(622e) Modified Cellulose Acetate to Sequester Acrolein for Neuropathic Pain Reduction

Modified Cellulose Acetate to Sequester Acrolein for
Neuropathic Pain Reduction

Introduction

The burden of spinal cord injuries (SCIs) is not only
impacted by the loss of motor function but also by the persistence of
neuropathic pain (NP) for decades after injury.  Despite much recent research, effective
analgesic treatments are not currently available for NP after SCI¹. 
Evidence suggests that NP can be affected by exogenous factors such as
cigarette smoke.  Several case studies have reported that cigarette smoke
exacerbates NP in people who have suffered an SCI^2,3. 
However, lifestyle changes involving cessation of cigarette smoking may be
difficult for a paraplegic due to the confinement, and depression in many
cases, they experience from the paralysis.  Further, this phenomenon of
increased pain due to cigarette smoke may lead to the discovery or confirmation
of theories regarding how NP develops and how it is intensified after spinal
cord injury. 

Cigarette smoke contains high concentrations of acrolein, a
neurotoxin that has been implicated in playing a major role in the pathogenesis
of SCI^4,5.
 In particular, endogenous concentrations of acrolein are significantly
elevated after spinal cord injury and acrolein inhalation in rodents, as well
as after cigarette smoking in humans^6,7. 
Acrolein is a direct agonist of the transient receptor protein ankyrin 1
(TRPA1) channel⁸,
which is a cation channel on sensory fibers that is crucial to the  perception
of thermal, chemical, and mechanical pain that has been shown to have increased
mRNA expression after SCI⁹. 
Therefore, it's very likely that cigarette-based acrolein ingestion is an
exogenous algesic factor contributing to NP.  As such, reducing acrolein uptake
is likely an effective way to curtail post-SCI pain.

The development of a filter to remove acrolein from
cigarette smoke may be beneficial to smokers who may be unable to quit and also
may provide useful information about the specific involvement of acrolein in
the exacerbation of NP as opposed to the collective involvement of the many
components of cigarette smoke.  Current cigarette filters are composed of
cellulose acetate and are mainly intended to capture particulate matter
including tar.  Previous studies have demonstrated the ability to perform
surface modification cellulose acetate with 3-aminopropyltriethoxysilane
(APTES)¹⁰. 
This study proposed that the stable surface attachment of APTES resulted a
multilayer formation, with exposed amine functional groups.  Amines have also
been shown to actively scavenge acrolein in many in vitro investigations¹¹. 
Therefore, it is reasonable to consider that chemically modifying a cellulose
acetate filter would result in a reduction of acrolein in cigarette smoke. 

The aim of this study is quantify the exacerbation of NP due
to acrolein inhalation after injury and to chemically modify cellulose acetate
in order to capture acrolein before entering the body to offer protection to
those who may be unable to quit smoking.  Spinal cord injury leaves the body
susceptible to acrolein-mediated damage, and increased exposure to acrolein
through cigarette smoke will result in sensory hypersensitivity.  Our
hypothesis is that this could be mitigated by eradicating acrolein through the
use of an acrolein filter. 

Materials and Methods

Cellulose Acetate Modification Prior to
polymer modification, mass spectrometry (MS) analysis was used to confirm
possible APTES-Acrolein interaction in a dilute IPA solution.  Cellulose
acetate films were modified using a procedure similar to one which was
previously reported ¹⁰. 
Briefly, 10% cellulose acetate films were suspended in anhydrous toluene to
which APTES (1% vol, final) was added under flowing nitrogen.  The reaction was
allowed to proceed for 48 hours.  X-ray photoelectron spectroscopy elemental
analysis was used to confirm APTES deposition onto the cellulose acetate
films.  To deposit APTES on actual cigarette filters, the filters from 3R4F
reference cigarettes were removed and opened to expose the fibrous cellulose
acetate.  The fibers were immersed in anhydrous toluene and the reaction was
allowed to be carried out as previously described.  Gas chromatography/mass
spectrometry and changes in the reduction of pain thresholds during animal
studies were used to quantify acrolein sequestering with the modified cellulose
acetate filter during inhalation.  

Animal Studies In order to test the efficacy
of the filters, animal studies were performed.  Briefly, moderate contusion
injuries were performed with an NYU-style impactor at the T-10 level on Sprague
Dawley male rats in accordance with Purdue Animal Care and Use Committee protocols. 
Mechanical pain testing occurred every other day beginning 14 days post injury
using von Frey filaments (0.01 to 15g) with the up-down method¹². 
Acrolein inhalation sessions were performed using aerosolized acrolein (300
ppm) in combination with compressed air fed at flow rates that resulted in a
final acrolein concentration of 1.5 ppm, which is in the range of acrolein
concentrations in actual cigarettes.  Inhalation of acrolein began 22 days
after injury and proceeded until 36 days post-injury, with sessions occurring
twice daily for 30 minutes.  Urine collection occurred once weekly beginning
two weeks after injury.  Liquid chromatography with tandem mass spectroscopy
(LC/MS/MS) was used to quantify the amount of 3-HPMA in urine⁷. 
Immediately after the 14 day inhalation period or one week after inhalation,
animals were sacrificed and spinal dorsal horn, dorsal root ganglia, and paw
skin tissues were harvested for RT-PCR and immunoblotting analysis for TRPA1
mRNA expression and acrolein-protein adduct quantification, respectively⁹. 

Preliminary Results       

In our preliminary studies, we have shown that acrolein
inhalation does indeed decrease the paw withdrawal threshold of animals after
SCI, indicating an increase in neuropathic pain.  Specifically, Figure 1 shows
that the paw withdrawal thresholds decreased by up to 80% for days 3-15 after
the start inhalation period (p<0.05) and were not significantly different
from the baseline results for days 17-21 (p>0.05).  Further, in these
animals, TRPA1 mRNA expression was increased during the inhalation period.  This
data supports the hypothesis that acrolein alone can exacerbate neuropathic
pain after SCI.  Acrolein's ability to both up-regulate and activate the TRPA1
channel make it a dual threat in the potentiation of neuropathic pain after
SCI.  To this end, removing acrolein, either endogenously through acrolein
scavengers or exogenously through acrolein filters, could alleviate neuropathic
pain for victims of SCI.  

Figure 1: Change in paw withdrawal thresholds to mechanical
stimuli from baseline over the course of 2 weeks of acrolein inhalation.

Preliminary mass spectrometry results indicate that there is
covalent bonding between acrolein and APTES in a dilute IPA solution (.025 vol%
APTES, 0.005 vol% acrolein).  The spectrum for APTES (MW=222) incubated with
acrolein (MW= 56) exhibited additional peaks at 278, 310, 366, and 398 m/z.   A
slight ion peak at 260 also existed in the spectrum.  The expected structure of
acrolein binding with APTES would have a molecular weight of 260.  The
additional peaks are a result of APTES-acrolein interactions in greater than a
1:1 stoichiometric fashion.  The ability of acrolein to covalently bind to
APTES indicates that the removal of acrolein with an APTES-modified cigarette
filter is feasible.  The sequestering of acrolein in this method could prevent
the ingestion of acrolein and offer protection from increased neuropathic pain
for those who have suffered spinal cord injuries.

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