Introduction
Parkinson’s disease (PD) is the second most common neurodegenerative and age-dependent disease. It is characterized by muscular rigidity, resting tremor, postural imbalance, and slowed movement (bradykinesia). These symptoms arise when nerve cells in the substantia nigra are destroyed or damaged and can no longer produce dopamine, a neurotransmitter that helps to control muscle motor movements (Hwang, 2013). Although the cause of this disease remains unknown, there are numerous factors including genetic and environmental factors that contribute to the onset of the disease. There has been recent evidence suggesting that oxidative stress may be a contributor. In addition, mitochondrial dysfunction causes an increase in reactive oxygen species (ROS) which may also trigger the onset of PD. This is because ROS begins to destroy proteins and cellular DNA, leading to structural damage, loss of energy supply, and neuronal death (Hwang, 2013).
There is currently no preventable treatment or cure to Parkinson’s disease. However, there are surgical treatments, medicines, and other therapies that help to alleviate symptoms. These treatments do not reverse the effects or treat the neurodegeneration of the disease. For the patient to maintain their motor function, it is vital to reduce cell loss as early as possible because neurons do not regenerate quickly.
A recent experiment, “Tianma Gouteng Yin, a traditional Chinese Medicine decoction, exerts neuroprotective effects in animal and cellular models of Parkinson’s disease,” was conducted by numerous researchers to further examine this compound. The purpose of this experiment was to test the ability of TGY to reverse the effects of rotenone in a model of PD. Rotenone is a compound that induces specific degeneration of dopaminergic neurons, resulting in PD-like symptoms. Drosophila flies were exposed to either 10mg, 50mg, or 100mg of TGY and 42L rotenone, or rotenone only for 14 days. Then the climbing assay, the amount of time it took for the flies to cross the finish line, was performed to examine the motor function of the flies. It revealed that flies exposed to rotenone only could not reach the finish line. On the other hand, in the absence of rotenone, it took only 15 seconds for the flies to cross the finish line. In addition, the motor movement deficits induced by rotenone were partially reversed by TGY which was indicated by the improvement of the climbing score (Liu et al., 2015). The hypothesis that the motor impairment induced by rotenone on drosophila flies is reversed by exposure to the neuroprotective effects of the compound Tianma Gouteng Yin (TGY), resulting in an increase in motility, is supported by this data.
TGY is prescribed to treat Parkinson’s disease-like symptoms, such as motor deficits and tremors. TGY works by reducing the level of -synuclein and preventing degeneration of dopaminergic neurons, resulting in neuroprotective effects. This study showed that this treatment is effective in rotenone-induced PD models because it improved survival rate, increased motility, lessened impaired motor function, and lessened apoptotic cell death (Liu et al., 2015). The average person should care about the results of this experiment because it reveals the neuroprotective effects that this natural substance has on patients with PD and how it can be used to treat symptoms associated with this disease.
Methods
Drugs used
Tianma Gouteng Yin (TGY) is an herbal Chinese medicine, while rotenone is a natural compound that is found in stems and seeds of several plants. The concentration of rotenone, 62.5mM, used was based on the previous conducted drosophila experiment that produced significant data and showed deficits in motility. It was made previously by the instructors at the Michigan State University Neuroscience Lab by dissolving 0.005g of rotenone into 200L DMSO. DMSO is a compound used to dissolve rotenone. The TGY capsule contained 500mg of the compound.
Vial Preparation
One week prior to motility testing, two sets of four different vials were made. The four different conditions included: control, rotenone only, rotenone + TGY, and TGY only. To make the TGY solution, we mixed 500mg of TGY capsule with 50mL of water and swirled it until it dissolved. The control vial contained 10mL of water and 10L of DMSO. The rotenone vial contained 10mL of water and 10L of the rotenone solution. The rotenone + TGY vial contained 10mL of TGY solution and 10L of the rotenone solution. Finally, the TGY only vial contained 10mL of TGY solution and 10L of DMSO. DMSO was added to the control and TGY vials in order to keep that variable constant throughout all of the vials. After each of the solutions were added to the vials, food flakes were added. Flies of all ages and both sexes were then anesthetized with CO2 and 25 flies were added to each of the eight vials. The flies were kept in the vials for 7 days, consuming the food and substances that were added to them.
Data Collection and Analysis
Prior to motility testing, two out of the four researchers transferred the flies into empty vials. This was to ensure that at least half of the researchers were blind to the experiment. Each vial was capped with another vial and were then taped together to form one long tube. The assay was done by tapping the flies to the bottom of the tube and placed against a trifold board with measurements in centimeters were on it. The one of the researchers who were blind to the conditions counted to 15 seconds and counted the number of flies that climbed above the 4 cm mark. This process was completed three times for each of the four tubes.
To analyze the data collected, an ANOVA analysis and LSD post-hoc test were completed using SPSS software. The ANOVA test was chosen because it determines whether there is a significant main effect, whereby if manipulating the independent variable significantly influences the dependent variable. After examining the results, there was no significance between the control and rotenone motility data so we had to conduct the experiment again with the second set of vials that were made. The new data was analyzed using the same method and software.
Results
The first set of vials provided data that was not sufficient and produced data that was unexpected. This could have been due to a variety of factors. Interestingly, the data showed that the flies exposed to rotenone only had much higher motility compared to the TGY + rotenone condition.
Figure 1. The effects of 7 days of TGY exposure on the motility of drosophila in a rotenone model of Parkinson’s disease. An ANOVA analysis revealed a significant main effect on motility. An LSD post-hoc test indicated that there is significantly higher motility in rotenone only flies compared to TGY + rotenone flies. * indicates p < .05
The second set of vials produced more accurate and significant data. As predicted, there was a significant increase in motility observed in the rotenone only condition compared to the TGY + rotenone condition. In addition, the control group had a substantially greater motor movement compared to the rotenone only, TGY + rotenone, and TGY only.
Figure 2. The effects of 7 days of TGY exposure on the motility of drosophila in a rotenone model of Parkinson’s disease. An ANOVA analysis revealed a significant main effect on motility. An LSD post-hoc test indicated that TGY + rotenone significantly increased motility compared to flies only exposed to rotenone but did not restore full motility. In addition, the control flies had significantly higher motility compared to rotenone only, TGY + rotenone, and TGY only conditions. * indicates p < .05
Citations:
Hwang, O. (2013, March 31). Role of Oxidative Stress in Parkinson’s Disease. Retrieved November 25, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3620453/
Liang-Feng Liu, Ju-Xian Song, Jia-Hong Lu, Ying-Yu Huang, Yu Zeng, Lei- Lei Chen, Siva Sundara Kumar Durairajan, Quan-Bin Han & Min Li. (2015). Tianma Gouteng Yin, a Traditional Chinese Medicine decoction, exerts neuroprotective e ects in animal and cellular models of Parkinson’s disease. Sci. Rep. 5, 16862