Team:Georgia State/Integrated Practices
PROJECT
Following our protein expression of the Mambalgin-1 protein , our next steps will include testing the analgesic properties in an animal model. Mambalgin can provide a non-addictive opioid alternative to morphine.We know that Acid-sensing ion channels (ASICS) are involved with nociception, depression and inflammation. ASICS are activated due the lower ph levels of the extracellular space. We also know that ASIC1 & ASIC2 are expressed in the central nervous and peripheral nervous system. (Salinas, et. al 2014) These neurons are connected to the dorsal root ganglion, which we know to bring in sensory information. We will be partnering with the Neuroscience Institute at Georgia State using Sprague-dawley rats to test our introduction of mambalgin to the ASICs. We will inject rats with the expressed mambalgin intra planarly and introduce thermal pain. We will be using the paw thermal stimulator to assess nociception. We will assess results by measuring paw withdrawal latency. We will have one experimental group and two control groups. Saline/vehicle will be our negative control, and our positive will be manufactured mambalgin. Our experimental group will be for our expressed mambalgin in Pichia pastoris. Salinas, M., Besson, T., Delettre, Q., Diochot, S., Boulakirba, S., Douguet, D., & Lingueglia, E. (2014). Binding Site and Inhibitory Mechanism of the Mambalgin-2 Pain-relieving Peptide on Acid-sensing Ion Channel 1a. The Journal of Biological Chemistry, 289(19), 13363–13373. http://doi.org/10.1074/jbc.M114.561076 Experimental Design We expect mambalgin to provide analgesic properties in the presence of the thermal stimulus. We should see a longer Paw Withdrawal Latency (PWL) for the rats that were administered mambalgin. Sprague Dawley Rats subjects ( Subjects are acclimated to device twice 30 minutes a day for 3 days.) Administer expressed Mambalgin (.34 nmoles) every 20 minutes Negative control rats given saline vehicle Intraplanar injection to hindpaw Positive control rats given mambalgin protein provided by manufacturer A paw thermal stimulator will be used to measure paw withdrawal latency 15 minutes after each injection. PWL baseline will be measured after the first 15 minutes PWL is the time that it takes for the rat to remove its paw from the thermal stimulus PWLs will be quantified using Graph Pad Software ANOVA and Fisher Post Hoc test will be used to determine specific group differences. ( Edison and Murphy 2013) Eidson, L. N., & Murphy, A. Z. (2013). Blockade of Toll-Like Receptor 4 Attenuates Morphine Tolerance and Facilitates the Pain Relieving Properties of Morphine. The Journal of Neuroscience, 33(40), 15952–15963. http://doi.org/10.1523/JNEUROSCI.1609-13.2013
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