Bio in Action Q2: Eriocaulon buergerianum Extract Protects PC12 Neurons in Zebrafish Against 6-hydroxydopamine-induced DamageThe five scientists who were researching Eriocaulon buergerianum were investigating the neuroprotective effects and underlying action mechanism of the herb’s ethanol extract. They are specifically trying to find any effects of Eriocaulon buergerianum on 6-hydroxydopamine-induced damaged neurons. This experiment consisted of seven main methods: (1) analyzing the viability of dopaminergic neurons by utilizing anti-tyrosine hydroxylase whole mount immunostaining, (2) examining the zebrafishes’ movement with a digital video tracking system, (3) assessing the viability of the PC12 cells with MTT assays, (4) checking PC12 cellular damage with LDH assays, (5) evaluating the nuclear morphological changes in apoptotic cells with DNA staining utilizing Hoechst 33342 dye, (6) quantifying intracellular nitric oxide with DAF-FM diacetate staining, and (7) determining the expression of inducible nitric oxide synthase with Western blot. First, the five scientists cultured fertilized zebrafish eggs by keeping them in an embryo medium. To prevent the coloration of the embryos, phenylthiourea was added and the zebrafish were exposed to 6-OHDA and different concentrations of EBE for 24 hours, both two days after fertilization. The positive control was zebrafish treated with both 6-OHDA and Nomifensin/L-nitroarginine methylester. To evaluate the viability of dopaminergic neurons in the zebrafishes’ brains, anti-tyrosine hydroxylase whole mount immunostaining was performed. The PC12 cells were then plated. Different concentrations of EBE were added to pretreat the PC12 cells incubated in F-12 K medium. Cells pretreated with L-NAME acted as the positive controls. Then, the mediums were discarded and the cells incubated with 6-OHDA dissolved in heat-inactivated horse blood. There was also a control group of untreated cells. Then, the viability and cellular damage of the PC12 cells were detected by the MTT and LDH assays. There were four main results: (1) ECE allowed the zebrafish to be able to move greater distances than without the ECE, (2) ECE increased the 6-OHDA-damaged PC12 cells’ ability to live, (3) ECE protected cells against 6-OHDA-induced nuclear fragmentation and accumulation of apoptotic bodies, and (4) ECE anti-oxidative activities such as the inhibition of NO production and iNOS expression in PC12 cells in vitrio are related to its neuroprotective effects in 6-OHDA-induced dopaminergic neuron damage. There were two main conclusions: (1) ECE does neuroprotective activities in zebrafish such as recovery of dopaminergic neuron loss caused by 6-OHDA and increase of total distance in movement in zebrafish and (2) the iNOS-NO pathway might be involved. The five scientists that did this research were Weiwei Wang, Zaijun Zhang, Lorita Chi-Veng Cheang, Simon MIng-Yuen Lee, and Zhixiu Lin. Meiwei Wang, Zaijun Zhang, Lorita Chi-Veng Cheang, and Simon Ming-Yuen Lee worked in the State Key Laboratory of Quality Research in Chinese Medicine in the Institute of Chinese Medical Sciences at the University of Macau under their advisor Padre Tomas Pereira in Taipa, Macao, China. Zhixiu Lin collaborated with them from the School of Chinese Medicine at the Chinese University of Hong Kong in Shatin, NT, Hong Kong, China. The research was made possible by grants from the Science and Technology Development Fund of Macao SAR and the Research Committee of the University of Macao. Zaijun Zhang and Meiwei Wang performed the experiments. Meiwei Wang wrote the manuscript. Zhixiu Lin reviewed the study design and literature. Lorita Chi-Veng Cheang revised the manuscript. Simon Ming-Yuen Lee supervised the study. All five scientists read and approved the final manuscript. The research was published on April 28, 2011. This work relates to the specific material from Quarter Two in class. For example, 6-hydroxydopamine-induced damage consists of apoptosis. In other words, exposure to 6-OHDA can cause a PC12 cell or a neuron in a zebrafish to undergo programmed cell death. We learned about apoptosis, in which cells are dismantled and digested, in the Cells unit (specifically in the Membrane Transport Signaling subunit) this quarter. A cascade of “suicide” proteins called caspases is activated in the cells undergoing apoptosis. This experiment also concluded that there is a possibility that the iNOS-NO pathway has an effect on how zebrafish cells react to Eriocaulon buergerianum ethanol extract because NO participates in cellular signaling pathways, and regulates brain functions such as neuronal apoptosis and neuronal cell damage by disrupting that particular neuron’s mitochondrial electron transport chain function. We learned about cellular signaling in the Cells unit (specifically in the Membrane Transport Signaling subunit) this quarter. In addition, while maintaining the zebrafish in the experiment, wild-type embryos were used, and we learned about wild-type phenotypes in the Heredity unit (specifically in the Genetics subunit). Wild-type is the phenotype that is most ubiquitous for that particular trait. This term was identified and named by embryologist Thomas Hunt Morgan. In addition, the five scientists used anti-tyrosine hydroxylase whole mount immunostaining to determine if the dopaminergic neurons in the zebrafish brains were viable.