Last but not least, I want to thank my family and friends for their support. Epilepsy affects approximately 50 million people worldwide, therefore improving treatment efficacy and safety for epileptics is imperative. In this study we sought to check the effectiveness and safety of cannabis components (delta-9-tetrahydrocannabinol and cannabidiol) and Tapinanthus globiferus extracts in the treatment of epilepsy.
Our results showed that Tapinanthus globiferus reduced seizure activity in a dose-dependent manner, and no toxicity was observed at the concentrations used. In contrast, delta-9-tetrahydrocannabinol and cannabidiol did not significantly reduce seizure activity and some toxicities were observed at the higher concentrations tested. We conclude that extracts of Tapinanthus globiferus show promise as anticonvulsants and that further research is needed to identify the active constituents and their pharmacological properties.
INTRODUCTION
- Epilepsy
- Cannabis
- Tapinanthus globiferus
- Zebrafish as a model
- Study goals
About 80% of DS cases are due to a mutation of the SCN1A gene that causes impaired voltage-gated sodium channels (Escayg & Goldin, 2010). The frequent attacks caused by DS are prolonged (> 5 minutes) and can be triggered by a slight increase in temperature, vaccines, illness, excitation and/or certain light. These seizures are also very resistant to medication, and if an SCN1A mutation is present, some of the common antiepileptic drugs will worsen seizures if administered.
THC can be produced synthetically or it can be extracted from the cannabis plant as an oil (Sharma, et al., 2012). More research should be done on new compounds to treat epilepsy so that effective and safe treatments can be more available. Figure 1: (A) delta-9-tetrahydrocannabinol (THC) and (B) cannabidiol (CBD). While the farming community sees TG as a threat to their livelihoods, it has beneficial uses as it has traditionally been used as a form of herbal medicine for several disorders, including epilepsy, but there are no current publications on its effects on epilepsy (Adesina et al. ., 2013).
Doctors used a different variety of mistletoe (Viscum album) to treat a patient with unresponsive childhood absence epilepsy, a drug-resistant version of epilepsy, and successfully eliminated her seizures (von Schoen-Angerer et al., 2015). As a traditional medicine, TG leaves are usually crushed, soaked in cold water or beer, and then the liquid is consumed orally (Adesina, et al., 2013). Since TG has traditionally been used to treat epilepsy to some extent and other mistletoe has been shown to be successful in treating epilepsy, we were.
Zebrafish (Danio rerio) are small fish from the Indian subcontinent that have been used as a model organism in research (Encyclopedia of Life, n.d.). They have a high fecundity, mature quickly, share most of their genome with humans, have embryos that develop outside the mother organism, and are easily observed under a microscope during development (Burke, 2016). Furthermore, the zebrafish has been shown to be an effective model in the fields of epilepsy, chemical toxicity, and development (Baraban et al. 2005; Hill et al., 2005; Veldman & Lin, 2008).
In addition to the ability to assess behavioral aspects of seizures in zebrafish via the ViewPoint Zebrabox, c-fos expression can be used to further confirm seizure activity. Zebrafish can also be used as a model that mimics DS by knockdown of the SCN1A gene (Rosen et al., 2009). This DS model has been used to identify both fenfluramine and clemizole as promising drugs for the treatment of this form of epilepsy (Zhang et al., 2015; Baraban et al., 2013).
METHODS AND MATERIALS
- Zebrafish culture and egg production
- Exposure procedures
- Seizure induction
- Viewpoint data collection and analysis
- RNA extraction, reverse transcription, and qPCR
Unwanted debris and unfertilized/dead eggs were removed using a transfer pipette from the collected eggs. The cleaned eggs were then transferred to a petri dish containing zebrafish water (60 ppm Instant Ocean, pH 7.5) and placed in an incubator at 28°C for 5 days. At 120 hours post-fertilization (hpf), larvae were transferred to 96-well plates with one larva in each well.
The larvae were selected based on a lack of deformities and the presence of an inflated swim bladder. Once dosing was complete, the plates were placed back into the incubator and lightly covered with aluminum foil to protect the compounds from light. After 24 hours of exposure to the dosing solutions, the larvae were observed to determine if TG, THC or CBD had any toxic side effects: a lack of response to touch, pericardial edema, yolk sac edema, a curved body axis and/or an uninflated swim bladder.
After adding PTZ to induce seizures, the plate is placed in the ViewPoint ZebraBox and the larvae are acclimated for 5 min before behavioral recording. The ViewPoint ZebraBox tracks larval movement for 15 min with lights on at 100% with a threshold value of 27 and small and large activity parameters set to 5-9 and >9 mm/s, respectively. Movement meeting the major activity requirements is indicative of seizure activity and was the data used to test for a compound's effectiveness as an anticonvulsant.
Therefore, PTZ significantly increases the duration of large activity compared to control, and a compound with anticonvulsant properties would significantly reduce the PTZ-induced large activity compared to PTZ alone and ideally would not. The data collected from the ViewPoint ZebraBox were analyzed using GraphPad Prism 5.0 (La Jolla, CA). If the data pass the Kolmogorov-Smirnov test for normality, statistically significant differences compared to PTZ were determined using a t-test.
If the data did not pass the Kolmogorov-Smirnov test for normality, then a Mann-Whitney test was used for analysis. To determine changes in gene expression caused by the addition of compounds, c-fos mRNA expression (higher expression is associated with more epileptic activity) is measured using qPCR. The abundance of c-fos gene expression was normalized to the expression of the housekeeping 18S gene and determined using qPCR with SYBR Green on a GeneAmp 7500 sequence detection system (Applied Biosystems).
RESULTS
Large activity post-PTZ exposure
Eleven different fractions of TG extracts were tested and two of these fractions (AF.1.10.TG.9 and AF.1.10.TG.14) showed a significant reduction in gross motility compared to 5 mM PTZ. The behavior of zebrafish larvae was analyzed using a Viewpoint Zebrabox (15 min recording with 100% light) to record the duration of high activity (> 9 mm/s) after a 24 h exposure (120-144 hpf) to natural compounds induced by 5 mM PTZ to determine whether these natural compounds have anticonvulsant properties (n=22-36 per treatment; Student's t-test; p≤0.05). The AF.16.TG.3 data are shown in Figure 4 as an example of one of the fractions that did not show anticonvulsant activity.
Gene expression
The results in Figure 6 show a dose-dependent increase in c-fos expression for both THC and CBD. Expression of c-fos was determined by qPCR for each dose group of the zebrafish larvae. The p-value above each bar shows that group compared to control, and the p-value above the horizontal bars shows that group compared to PTZ using a Student t-test.
Toxicity
DISCUSSION
Globimetula braunii extract prevented PTZ seizures in 83.33% of mice given 150 mg/kg ethyl acetate fraction (Aliyu et al., 2014). These studies also confirm that the dose of TG extracts should be increased in future trials to determine whether higher concentrations will continue to show greater reduction. Gene expression analysis was performed on the most promising potential therapeutics from TG (AF.1.10.TG.9), THC and CBD.
Our results showed an unexpected dose-dependent increase in c-fos expression for all three of the compounds. However, we measured not only the c-fos expression of larval brains, but a pool of 10–12 whole larvae (only three biological replicates; n=3). Future studies should be performed with AF.1.10.TG.9 in larger animal models so that changes in brain c-fos expression can be measured alone or by in situ hybridization of whole mounts on individual zebrafish larvae.
Another advantage of the zebrafish test is that toxicity can be assessed for each organism prior to behavioral analysis. The highest THC concentration of 11.2 µM caused death in more than half of the zebrafish, but rates of other deformities remained fairly low at concentrations between 0.02 and 2.23 µM. In contrast, CBD caused less than 10% death or body deformities at any of the concentrations tested.
Further testing should be done to determine the highest concentrations of TG extracts that can be considered safe. Additionally, long-term exposures should be conducted to determine whether acute or chronic exposures cause toxicity. While cannabis did not show any effectiveness for treating generalized seizures in this study, further studies should be conducted using a DS model to determine its efficacy in reducing seizures in drug-resistant epilepsy.
TG may also be useful in the treatment of drug-resistant varieties of epilepsy, so further research using DS models should be carried out. Because AF.1.10.TG.9 was not toxic at the highest concentration tested and PTZ-induced seizure activity was not completely reduced to control levels, higher concentrations of this extract should be tested and may be more effective. Individual chemicals from the extract can then be tested and can be more effective than the AF.1.10.TG.9 mixture.
Why use zebrafish to study human diseases?. 2014a) Effects on specific promoter DNA methylation in zebrafish embryos and larvae following exposure to benzo[a]pyrene. Localization and induction of c-fos protein-like immunoreactive material in the nuclei of mature mammalian neurons. Retrieved March, 2017, from https://www.jove.com/video/1115/microinjection-of-zebrafish-embryos-to-analys-gene-function.
Retrieved March, 2017, from http://www.epilepsy.com/learn/treating-seizures-and-epilepsy/seizure- and-epilepsy-medicines/side-effects.
