Developing a potential drug model for demyelination

diseaseA study on dielectric strength liquid-alpha m beta 2 complex

on the neurotransmission increase in demyelinated gray matter in Lutjanus buccanella

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.

 

Introduction

Neuron Diagram , Source: www.biomed.brown.edu

Oligodendrocyte making myelin, Source: ncbi.nlm.nih.gov

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.

Purpose

Myelin sheaths and Schwann cells in PNS, Source: http.sciencedaily.com.releases.htm

Dissection of Lollingucula brevis, Source: pleasanton.k12.ca.us

HypothesisAs 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.

Null HypothesisThere 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).

Action potential schematic, Source: colorado.edu/actionpotentialincharcot-tooth

Glycerol 3-D ball and stick model, Source: pubchem.ncbi.gov/glycerol3D

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

Molecule Type

Dielectric Strength in vivo

3D ball and stick representation

Toxicity Levels by PubChem library in NCBI

Cresol Red 10.6 0-20% (3 months and over)

Propylene glycol

11.9 0-25% (3 months and over)

Glycerol 42.5 0-20% (in 3 months – 98 years)

Dielectric Liquids (used in project)

Molecule Type

Dielectric Strength in vivo

3D ball and stick representation

Toxicity Levels by PubChem library in NCBI

Ethanol 24.3 0-10% with 95% of the dose leaving in 1st hour

Methanol 33.1 0-2.5% with 75% emitted in air within first 2 hours

Furfural 42.0 0-3% with 90% excreted within 2 hours

Ch 1 Ch 2

Pulse SourceSquare Pulse emitted

Decade Box

Sample

Saline Bath

Oscilloscope (measures membrane potential)The goal is to measure the pulse transmission delay and amplitude delay in the sample.

Probe ends – insulated wire so only the conductor is inside the sample.

The following materials were used for experimentation:1. 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; 2. Red Sharps-Disposal bin; Cranial Surgery Microscope; 500mL glycerol solution, cresol red laboratory grade, propylene3. Oscilloscope with Square Pulse Source (up to 70 millivolts)4. Lollingucula brevis (Common Southwestern Atlantic Squid)5. Semi-permeable membrane with permeability to Na+ and K+ ions (Gortex® Fabric); Closed circuit (wiring, clip leads);

Resistance Decade Box (1-1000mV)6. Lutjanus buccanella (Southwestern Atlantic Snapper Fish) spinal cord site for in situ dissection and demyelinating fluid

(.5L)

Materials

Diagram of Setup: Phase I & III

340

360

380

400

420

440

460

0% 20% 40% 60% 100%

Propylene

Glycerol

Cresol Red

Solution Concentration of substance + 5%

Time D

elay in Signal Transfer + 5 ns

Dielectric Strength vs. Neurotransmission Rate

40% Rehabilitation (with glycerol)

Concentration of Substance + 5%

Time Delay in Signal Transfer + 5 ns

Dielectric Strength vs. Neurotransmission Rate

Rate of Tim

e Delay in nanoseconds + 5ns

Rate Graph: Glycerol Concentration over the Time Delay

Glycerol Solution Concentration (+ 5%)

MnCl2 (catalyst) concentration in µg/mL (+ 5E-4 µg/mL)Percent Adhesion w

ith Glycerol

Percent Adhesion of Glycerol-AlphamBeta2 integrand

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)

340

360

380

400

420

440

460

480

500

Absence of ligand-gylcerol complex Presence of ligand-glycerol complex

Absence vs. Presence of 20% glycerol-alpham beta2 complex

Time D

elay in Signal Transfer + 5 ns

Effect of Drug-Ligand on Neurotransmission Rate in situ

80% Rehabilitation

Phase I 0% 20% 40% 60% 100%T-Test

1.8E-17 5.0E-30 2.4 E-10 3.1E-20 3.4E-14Avg. 454 400 391 386 383SD 18 13 14 16 12Max 480 430 420 420 490Min 420 380 360 30 309

 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.

Discussion

Phase III Absence PresenceT-Test N/A 1.40E-57

Avg. 454.08 348.2SD 12 11Max 480 390Min 420 330

ConclusionIt 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

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.

Future Research

Autoimmunity affecting myelination in Charcot-Marie Tooth, Source: Grays Anatomy. Pub, 2010. Print

Demyelination schematic, Source: Grays Anatomy. Arcturus Pub, 2010. Print

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

ReferencesMore complete bibliography of referenced materials can be found here:

Full Bibliography and Acknowledgements

1. 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

2. 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

3. 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.\

4. 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

More Information and Complete Research Paper

• For more information on the project, please see the Research Paper– Full Version of Research Paper - VSF Sc

ience Fair 2011-2012• A general listing of all resources,

information, and all research related to project can be found below:– Repairing Myelin: Rehabilitating Demy

elination and potential cures - Full project