Developing a potential drug model for demyelination disease

1. Developing a potential drug model for demyelination disease A study on dielectric strength liquid-alpha m beta 2 complex on the neurotransmission increase in demyelinated gray matter in Lutjanus buccanella

2. Introduction Demyelination disorders such as Multiple Sclerosis, Optic Neuritis, and Guilliane Barr syndrome, and other Leukodystrophies are prevalent in an aging society and especially in the industrial northern hemisphere. Between 300,000 and 460,000 individuals are estimated to be suffering from de and dismyelination in the United States alone – an amount equal to about 200 patients a week that are newly diagnosed. Although the exact etiology is not known, it is thought that demyelination originates from a autoimmune response developing a decay in the myelin sheathing, which covers the axons of neurons and is produced by glial cells wrapping around the neuron from fetus to developmental stages. The result of the autoimmune response is a reduction of neurotransmission efficiency due to the lack of saltatory conduction, leading to motor dysfunction and cognitive problems in CNS demyelination like Multiple Sclerosis. Although research continues today in pharmaceuticals to reduce the immune response or in physiology of the myelin sheath to strengthen itself, the researcher used an alternate approach focused on a prior year’s findings on the insulative property of liquids and tailored this optimized material to create a fully functioning artificial myelin and permanent anchor mechanism in drug delivery. Neuron Diagram , Source: www.biomed.brown.edu Oligodendrocyte making myelin, Source: ncbi.nlm.nih.gov

3. Purpose • The experiment aims at developing a potential drug model for demyelinating diseases using myelin membrane specific ligand-binding mechanisms on Major Basic Protein (MBP). To do so, a dielectric fluid was selected that best increased neurotransmission and was biocompatible within a tolerance interval (corroborated by the PubChem database from the National Center for Biotechnology Information). The second major aim is to find an “anchor” mechanism that has specificity as well as high adhesion potential to myelin sheath surface membrane proteins, like the Major Basic Protein (MBP) and to the polar dielectric liquid. The experiment consists of three phases: • Phase I: Optimization of Neurotransmission efficiency using dielectric liquids under human tolerance ratio • Phase II: Maximization of optimal dielectric liquid adhesion to the alpha m beta 2 integrand (ligand) • Phase III: Testing of optimized dielectric liquid-alpha m beta 2 complex on demyelinated tissue in an in situ model of Lutjanus bucanella. Myelin sheaths and Schwann cells in PNS, Source: http.sciencedaily.com.releases.htm Dissection of Lollingucula brevis, Source: pleasanton.k12.ca.us

4. Hypothesis Null Hypothesis As the concentration of the alpham beta2 integrand-optimized dielectric liquid complex increases, the rate of neurotransmission in demyelination induced white matter of Lutjanus buccanella will also increase. The rationale is that inhibition of ion movement by dielectric liquid produces a natural increase in neurotransmission efficiency in saltatory conduction. There exists no correlation between the presence of the alpham beta2 dielectric liquid complex and the resultant time delay in neurotransmission. Thus, any small change in transmission results in the randomness of the system (the opportunity for sodium and potassium transfers simply translate into change in voltage drops across membrane). Glycerol 3-D ball and stick model, Source: pubchem.ncbi.gov/glycerol3D Action potential schematic, Source: colorado.edu/actionpotentialincharcot-tooth

5. Review of Goals for Drug Model • Increased Neurotransmission • By acting as a myelin sheath for the cells (saltatory conduction viability) • Minimizing Time Delay of Stimulus and Response • Low Toxicity • Minimization of toxicity parameter given by NCBI • So we only use the tolerance interval of the human body

6. Dielectric Liquids (used in project)

8. Materials • The following materials were used for experimentation: • Sterilized Goggles, Biogel Gloves, Clean and Sterilized Lab Apron; Sterilized scalpel; Sterilized grasper; 300ml, (0.91%) physiological saline; Fluorescence analyzer; MnCl2 up to 1.0 µg/mL; k652 cell lines; • Red Sharps-Disposal bin; Cranial Surgery Microscope; 500mL glycerol solution, cresol red laboratory grade, propylene • Oscilloscope with Square Pulse Source (up to 70 millivolts) • Lollingucula brevis (Common Southwestern Atlantic Squid) • Semi-permeable membrane with permeability to Na+ and K+ ions (Gortex® Fabric); Closed circuit (wiring, clip leads); Resistance Decade Box (1-1000mV) • Lutjanus buccanella (Southwestern Atlantic Snapper Fish) spinal cord site for in situ dissection and demyelinating fluid (.5L) Diagram of Setup: Phase I & III Oscilloscope (measures membrane potential) The goal is to measure the pulse transmission delay and amplitude delay in the sample. Ch 2 Ch 1 Decade Box Saline Bath Pulse Source Square Pulse emitted Probe ends – insulated wire so only the conductor is inside the sample. Sample

9. Solution Concentration of substance + 5% Dielectric Strength vs. Neurotransmission Rate Time Delay in Signal Transfer + 5 ns 40% Rehabilitation (with glycerol)

10. Concentration of Substance + 5% Rate Graph: Glycerol Concentration over the Time Delay Dielectric Strength vs. Neurotransmission Rate Time Delay in Signal Transfer + 5 ns Rate of Time Delay in nanoseconds + 5ns Glycerol Solution Concentration (+ 5%)

11. MnCl2 (catalyst) concentration in µg/mL (+ 5E-4 µg/mL) Percent Adhesion of Glycerol-AlphamBeta2 integrand Percent Adhesion with Glycerol 0 µg/mL 0.001 µg/mL 0.01 µg/mL 0.1 µg/mL 1.0 µg/mL MnCl2 (catalyst) concentration in µg/mL (+ 5E-4 µg/mL)

12. Absence vs. Presence of 20% glycerol-alpham beta2 complex Effect of Drug-Ligand on Neurotransmission Rate in situ Time Delay in Signal Transfer + 5 ns 80% Rehabilitation

13. Discussion  In phase I, using Lollingucula brevis (squid) nerve tissue in an in vitro model, glycerol, as predicted from the Nernst equation and the insulation model of the myelin sheath, was found to produce significant neurotransmission improvement within the human biocompatibility tolerance limit of 20%. Thus the increase in dielectric strength, glycerol (42.5), propylene glycol (11.9), and cresol red (10.9) solution, provided a greater rate of increase. In phase II, glycerol was linked with a T-cell surface protein (alpha m beta 2 integrin protein) using Manganese (II) Chloride, optimized at 1.0 micrograms per microliter. In phase III, using a cadaveric in situ Lutjanus buccanella nerve, the modified glycerol-alpha m beta 2 was introduced to nerve, bringing attached glycerol into the ruptured myelin surface environment. The R2 value of 0.9324 showed high correlation between impulse rate and glycerol amount while regression models for rate graphs reflect both high correlation and optimized neurotransmission under glycerol and catalyst saturation. After a direct comparison analysis using t-test (p-value being < 0.05), it was seen that the ligand-drug presence in situ was the cause of the an 80% electrical rehabilitation of transmission.

14. Conclusion It was found that there was 1) an increase in neurotransmission came from a biocompatible, larger dielectric strength liquid: 20% glycerol, 2) adhesion of alpham beta2 integrand and glycerol increased until saturation point of MnCl2 of 1.0 µg/mL and 3) that there was a tremendous net gain in neurotransmittance rehabilitation in the PNS of a vertebrate animal in situ, in other words the bound glycerol-alpha m beta 2 complex had created an 80% increase in the electrical neurotransmission speed, exceeding the 40% increase observed using glycerol only as a liquid coating, and did so with specific surface attachment. As a result a potential solution to demyelination was found, tested, and successfully rendered– the use of glycerol-alpha m beta 2 integrin complex as a viable myelin substitute. Diagram of procedure and Oscilloscope sampler, Source: Serway, Raymond A. and Jerry S. Faughn. College Physics. CA: Cengage Learning, 2009

15. Future Research Further research should be conducted in vivo with mammalian species bringing more complexity to drug delivery and MBP adhesion rates as well as blood brain barrier permeability of drug model with large protein. Moreover, T-cell stimulated secretions of alpham beta2 – glycerol can develop more involved groupings. This research can be ultimately used for surgical and pharmaceutically administered replacement of demyelinating disorders of the CNS like Multiple Sclerosis. Local PNS access through recognition protein, alpham beta2 integrand, provides ways to treat PNS based disorders like Charcot-Marie Tooth and Guillain-Barre syndrome with increased specificity. In short, the complex allows for a recognition mechanism for ruptured myelin sites through MBP and results in a large electrical rehabilitation. Autoimmunity affecting myelination in Charcot-Marie Tooth, Source: Grays Anatomy. Pub, 2010. Print Demyelination schematic, Source: Grays Anatomy. Arcturus Pub, 2010. Print

16. Acknowledgements • I would like to acknowledge the following faculty and organizations in giving me lab space and access to Flourescence analyzers and k652 cell lines • Bruce Nappi, M.S. Director of Simulation Center at University of Florida Medical School • I would like to thank Ms. Teryn Romaine and Ms. Cloran for assistance and mentoring for presentations and information • More complete references and acknowledgements can be found at: • Full Bibliography and Acknowledgements

17. References More complete bibliography of referenced materials can be found here: • Full Bibliography and Acknowledgements • A. Shibata, M. V. Wright, S. David, L. McKerracher, P. E. Braun and S. B. Kater. Unique Responses of Differentiating Neuronal Growth Cones to Inhibitory Cues Presented by Oligodendrocytes. The Journal of Cell Biology. Vol. 142, No. 1 (Jul. 13, 1998), pp. 191-202 • B. A. Strange, P. C. Fletcher, R. N. A. Henson, K. J. Friston and R. J. Dolan. Segregating the Functions of Human Hippocampus. Proceedings of the National Academy of Sciences of the United States of America. Vol. 96, No. 7 (Mar. 30, 1999), pp. 4034-4039 • Bunge, R., Salzer, J. (1980). Studies of Schwann Cell proliferation: I. An Analysis of Tissue Culture proliferation during Development, degeneration, and direct injury. The Journal of Cellular Biology, 84, 739-752.\ • C. Lubetzki, C. Demerens, P. Anglade, H. Villarroya, A. Frankfurter, V. M.-Y. Lee and B. Zalc. Even in Culture, Oligodendrocytes Myelinate Solely Axons. Proceedings of the National Academy of Sciences of the United States of America. Vol. 90, No. 14 (Jul. 15, 1993), pp. 6820-6824

18. More Information and Complete Research Paper • For more information on the project, please see the Research Paper • Full Version of Research Paper - VSF Science Fair 2011-2012 • A general listing of all resources, information, and all research related to project can be found below: • Repairing Myelin: Rehabilitating Demyelination and potential cures - Full project