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. 2018 Nov 1;35(21):2530-2539.
doi: 10.1089/neu.2018.5675. Epub 2018 Jul 5.

Increased Levels of Circulating Glial Fibrillary Acidic Protein and Collapsin Response Mediator Protein-2 Autoantibodies in the Acute Stage of Spinal Cord Injury Predict the Subsequent Development of Neuropathic Pain

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Increased Levels of Circulating Glial Fibrillary Acidic Protein and Collapsin Response Mediator Protein-2 Autoantibodies in the Acute Stage of Spinal Cord Injury Predict the Subsequent Development of Neuropathic Pain

Georgene W Hergenroeder et al. J Neurotrauma. .
Free PMC article

Abstract

Neuropathic pain develops in 40-70% of spinal cord injury (SCI) patients and markedly compromises quality of life. We examined plasma from SCI patients for autoantibodies to glial fibrillary acidic protein (GFAP) and collapsin response mediator protein-2 (CRMP2) and evaluated their relationship to the development of neuropathic pain. In study 1, plasma samples and clinical data from 80 chronic SCI patients (1-41 years post-SCI) were collected and screened for GFAP autoantibodies (GFAPab). Results from study 1 indicated that GFAPab were present in 34 of 80 (42.5%) patients, but circulating levels did not correlate with the occurrence of neuropathic pain. In study 2, longitudinal plasma samples and clinical data were collected from 38 acute SCI patients. The level of GFAPab measured at 16 ± 7 days post-SCI was found to be significantly higher in patients that subsequently developed neuropathic pain (within 6 months post-SCI) than patients who did not (T = 219; p = 0.02). In study 3, we identified CRMP2 as an autoantibody target (CRMP2ab) in 23% of acute SCI patients. The presence of GFAPab and/or CRMP2ab increased the odds of subsequently developing neuropathic pain within 6 months of injury by 9.5 times (p = 0.006). Our results suggest that if a causal link can be established between these autoantibodies and the development of neuropathic pain, strategies aimed at reducing the circulating levels of these autoantibodies may have therapeutic value.

Keywords: CRMP2; GFAP; autoantibodies; neuropathic pain; spinal cord injury.

Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
Capillary electrophoresis assay to measure GFAP antibodies. (A) Image of a representative capillary western showing the relative immunoreactivities of different concentrations of a commercial anti-GFAP antibody against a fixed amount (160 ng) of purified recombinant GFAP protein. No immunoreactivity was observed when the antibody was eliminated from the incubation mixture. Positive immunoreactivity was observed with the plasma of an SCI subject used as the primary antibody. (B) Summary data showing the relationship between immunoreactivity and concentration of the antibody, indicating the linearity of the detection. When the plasma (1:50 dilution) of an SCI study subject was used as the primary antibody, immunoreactivity within the linear range of the assay was observed (white square). Ab, antibody; anti-GFAP, antibody to GFAP; GFAP, glial fibrillary acidic protein; GFAPab, autoantibodies to GFAP; SCI, spinal cord injury.
<b>FIG. 2.</b>
FIG. 2.
Circulating levels of GFAPab in chronic SCI. (A) Representative image of capillary western showing the specificity of the GFAPab immunoreactivity. Plasma (1:50 dilution) was pre-incubated with either 160 ng/uL of GFAP or an equimolar amount of CRMP2. GFAPab immunoreactivity was abolished after pre-incubation of plasma with recombinant GFAP, but not recombinant CRMP2. No immunoreactivity was detected when the primary antibody was eliminated (lane 4, negative control). The commercial anti-GFAP antibody (lane 5) is shown as a positive control. (B) Box plot (25th and 75th percentile) showing the relative levels of GFAPab in healthy volunteers (HV) and chronic SCI subjects (cSCI). (C) Box plot showing the relative levels of GFAPab in chronic SCI patients with neuropathic pain (NP) and those without neuropathic pain (No NP). Error bars are the 90th and 10th percentile. Outliers are represented as white circles. Horizontal line indicates the median. Ab, antibody; anti-GFAP, antibody to GFAP; CRMP2, collapsin response mediator protein-2; GFAP, glial fibrillary acidic protein; GFAPab, autoantibodies to GFAP; SCI, spinal cord injury.
<b>FIG. 3.</b>
FIG. 3.
Circulating levels of GFAPab in acute SCI predict the development of neuropathic pain. (A) Box plots showing the relative levels of GFAPab in plasma from healthy volunteers (HV) and acute SCI (aSCI) study subjects. Levels of GFAPab were significantly elevated in plasma of aSCI subjects compared to healthy volunteers. (B) Box plots showing relative levels of GFAPab in SCI subjects who subsequently developed neuropathic pain (NP) and those who did not (No NP). Acute SCI subjects who subsequently developed neuropathic pain had significantly higher GFAPab levels than those without neuropathic pain. Error bars are the 90th and 10th percentile. Outliers are represented as white circles. Horizontal line indicates the median. (C) Time course of GFAPab development for a subset of 13 SCI patients with and without neuropathic pain who had plasma samples available at four time points from 1.7 ± 0.7 to 96 ± 54 days. Longitudinal samples were not available for all of our study subjects. However, we did not identify any subjects with GFAPab-negative results at the 16- ± 7 d time point that were GFAPab positive at earlier or later time points. GFAP, glial fibrillary acidic protein; GFAPab, autoantibodies to GFAP; SCI, spinal cord injury.
<b>FIG. 4.</b>
FIG. 4.
Identification of CRMP2 autoantibodies. (A) Representative images of western blots from 2D gels showing immunoreactivity when <3-day and 16-day plasma from the same patient was used as the primary antibody (1:1000). A marked increase in immunoreactive spots can be seen when the membrane was probed with the 16-day plasma. (B) Image of a 2D gel membrane that was double-labeled with 16-day post-SCI plasma (top, CRMP2ab; green) and a commercially available CRMP2 antibody (middle, anti-CRMP2; red). Merged image (bottom) showing colocalization of the CRMP2ab and the commercial anti-CRMP2 antibody. White arrows point to CRMP2ab spots not detected by the commercial anti-CRMP2 antibody. (C) Representative western blot showing CRMP2ab immunoreactivity after depletion of CRMP2 from human brain homogenate sample. CRMP2ab immunoreactivity (open arrowhead, brain PRE) is markedly reduced after CRMP2 immunodepletion (brain FT). (D) Representative image of capillary western showing the specificity of the CRMP2ab immunoreactivity. Plasma (1:100 dilution) was pre-incubated with either 36 ng/uL of CRMP2 or an equimolar amount of GFAP. CRMP2ab immunoreactivity was markedly decreased after pre-incubation of plasma with recombinant CRMP2, but not recombinant GFAP. No immunoreactivity was detected when the primary antibody was eliminated (lane 4, negative control). A strong signal was observed with the commercial CRMP2 antibody (lane 5) used as the primary antibody. 2D, two-dimensional; Ab, antibody; anti-CRMP2, antibody to CRMP2; CRMP2, collapsin response mediator protein-2; CRMPab, autoantibodies to CRMP2; GFAP, glial fibrillary acidic protein; SCI, spinal cord injury.

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