Biomarkers for severity of spinal cord injury in the cerebrospinal fluid of rats

Abstract

One of the major challenges in management of spinal cord injury (SCI) is that the assessment of injury severity is often imprecise. Identification of reliable, easily quantifiable biomarkers that delineate the severity of the initial injury and that have prognostic value for the degree of functional recovery would significantly aid the clinician in the choice of potential treatments. To find such biomarkers we performed quantitative liquid chromatography-mass spectrometry (LC-MS/MS) analyses of cerebrospinal fluid (CSF) collected from rats 24 h after either a moderate or severe SCI. We identified a panel of 42 putative biomarkers of SCI, 10 of which represent potential biomarkers of SCI severity. Three of the candidate biomarkers, Ywhaz, Itih4, and Gpx3 were also validated by Western blot in a biological replicate of the injury. The putative biomarkers identified in this study may potentially be a valuable tool in the assessment of the extent of spinal cord damage.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Experimental Outline.

Figure 2. Injury assessment.

(A) Influence of 1.3-mm and 1.7-mm displacement injuries at C5 on forelimb usage during rearing. A contusion injury with a tissue displacement of 1.3-mm produced a ‘mild’ injury with forelimb motor deficits that recovered over time. The more ‘severe’ 1.7-mm displacement injury produced nearly complete loss of forelimb usage that was sustained until the end of the study. Error bars indicate standard error of the mean (SEM). “*” significantly different from sham group, p≤0.05; # 1.7-mm groups significantly different from 1.3-mm group, p≤0.05. (B) Histological outcome to the spinal cord following contusion injury with 1.3-mm or 1.7-mm tissue displacement 6–10 weeks after injury. Example image of eriochrome cyanine- and neutral red-stained coronal section of the injury at the epicenter. Scale bar: 500 µm.

Figure 3. Quantitative proteomic analysis of CSF from injured animals.

(a) Relative protein abundances for all proteins detected in rat CSF 24 h after sham, moderate or severe SCI. Labeled data points represent potential biomarkers of SCI severity (Tables 3 and 4). Error bars represent standard error of the mean (SEM). (b) Representative examples of MS spectra from the indicated peptides. Open arrowheads point to the monoisotopic peaks from light (sham), medium (moderate) and heavy (severe) conditions.

Figure 4. Western blot validation of candidate biomarkers of spinal cord injury.

(a) pooled sample of CSF from 12 sham injury rats (C); pooled sample of CSF from 12 moderate injury rats (M); pooled sample of CSF from 12 severely injured rats (S). The chart on the right shows quantification of the Western blots of moderately and severely injured rats relative to sham injury, (b) three pools of CSF from four sham, moderate and severe injury rats, C1–C3, M1–M3, S1–S3, respectively. The chart on the right shows quantification of the Western blots relative to sham injury. Each data point represents mean of three samples; error bars – standard error of the mean.

Figure 5. Results of LC-MS/MS data analysis using core module of the Ingenuity Pathways Analysis (IPA) software package.

The LC-MS/MS data set was filtered for proteins present in CSF only. The proteins from this filtered dataset that met the 1.5 fold cut-off criteria and were associated with biological functions in the IPA Knowledge Base were considered for the analysis. A p-value determining the probability that each biological function assigned to that data set is due to random chance alone was calculated using right-tailed Fischer's exact test. P-values