Introduction
Parkinson’s disease is a complex disease that includes a whole system of symptoms and predictors, each of which must be investigated individually. The biological pathways leading to such disease dysfunctions are under study, and therefore the dependence and correlation with many characteristics may not be apparent. In this regard, this study is being conducted to clarify the existence of sex differences in the development of ultrasonic vocalization and anxious behavior, which in turn portend motor deficits, which are a direct symptom of Parkinson’s. The authors drew on previous studies where a similar relationship had already been established and made differences in the long term of the experiment, taking young rats as a basis and tracking the dynamics over a long distance.
Accordingly, the leading independent variable is the sex and genotype of rats, which is divided into WT (wild) and Pink1, while the dependent variable is a whole set: rat weight, cylinder forelimb, cylinder hindlimb, rears and lands, ultrasonic sound total calls, open field time /movements and number of entries in the central zone. In simpler terms, vocalization, the anxiety of rats, weight, and their motor activity were assessed, which were just calculated thanks to the listed technologies and approaches. Accordingly, the null hypothesis, which was rejected with a statistical significance of p=0.05, stated: “differences in the genotype and gender of rats do not affect the predicates of motor deficits as a symptom of Parkinson’s disease, namely, anxiety and ultrasonic vocalization.”
The hypothesis was: “differences in the genotype and gender of rats affect the predicates of motor activity deficit as a symptom of Parkinson’s disease, namely anxiety and ultrasonic vocalization.”
The independent variables were: genotype and sex.
The dependent variables were: rat weight, cylinder forelimb, cylinder hindlimb, rears and lands, ultrasonic sound total calls, open field time/movements, and the number of entries in the central zone.
Experimental Design
For the experiment’s adequacy, the participants were rats of two species and different sexes in equal proportions: 24 per species, 12 males and 12 females among them. A group of stimulated rats was also allocated to create a controlled amount during testing the effect of USV; however, they were not included in the main experiment. Another critical factor in selecting rats was the age of 2 months or 4 to 6 weeks. The study was conducted according to exceptional standards for the maintenance of participants in the experiment.
First, the rats were put on a 12-hour light cycle. This fact means they spent 12 hours in the light and the next 12 hours in the dark. A red glow was used to assess rats’ behavior in this cycle. Food and water were supplied on time and without restrictions. It is crucial to obtain experimental design approval from all relevant authorities by the Animal Care and Use Committee of the University of Wisconsin-Madison School of Medicine and Public Health and in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.
The weight of the rats was assessed using appropriate equipment with a digital scale. A literature review conducted by the authors showed that ultrasonic vocalization is activated during the estrous cycle of females. Consequently, female behavioral tests were performed during this period, accompanied by more significant anxiety. The cycle itself was detected both by behavioral signs and by cytodiagnostics. Moreover, this cycle consists of four stages, and the females were evaluated only during one of them; smears assessed the transitions.
The anxiety of rats is assessed by movement in an open field: the number of entrances to the central zone and the total distance traveled have been proven to be indicators of this behavior. For this assessment, video cameras were used and calculating these variables. A transparent cylinder, also equipped with cameras, was used to count the motor activity of the rats for 1 second. To do this, the evaluators were deprived of information about the genotype and sex of rats in terms of the number of dynamics of movement of the fore and hind limbs through the glass.
Ultrasonic vocalization was considered more complex and was differentiated into many other variables: duration, bandwidth, intensity, and peak frequency. At the same time, the number of calls was also taken into account; rats that issued less than 30 were not taken into account in the statistics at all. The obtained data were used to compile a Custom CodeSet consisting of mRNA of breed-differentiable genes. It singled out five types of central genes responsible for most of the dependent variables. These data were evaluated using special software for a variety of specific characteristics. This action is applied and is not directly related to the activities for testing the hypothesis.
Finally, statistical analysis was performed using special SigmaPlot software, according to the two-way ANOVA method. At the same time, variables that did not correspond to the normality distribution were transformed using the square root or the rank; in this case, the Shapiro-Wilk and Levene tests were used. The critical level of significance was constantly maintained at the level of 0.05 for each assessment.
Results
As a result of evaluating the results, many conclusions were drawn: Pink1 females turned out to be more anxious than males due to the more significant number of entries into the central zone, in the absence of statistically significant differences in the distance traveled, either by genotype or by sex. Again, Pink1 females had more significant motor activity in the cylinder score, and USV scores were higher for males, regardless of genotype. Then, using the NanoString approach, heat maps with genes and genotype differentiation were developed with further identification of the genes necessary for this work. Experimental rats were deeply anesthetized with isoflurane and subsequently euthanized. Samples of the brain and other body parts were frozen, placed in appropriate solutions, and isolated. Subsequently, a procession of RNA was carried out to analyze the necessary genes.
Conclusions
The initial hypothesis was supported by the fact that both gender and genotype have a statistically significant effect on the manifestation of specific symptoms of Parkinson’s disease. To varying degrees, it manifested itself with greater force in females, for example, anxiety, during ultrasonic vocalization in males. It is essential that the differences turned out to be significant and made it possible to identify the corresponding genes that are directly or indirectly responsible for these symptoms. The findings of this study made it possible to determine the future vector of work, confirmed earlier studies on the presence of a correlation between the sexes, and made it possible to apply NanoString technology to differentiate differences by genotype.
Personal Opinion
In my opinion, a sufficiently small sample for this experiment is leveled by the use of non-parametric comparison methods, such as the Mann-Whitney U-test. In this work, the competence of the evaluators was also assessed, and an integrated approach was applied to the consideration of statistical significance. In general, the results of the work were supplemented by further analysis of genes and genotypes, which not only increased the scientific significance of the work but also more accurately determined the vector of further research. It seems that this study meets all scientific requirements and is carried out in the best possible way.
Real World Application
Studying Parkinson’s disease is very important not only for the scientific community but for the world as a whole. Specifically, this study makes adjustments to the daily lives of those suffering from this disease and medical professionals who have to deal with it. For the rest of the population, the research may be educational about warning signs of anxiety and motor deficits as signals for medical evaluation and potential treatment.
Naturally, it should be taken into account that between experiments on rats and real studies on humans, there are several more scientific and applied work stages. However, already at this stage, there are revealed correlations, which are confirmed by more than the first experiment, judging by the literature review of this work. However, in the case of such studies, their impact on everyday life and professional activities is achieved only with the accumulation of knowledge and, as a rule, over a long distance, respectively, has a delayed effect.