4-Aminopyridine derivatives enhance impulse conduction in guinea-pig spinal cord following traumatic injury.

Abstract

4-Aminopyridine (4-AP), a potassium channel blocker, is capable of restoring conduction in the injured spinal cord. However, the maximal tolerated level of 4-AP in humans is 100 times lower than the optimal dose in in vitro animal studies due to its substantially negative side effects. As an initial step toward the goal of identifying alternative potassium channel blockers with a similar ability of enhancing conduction and with fewer side effects, we have synthesized structurally distinct pyridine-based blockers. Using isolated guinea-pig spinal cord white matter and a double sucrose gap recording device, we have found three pyridine derivatives, N-(4-pyridyl)-methyl carbamate (100 microM), N-(4-pyridyl)-ethyl carbamate (100 microM), and N-(4-pyridyl)-tertbutyl (10 microM) can significantly enhance conduction in spinal cord white matter following stretch. Similar to 4-AP, the derivatives did not preferentially enhance conduction based on axonal caliber. Unlike 4-AP, the derivatives did not change the overall electrical responsiveness of axons to multiple stimuli, indicating the axons recruited by the derivatives conducted in a manner similar to healthy axons. These results demonstrate the ability of novel constructs to serve as an alternative to 4-AP for the purpose of reversing conduction deficits.

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4-AMINOPYRIDINE DERIVATIVES ENHANCE IMPULSE CONDUCTION
IN GUINEA-PIG SPINAL CORD FOLLOWING TRAUMATIC INJURY
J. M. MCBRIDE,a1D. T. SMITH,bS. R. BYRN,b
R. B. BORGENSaAND R. SHIa*
aDepartment of Basic Medical Sciences, Center for Paralysis Re-
search, Purdue University, 408 South University Street, West Lafay-
ette, IN 47907, USA
bDepartment of Industrial and Physical Pharmacy, Purdue University,
West Lafayette, IN 47907, USA
Abstract—4-Aminopyridine (4-AP), a potassium channel
blocker, is capable of restoring conduction in the injured
spinal cord. However, the maximal tolerated level of 4-AP in
humans is 100 times lower than the optimal dose in in vitro
animal studies due to its substantially negative side effects.
As an initial step toward the goal of identifying alternative
potassium channel blockers with a similar ability of enhanc-
ing conduction and with fewer side effects, we have synthe-
sized structurally distinct pyridine-based blockers. Using
isolated guinea-pig spinal cord white matter and a double
sucrose gap recording device, we have found three pyridine
derivatives, N-(4-pyridyl)-methyl carbamate (100 ?M), N-(4-
pyridyl)-ethyl carbamate (100 ?M), and N-(4-pyridyl)-tertbutyl
(10 ?M) can significantly enhance conduction in spinal cord
white matter following stretch. Similar to 4-AP, the deriva-
tives did not preferentially enhance conduction based on
axonal caliber. Unlike 4-AP, the derivatives did not change
the overall electrical responsiveness of axons to multiple
stimuli, indicating the axons recruited by the derivatives con-
ducted in a manner similar to healthy axons. These results
demonstrate the ability of novel constructs to serve as an
alternative to 4-AP for the purpose of reversing conduction
deficits. © 2007 IBRO. Published by Elsevier Ltd. All rights
reserved.
Key words: axonal stretch, compound action potential, re-
fractory period, repetitive stimuli, myelin damage, conduc-
tion block.
The primary functional deficits of traumatic spinal cord
injury mainly stem from damage to axons of the white
matter. In most human injuries the spinal cord is not com-
pletely transected, rather the axons are strained, contused,
and/or compressed. Within the injured cord there is a
considerable amount of anatomically continuous yet phys-
iologically compromised axons (Blight, 1983a,b; Fehlings
and Tator, 1995; Hayes and Kakulas, 1997; Kakulas,
1999). Many of these axons undergo conduction block due
to ionic disturbance and progressive demyelination follow-
ing injury. Alleviating conduction block from a small popu-
lation of these physiologically compromised yet anatomi-
cally intact axons could produce substantial functional re-
covery. Therefore, identification of compounds capable of
restoring conduction in surviving axons is an effective op-
tion for treatment of spinal cord trauma.
Previous studies have indicated increased activity of
potassium channels as a contributor to conduction block in
demyelinated nerve fibers (Chiu and Ritchie, 1980; Sher-
ratt et al., 1980). There is increasing evidence supporting
this notion. For example, ?-subunits Kv1.1, Kv1.2, and
cytoplasmic ?-subunit Kv?2 which form the 4-aminopyri-
dine (4-AP) sensitive potassium channel have been doc-
umented in the juxtaparanodal region of spinal cord my-
elinated axons (Rasband and Trimmer, 2001; Karimi-
Abdolrezaee et al., 2004; Rasband, 2004). Due to their
location, these potassium channels are typically hidden
beneath the myelin sheath. Consequently, myelin damage,
a well-documented secondary injury subsequent to spinal
cord trauma (Blight, 1983a, 1985) will likely unmask these
otherwise silent channels leading to conduction block via
potassium ion efflux.
The ability of 4-AP, a potassium channel blocker, to
restore conduction in damaged axons has been studied
extensively (Targ and Kocsis, 1985; Blight and Gruner,
1987; Blight et al., 1991; Hayes et al., 1994, 2004; Shi and
Blight, 1997; Shi et al., 1997; Halter et al., 2000; Jensen
and Shi, 2003). While improvements in sensory and motor
function following application of 4-AP have been demon-
strated in both animal and human spinal cord injuries, the
overall therapeutic benefit of 4-AP remains modest (Don-
ovan et al., 2000; Halter et al., 2000). One obvious reason
is that the maximum tolerable blood level of 4-AP in both
animals and humans is only 0.5–1 ?M, while the most
effective concentration determined in vitro is 100 ?M (Shi
and Blight, 1997; Shi et al., 1997). Concentrations beyond
1 ?M produce side effects such as respiratory distress,
anxiety, and epileptiform seizures (Stork and Hoffman,
1994; Pena and Tapia, 1999, 2000). Possible reasons for
the negative side effects associated with higher doses of
4-AP are increased synaptic transmission or additional
blockade of potassium channel currents associated with
the resting membrane potential (Shi and Blight, 1997).
In order to overcome these limitations while continuing
to reverse conduction block, we have developed several
pyridine-based compounds whose structures are similar
to, yet distinct from, 4-AP. The goal of this line of study is
to search for compounds that are capable of enhancing
conduction effectively as 4-AP in the injured spinal cord
with fewer side effects. Here we report our preliminary
results, demonstrating that, as an initial step toward our
1Present address: Cleveland Clinic Lerner College of Medicine, 9500
Euclid Avenue NA/24, Cleveland, OH 44195, USA.
*Corresponding author. Tel: ?1-765-496-3018; fax: ?1-765-494-7605.
E-mail address: riyi@purdue.edu (R. Shi).
Abbreviations: CAP, compound action potentials; tBoc, tertbutyl car-
bamate; 4-AP, 4-aminopyridine.
Neuroscience 148 (2007) 44–52
0306-4522/07$30.00?0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2007.05.039
44

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goal, three newly identified analogs are capable of enhanc-
ing axonal conduction following spinal cord trauma.
EXPERIMENTAL PROCEDURES
Isolation of spinal cord
All animals used in this study were handled in strict accordance
with the U.S. National Institutes of Health’s Guide for the Care and
Use of Laboratory Animals and the experimental protocol was
approved by the Purdue Animal Care and Usage Committee.
Every effort was made to minimize the number of animals used
and their suffering. The method used to isolate the spinal cord was
similar to that described previously (Shi and Blight, 1996; Shi et
al., 1997; Shi and Pryor, 2002). A combination of ketamine
(80 mg/kg) and xylazine (12 mg/kg) was used to anesthetize adult
female guinea pigs weighing 350–500 g (obtained from Hilltop
Laboratory Animals, Scottdale, PA, USA). Following anesthesia
the animal was transcardially perfused with cold, oxygenated
Krebs’ (15 °C) solution to remove the blood and lower cord tem-
perature. Following perfusion the entire vertebral column was
excised rapidly and the spinal cord was removed from the verte-
brae. The cord was then subdivided to produce ventral white
matter strips that were subsequently incubated in fresh Krebs’
solution at room temperature for 1 h. The term “ventral white
matter strips” will be used interchangeably below with “cords” or
“spinal cords” for ease of description. The composition of the
Krebs’ solution was as follows (in mM): 124 NaCl, 2 KCl, 1.2
KH2PO4, 1.3 MgSO4, 1.2 CaCl2, 10 dextrose, 5.6 sodium ascor-
bate, and 26 NaHCO3, equilibrated with 95% O2–5% CO2to
produce a pH of 7.2–7.4.
Recording chamber
Numerous variations of the recording chamber have been de-
scribed in previous publications (Shi and Blight, 1996; Shi and
Borgens, 1999; Shi and Pryor, 2002). As displayed in Fig. 1 a strip
of spinal cord white matter 45 mm in length and 2 mm in diameter
was placed across the chamber with the central compartment
(volume: 3.6 mL) receiving a continuous perfusion of oxygenated
Krebs’ solution (2 mL/min). This portion of the chamber is also the
site where the tested analogs were introduced. The ends of the
spinal cord strip were placed across the sucrose gap channels
(volume: 0.28 mL) to side compartments filled with isotonic potas-
sium chloride (120 mM). The sucrose gap was perfused with
isotonic sucrose solution (320 mM) at a rate of 1 mL/min. To
prevent the exchange of solutions the white matter strip was
sealed with a thin plastic sheet and vacuum grease on either side
of the sucrose gap channels. Chamber temperature was main-
tained at 37 °C via an in line heater and temperature probe
(Warner Instruments, Hamden, CT, USA). Electrodes were
present in the central and outer two wells to monitor conduction
through each solution. The electrodes were not in direct contact
with the spinal cord and the compound action potentials (CAP)
conduction distance is in fact through the central well of the
chamber. The cord was stimulated by a 0.1 ms constant current
unipolar pulse with CAP recorded at opposite ends of the strip.
Recordings were made using a bridge amplifier (Neurodata In-
struments, Delaware Water Gap, PA, USA) and subsequent anal-
ysis was performed using custom Labview®software (National
Instruments, Austin, TX, USA) on a Dell PC. Further details and a
description of the original chamber can be found in our previous
publications (Shi and Blight, 1996; Shi and Borgens, 1999; Shi
and Pryor, 2002).
CAP amplitude
Recordings of axonal conduction were made by analysis of CAP
which are formed by the spatio-temporal summation of many
single unit action potentials fired by individual axons. To record the
CAP amplitude, a supramaximal stimulus (110% of the maximal
stimulus) was delivered at a frequency of one stimulus every 3 s.
The CAP was recorded continuously and stored in the computer
for future analysis. To assess axonal conduction during each
experimental condition averages were recorded at designated
time points throughout the experiment. In addition, a real time plot
of CAP amplitude was displayed throughout the experiment.
Stretching
The injury device as well as estimation of the magnitude of stretch
or strain (the degree of elongation from the initial length) is de-
scribed in our previous publication (Shi and Pryor, 2002). As
displayed in Fig. 1 a flat raised surface with a small hole was
placed in the central compartment. The ventral white matter strip
was laid across this surface and immobilized with a nylon mesh
stabilizer on either side of the elongation site. The placement of
the nylon mesh had no significant effect on action potential am-
plitude (J. M. McBride and R. Shi, unpublished observations). A
Plexiglas stretch rod attached to a micromanipulator was sus-
pended above the white matter strip. During stretch injury the rod
was released traveling at a rate of 1.5 m/s from a pre-measured
distance and removed from the cord immediately after application.
This device produced a strain of 50% on the isolated tissue which
Fig. 1. Characterization of spinal cord tissue extraction, recording device, and conduction changes in response to stretch injury. (A) Drawing of tissue
isolation from an extracted adult female guinea-pig spinal cord. (B) Double sucrose gap recording chamber with an isolated spinal cord sample
mounted in the apparatus. The central chamber is continuously perfused with oxygenated Krebs’ solution; this is also the site of oxygenated pyridine
compound administration. The ends of the tissue were placed in separate wells filled with 120 mM isotonic KCl, which were separated from the central
chamber by two smaller chambers containing 230 mM isotonic sucrose. (C) Example of CAP amplitude 30 min after injury to the isolated spinal cord
white matter.
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indicates that the tissue is being elongated 50% of its original
length.
Derivative synthesis
Examples of three chemical derivative classes of 4-AP, amides,
carbamates, and ureas, were synthesized to determine if any of
these compounds were biologically active in spinal cord injury
(Smith et al., 2005). All prospective analogues were chosen that
would not completely eliminate the pyridine–pyridinium equilib-
rium responsible for both transport and biological activity. In ad-
dition the pyridine nitrogen atom was not altered since the pro-
posed mechanism of blockade arises from the ability of hydrogen
bonds to form between the pyridine nitrogen and the channel pore.
These compounds were synthesized to test steric requirements of
the active site as well as bonding interactions between the deriv-
atives and the channel. For this paper we will focus on conduction
changes in the injured cord following application of the carbamate
derivatives (Fig. 2).
Carbamates were synthesized from 4-AP which was pur-
chased from Richman Chemical Co., Lower Gwynedd, PA, USA.
Melting points were determined in capillary tubes using a Thomas
Hover melting point apparatus. NMR spectra were obtained on a
Bruker ARX-300 instrument using the indicated solvent. Please
refer to Smith et al. (2005) for the detailed protocol of synthesizing
methyl carbamate, ethyl carbamate, and tertbutyl carbamate
(tBoc).
All three 4-AP analogs were added to oxygenated Krebs’
solution and introduced to the injured cord through the central well
of the recording chamber. For initial testing all analogs were
presented to the cord at a concentration of 100 ?M, the most
effective concentration of 4-AP in vitro.
Statistical analysis
Throughout the paper, Student’s t-test was used to compare
electrophysiological data. Statistical significance was attributed to
values P?0.05. Averages were expressed as mean?standard
error.
RESULTS
After placement in the recording chamber the ventral white
matter strip was monitored for approximately 30–45 min or
until the CAP amplitude had maintained a stable baseline
recording. An additional 10 min was utilized to record
baseline measurements before the injury device was low-
ered. Immediately after inducing injury the amplitude of the
CAP was completely eliminated. CAP response subse-
quently began to increase, reaching a plateau approxi-
mately 30–45 min after injury (Fig. 1C).
CAP response following derivative administration
Initial testing revealed that these compounds had no sig-
nificant effect on CAP amplitude in the uninjured cord (data
not shown). During presentation of the derivatives to the
injured cord we observed an increase in CAP response
which plateaued 30–45 min after the initial application.
Fig. 3 displays the CAP response following administration
of ethyl, methyl, and tBoc. Ethyl carbamate at a concen-
tration of 100 ?M resulted in a gradual increase in the CAP
response which slowly declined after drug had been re-
moved (Fig. 3A). The trend line following application of
100 ?M methyl carbamate displayed a steeper CAP incli-
nation which also progressively declined after wash was
initiated (Fig. 3B). The most striking increase in CAP am-
plitude was observed after presentation of 10 ?M tBoc
(Fig. 3C). This lower concentration of 10 ?M was chosen
after a previous study indicated that 100 ?M tBoc ex-
hibited a significant decrease in CAP response (Smith et
al., 2005). Similar to the other two compounds, the
increase of CAP amplitude was largely reversible after
tBoc had been removed. Imposed above the trend lines
are representative action potentials for three stages of
the experiment: pre-drug, drug, and wash. These indi-
vidual action potential profiles showed no appreciable
difference for any of the compounds in the three dis-
played conditions (Fig. 3).
Overall, 100 ?M methyl carbamate improved CAP am-
plitude 16.27%?3.15 (n?7, P?0.001), 100 ?M ethyl car-
bamate improved CAP amplitude 7.86%?1.90 (n?7,
P?0.002), and 10 ?M tBoc produced similar results in-
creasing CAP response 14.88%?2.89 (n?6, P?0.001)
(Fig. 4). After examining the single CAP response following
administration of the derivatives, multiple response analy-
Fig. 2. Molecular structure demonstrating the equilibrium of 4-AP in aqueous solution and the tested derivatives tBoc, ethyl carbamate, and methyl
carbamate. Each of the derivatives is similar in structure to 4-AP however, the side groups are modified.
J. M. McBride et al. / Neuroscience 148 (2007) 44–5246

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sis was performed to further define the effect of these
compounds on basic nerve function.
Derivatives had no significant effect on
activation threshold
Superimposed images in Fig. 5A exhibit the changes of
CAP amplitude in response to 100 ?M methyl carbamate
at different stimulus intensities before and after drug ap-
plication. During a wide range of stimulus intensities (1.85–
6.5 V), CAP amplitude in the presence of methyl carbam-
ate is proportionally higher than before methyl carbamate
application (Figs. 4, 5). This relationship is demonstrated in
absolute terms in Fig. 6. The near unity of the slope indi-
cates that the enhancement in conduction in response to
methyl carbamate application was not biased toward ax-
ons with low or high thresholds (Fig. 6). Similar results
were found when 100 ?M ethyl carbamate and 10 ?M tBoc
were administered. Specifically, R2values for methyl car-
bamate, ethyl carbamate, and tBoc are 0.95, 0.99, and
0.99 respectively.
Dual and multiple stimuli
Fig. 7A displays the relationship between the interstimulus
interval (0.5–13 ms) and the amplitude of the two elicited
CAPs. We have found that the ability of the cord to respond
to dual stimuli presented at different time intervals was not
altered after application of the derivatives. A plot of the
second CAP against the log of the interstimulus interval, in
response to 100 ?M methyl carbamate, illustrates this
point (Fig. 7B). The CAP response as a function of
stimulus interval completely overlapped during pre-drug,
drug, and wash periods (Fig. 7B). An analogous phe-
nomenon was observed in response to 100 ?M ethyl
carbamate and 10 ?M tBoc (data not shown). Further
examination indicates that the absolute and relative re-
fractory period in response to the three derivatives did not
change significantly during pre-drug, drug, and wash periods
(Fig. 8A–C).
Fig. 4. Increase in CAP amplitude as a percent of pre-drug response
after stretch injury to the guinea-pig spinal cord. All compounds in-
creased CAP response significantly. Ethyl carbamate administered at
a concentration of 100 ?M increased CAP amplitude 7.86%?1.90
(P?0.002). 10 ?M tBoc also increased CAP response 14.88%?
2.89 (P?0.0005). The most significant increase in CAP, 16.27%?3.15
(P?0.0003), was observed with application of methyl carbamate at a
concentration of 100 ?M.
Fig. 3. Trend line representation of CAP amplitude in response to
pyridine compound administration after mechanical injury to the guin-
ea-pig spinal cord. Trend lines indicate a record of CAP amplitude over
time. (A) CAP response to 100 ?M ethyl carbamate. Note the slow
rising phase after administration of ethyl carbamate, and gradual
decline during wash. (B) CAP response to 100 ?M methyl carbamate.
The CAP here exhibited a steeper increase in response to drug
application with a gradual decline after wash. (C) Similar trend line
representation of CAP response as in A and B however, here 10 ?M
tBoc displayed a marked increase in amplitude similar to B at a lower
concentration. A gradual decline in response was also noted after
wash had ensued. Shown above the figures are examples of wave-
forms pre-drug, during drug administration, and after wash. All wave-
forms were taken when the CAP response had stabilized.
J. M. McBride et al. / Neuroscience 148 (2007) 44–5247

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We also examined changes in the ability of the cord to
follow repetitive stimuli following application of the deriva-
tives. An example of CAP response to a train of stimuli
(500 Hz for 100 ms) is shown in Fig. 9A. The average
amplitude of the last four CAPs in response to a train of
stimuli at 500 Hz/100 ms or 1000 Hz/100 ms, in both
pre-drug and drug conditions is displayed (Fig. 9B–D). The
CAP response to stimuli of higher or lower frequency is not
affected by application of the tested derivatives (P?0.05 in
all comparisons, pre-drug vs. drug, Fig. 9B–D).
DISCUSSION
Stretch injury to spinal cord white matter
Stretch is an important component of mechanical injury to
the spinal cord (Blight and Decrescito, 1986; Shi and
Pryor, 2002). Compared with existing stretch models in live
animals and in monolayer tissue culture system (Maxwell
et al., 1991; Smith et al., 1999; Bain et al., 2001), the in
vitro, or so called ex vivo, spinal cord stretch model em-
ployed in this study has several advantages. First, com-
pared with the examination using monolayer cell culture;
this system analyzes a whole tissue sample, which is more
clinically relevant. Unlike monolayer tissue cultures, axons
within the spinal cord white matter are densely packed
together, a factor likely affecting the behavior of axons
subjected to stretch. By using isolated white matter strips,
we can injure the spinal axons in a preparation closer to an
in vivo condition. Therefore, the information derived from
the current model is relevant to the in vivo spinal cord
injury. Second, compared with in vivo stretch models, this
ex vivo stretch preparation provides increased environ-
mental control and greater accessibility of the cord. This
allows us to control the degree of stretch (strain) and the
speed of stretch (strain rate) in order to mimic stretch
injuries in various real life situations. Furthermore, by in-
creasing environmental control, this ex vivo preparation is
Fig. 5. Comparison of response amplitude at different stimulus inten-
sities following stretch injury in the untreated and 100 ?M methyl
carbamate treated cord. Each condition is represented (A) in the form
of superimposed recordings and (B) a line graph. The data points in B
represent an average response of seven white matter strips. Stimulus
intensities applied to the cord ranged from 1.85–6.5 V. The response
amplitude of the treated cords differed significantly from the untreated
cords (P?0.03) from stimulus intensities 2.5–6.5 V. Similar observa-
tions were made after application of 100 ?M ethyl carbamate and
10 ?M tBoc (data not shown).
Fig. 6. Normalized CAP response of the injured spinal cord plotted
before and after treatment with 100 ?M methyl carbamate. Original
data for this figure are the same as for Fig. 4B with seven white matter
strips exposed to stimulus intensities ranging from 1.85–6.5 V. Overall
axons with different stimulus thresholds display a similar response to
drug-mediated amplitude enhancement. A similar trend was observed
after application of 100 ?M ethyl carbamate and 10 ?M tBoc (data not
shown).
J. M. McBride et al. / Neuroscience 148 (2007) 44–5248