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
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
Neuron Diagram , Source: www.biomed.brown.edu
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
Oligodendrocyte making myelin, Source:
ncbi.nlm.nih.gov
create a fully functioning artificial myelin and permanent anchor mechanism in drug delivery.
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
Myelin sheaths and Schwann cells in PNS, Source:
http.sciencedaily.com.releases.htm
(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.
Dissection of Lollingucula brevis, Source: pleasanton.k12.ca.us
Hypothesis
Null Hypothesis
As the concentration of the alpham beta2 integrand-
There exists no correlation between the presence of the
optimized dielectric liquid complex increases, the rate
alpham beta2 dielectric liquid complex and the resultant
of neurotransmission in demyelination induced white
time delay in neurotransmission. Thus, any small change
matter of Lutjanus buccanella will also increase. The
in transmission results in the randomness of the system
rationale is that inhibition of ion movement by
(the opportunity for sodium and potassium transfers
dielectric liquid produces a natural increase in
simply translate into change in voltage drops across
neurotransmission efficiency in saltatory conduction.
membrane).
Glycerol 3-D ball and stick model, Source: pubchem.ncbi.gov/glycerol3D
Action potential schematic, Source: colorado.edu/actionpotentialincharcot-tooth
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
Dielectric Liquids (used in project)
Dielectric
3D ball and stick
Strength in
representation
vivo
10.6
Toxicity Levels by
PubChem library in
NCBI
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)
Molecule
Type
Cresol Red
Molecule
Type
Dielectric
3D ball and stick
Strength in
representation
vivo
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
Materials
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, propylene
3. 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)
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
Pulse Source
Square Pulse
emitted
Sample
Saline Bath
Probe ends – insulated wire so
only the conductor is inside
the sample.
Dielectric Strength vs. Neurotransmission Rate
Time Delay in Signal Transfer + 5 ns
460
40% Rehabilitation
(with glycerol)
440
420
Propylene
400
Glycerol
380
Cresol Red
360
340
0%
20%
40%
60% 100%
Solution Concentration of substance + 5%
Rate
Concentration of Substance + 5%
Strength
vs. Neurotransmission
RateTime
Graph: Dielectric
Glycerol
Concentration
over the
Time Delay in Signal Transfer + 5 ns
Rate of Time Delay in nanoseconds + 5ns
Glycerol Solution Concentration (+ 5%)
Delay
Percent Adhesion of Glycerol-AlphamBeta2 integrand
Percent Adhesion with Glycerol
MnCl2 (catalyst) concentration in µg/mL (+ 5E-4 µg/mL)
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)
Effect of Drug-Ligand on Neurotransmission Rate in situ
500
Time Delay in Signal Transfer + 5 ns
480
460
440
80% Rehabilitation
420
400
380
360
340
Absence of ligand-gylcerol complex
Presence of ligand-glycerol complex
Absence vs. Presence of 20% glycerol-alpham beta2 complex
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 (pvalue being < 0.05), it was seen that the ligand-drug presence in situ was the cause of the an 80% electrical rehabilitation of transmission.
Phase I
T-Test
Avg.
SD
Max
Min
0%
20%
40%
60%
100%
1.8E-17 5.0E-30 2.4 E-10 3.1E-20 3.4E-14
454
400
391
386
383
18
13
14
16
12
480
430
420
420
490
420
380
360
30
309
Phase III
T-Test
Avg.
SD
Max
Min
Absence
N/A
Presence
1.40E-57
454.08
12
480
420
348.2
11
390
330
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 alpha m
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
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
Autoimmunity affecting myelination in Charcot-Marie Tooth, Source: Grays Anatomy. Pub, 2010.
Print
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.
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
References
More complete bibliography of referenced materials can be found here:
 Full Bibliography and Acknowledgements
1.
2.
3.
4.
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
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