Modified Cellulose Acetate to Sequester Acrolein for Neuropathic Pain Reduction
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 SCI2,3. 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 SCI4,5. 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 humans6,7. 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.
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.
1. Hurlbert, R. J. Methylprednisolone for acute spinal cord injury: an inappropriate standard of care. J. Neurosurg. 93, 1–7 (2000).
2. Richards, J. S., Jr, S. C. K., Ness, T. J. & Oleson, C. V. Effects of Smoking on Neuropathic Pain in Two People With Spinal Cord Injury. 28, 330–332 (2005).
3. Botterell, E. H., Callaghan, J. C. & Jousse, A. T. Pain in Paraplegia. Proceeding R. Soc. Med. 47, 17–24 (1953).
4. Dong, J.-Z. & Moldoveanu, S. C. Gas chromatography–mass spectrometry of carbonyl compounds in cigarette mainstream smoke after derivatization with 2,4-dinitrophenylhydrazine. J. Chromatogr. A 1027, 25–35 (2004).
5. Chen, P. X. & Moldoveanu, S. C. Mainstream Smoke Chemical Analyses for 2R4F Kentucky Reference Cigarette. Contrib. to Tob. Res. 20, (2003).
6. Tully, M., Zheng, L., Acosta, G., Tian, R. & Shi, R. Acute systemic accumulation of acrolein through inhalation at a concentration relevant to cigarette smoke in mice. Neurosci. Bull. In Press, (2014).
7. Zheng, L. et al. Determination of urine 3-HPMA, a stable acrolein metabolite in a rat model of spinal cord injury. J. Neurotrauma 30, 1334–41 (2013).
8. Bautista, D. M. et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124, 1269–82 (2006).
9. Due, M. R. et al. Acrolein involvement in sensory and behavioral hypersensitivity following spinal cord injury in the rat. J. Neurochem. (2013). doi:10.1111/jnc.12500
10. Howarter, J. A. et al. Surface Modification of Polymers with 3-Aminopropyltriethoxysilane as a General Pretreatment for Controlled Wettability. 1128–1132 (2007).
11. Burcham, P. C., Kerr, P. G. & Fontaine, F. The Antihypertensive Hydralazine is an Effective Scavenger of Acrolein. Redox Rep. 5, 47–49 (2000).
12. Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M. & Yaksh, T. L. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55–63 (1994).
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division