of YSE between fli and cnr2-/- Using a one-way ANOVA, we assessed the mean percent incidence of YSE ± the standard error for each CBD treatment (n=5). Percent incidence of YSE between fli and cnr2-/- larvae: larvae exposed to fli 0.6 mg/l showed significantly higher percent YSE incidence than other treatment groups.
Percent incidence of PE between fli and cnr2-/- larvae: fli 0.6 mg/L exposed larvae showed significantly greater PE percent incidence than other treatment groups. Summary table of CBD malformations: Mean percent prevalence and standard error (mean ± SE) of each malformation found in both fli and cnr2-/- strain fish. CBD and 0.6 mg/L treatments did not show significant differences in duration of locomotion during dark periods from control.
CBD cnr2-/- average dark period locomotion: Using a one-way ANOVA, the average duration of activity (velocity ≥ 5 mm/s) was assessed for each fish (n=300) during dark exposure in larval zebrafish. The percentage occurrence of physical deformity in both fli and cnr2-/- fish was treatment dependent according to two-way ANOVA statistical tests. ANOVA, the mean percentage occurrence of YSE ± the standard error for each THC treatment (n=3) was assessed.
The percent incidence of YSE was significantly higher in fli and cnr2-/- in 2.5 mg/l exposed larvae, 1.25 mg/l exposed larvae and control larvae. Summary table of THC malformations: mean percent incidence and standard error (mean ± SE) of each malformation found in both fli and cnr2-/- fish. THC cnr2-/- mean locomotion in the dark period: Using a one-way ANOVA, the mean duration of activity (velocity ≥ 5 mm/s) during dark exposure in larval zebrafish was assessed in a dose-dependent manner (n=180). .
Discussion
- Lethality of THC and CBD
- CBD and THC morphology
- CB 2 and cardiac function
- CB 2 and hematopoiesis
- Light/dark behavioral assessment
- Dark phase behavioral assessment
- Conclusion
As in the study, Carty (2018) also noted a significant percentage incidence in yolk sac edema and pericardial edema, but at lower concentrations than those we recorded. First, Carty (2018) exposed embryos at an earlier stage of life, which increased the frequency of physical malformations (Bloch et al., 1986; Harbison et al., 1977). By acting on these receptors, cannabinoids stimulate cardiac function (Matsuda et al., 1990; Munro et al., 1993).
In cnr2-/- fish exposed to the same concentrations of CBD, this unfavorable outcome was not observed. The first site of hematopoiesis in zebrafish occurs in the intermediate cell mass, which later develops into the yolk sac and is comparable to the mouse amniotic sac (Kulkeaw and Sugiyama, 2012). CB2 is primarily localized on cells of hematopoietic origin across species and is therefore most likely found in the zebrafish yolk sac (Basu and Dittel, 2011).
CBD binding to CB2 may have caused uncontrolled growth of hematopoietic cells within the yolk sac. In cnr2-/- larvae, only those exposed to the highest sublethal concentrations of THC had a percent incidence similar to fli larvae at these concentrations. Control larvae also showed a significant increase in yolk sac edema; a further study with a larger sample size should be conducted to investigate this finding.
There was a dramatic increase in activity during the intermediate ten-minute assessment interval, which corresponded to the dark period. This trend indicates that higher THC concentrations induce stress and anxiety-like behavior (Ellis et al., 2012). It also suggests that a neurotoxic event induced by higher THC concentrations may trigger molecular signaling and alter behavior (Kim et al., 2013).
According to several studies, both THC and CBD act as stimulants at low concentrations (Achenbach et al., 2018; Akhtar et al., 2013).
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