Plasma Neurofilament Light Chain (NF-L) Is a Prognostic Biomarker for Cortical Damage Evolution but Not for Cognitive Impairment or Epileptogenesis Following Experimental TBI

Abstract

Plasma neurofilament light chain (NF-L) levels were assessed as a diagnostic biomarker for traumatic brain injury (TBI) and as a prognostic biomarker for somatomotor recovery, cognitive decline, and epileptogenesis. Rats with severe TBI induced by lateral fluid-percussion injury (n = 26, 13 with and 13 without epilepsy) or sham-operation (n = 8) were studied. During a 6-month follow-up, rats underwent magnetic resonance imaging (MRI) (day (D) 2, D7, and D21), composite neuroscore (D2, D6, and D14), Morris-water maze (D35−D39), and a 1-month-long video-electroencephalogram to detect unprovoked seizures during the 6th month. Plasma NF-L levels were assessed using a single-molecule assay at baseline (i.e., naïve animals) and on D2, D9, and D178 after TBI or a sham operation. Plasma NF-L levels were 483-fold higher on D2 (5072.0 ± 2007.0 pg/mL), 89-fold higher on D9 (930.3 ± 306.4 pg/mL), and 3-fold higher on D176 32.2 ± 8.9 pg/mL after TBI compared with baseline (10.5 ± 2.6 pg/mL; all p < 0.001). Plasma NF-L levels distinguished TBI rats from naïve animals at all time-points examined (area under the curve [AUC] 1.0, p < 0.001), and from sham-operated controls on D2 (AUC 1.0, p < 0.001). Plasma NF-L increases on D2 were associated with somatomotor impairment severity (ρ = −0.480, p < 0.05) and the cortical lesion extent in MRI (ρ = 0.401, p < 0.05). Plasma NF-L increases on D2 or D9 were associated with the cortical lesion extent in histologic sections at 6 months post-injury (ρ = 0.437 for D2; ρ = 0.393 for D9, p < 0.05). Plasma NF-L levels, however, did not predict somatomotor recovery, cognitive decline, or epileptogenesis (p > 0.05). Plasma NF-L levels represent a promising noninvasive translational diagnostic biomarker for acute TBI and a prognostic biomarker for post-injury somatomotor impairment and long-term structural brain damage.

Keywords: ROC analysis; fluid-percussion injury; post-traumatic epilepsy; rat; single molecule array (SIMOA).

Figure 1

Plasma NF-L concentrations at different time-points after a sham operation or TBI. (A) Box and whisker plots (whiskers: minimum and maximum; box: interquartile range; line: median) showing plasma NF-L levels (y-axis) in different groups (x-axis). Samples collected at baseline (n = 34) before any operation (BL on D-6) were considered comparable to naïve samples. In the sham group (n = 8, blue), plasma NF-L levels were analyzed on D2 post-operation only. In the TBI group (n = 26, orange), plasma NF-L levels were assessed on D2, D9, and D176 after TBI. Each dot represents 1 animal. Note the slightly elevated plasma NF-L levels in the sham-operated animals on D2 compared with their baseline values (** p < 0.01, Wilcoxon). In the TBI group, the NF-L levels were elevated on all testing days as compared to their baseline values [Friedman test (p < 0.001) followed by post hoc analysis with Wilcoxon: ***, p < 0.001]. On D2 and D9, the average NF-L levels were higher in the TBI group than in the sham group ($$$, p < 0.001, Mann-Whitney U test). (B) Dynamics of NF-L concentrations in individual TBI animals over time [Friedman test (p < 0.001) followed by post hoc analysis with Wilcoxon: ###, p < 0.001 compared with D2; +++, p < 0.001 compared with D9]. (C) Change in plasma NF-L levels as a percentage over time (D2 marked as 100%) [Friedman test (p < 0.001) followed by post hoc analysis with Wilcoxon: ###, p < 0.001 compared with D2; +++, p < 0.001 compared with D9]. Abbreviations: BL, baseline; D2, day 2 after TBI; D9, day 9; D176, day 176 (6 months); NF-L, neurofilament light chain; TBI, traumatic brain injury.

Figure 2

Plasma NF-L and cortical lesion severity in quantitative T₂ magnetic resonance imaging (MRI). (A) Box and whisker plots (whiskers: minimum and maximum; box: interquartile range; line: median) showing the total volume of abnormal pixels (cortical T₂ signal, y-axis) in rat brain MRI on D2, D7, and D21 after TBI (n = 26) or sham operation (n = 8). The TBI group included 13 rats without epilepsy (TBI−) and 13 rats with epilepsy (TBI+). Each dot represents 1 animal. (B) Spearman correlation between the plasma levels of NF-L (y-axis) and volume of abnormal T₂ area (x-axis) in MRI (TBI group only) on D2, D7, and D21. Note that on D2, the higher the NF-L level, the greater the volume of the abnormal T₂ area. No correlations were detected at later time-points. Statistical significance: ***, p < 0.001 as compared with the sham group (Mann-Whitney U test). Abbreviations: D2, day 2 after TBI; D9, day 9; D21, day 21; NF-L, neurofilament light chain; ns, not significant; TBI, traumatic brain injury.

Figure 3

Plasma NF-L as a diagnostic biomarker for sham operation and TBI. (A) ROC analysis indicated that NF-L levels on D2 distinguished sham-operated rats (n = 8) from the naïve condition (baseline samples, n = 34) with 100% sensitivity and 100% specificity (AUC 1.0, p < 0.001, cut-off 49 pg/mL (dashed line)]. (B) NF-L levels on D2 distinguished TBI rats (n = 26) from the naïve condition (n = 34) with 100% sensitivity and 100% specificity (AUC 1.0, p < 0.001, cut-off: 2201 pg/mL). (C) NF-L levels on D2 distinguished TBI (n = 13) and sham-operated rats (n = 13) with 100% sensitivity and 100% specificity (AUC 1.0, p < 0.001, cut-off: 2201 pg/mL). Abbreviations: AUC, area under the curve; BL, baseline; D2, day 2 post-TBI, NF-L, neurofilament light chain; ROC, receiver operating characteristics; TBI, traumatic brain injury.

Figure 4

Plasma NF-L and somatomotor performance after TBI. (A) Evolution of the neuroscore in each TBI rat (n = 26) over the 14-day follow-up (D2, D6, and D14) after TBI. Note that in the sham-group, the average neuroscore was 26.5 on D2, 27.4 on D6, and 27.5 on D14. (B) The optimal cut-off and ROC analysis indicated that the D2 neuroscore differentiated TBI (n = 26) and sham-operated rats (n = 8) with 100% sensitivity and 100% specificity [AUC 1.0, p < 0.001, cut-off 13.0 (dashed line)]. (C) Spearman’s correlation revealed that the higher the plasma NF-L levels on D2, the lower the neuroscore on D2 post-TBI (ρ = −0.480, p < 0.05). (D) Improvement of the neuroscore (recovery index) for each TBI rat (n = 26), compared with the neuroscore on D2. Statistical significances: ***, p < 0.001 compared to D2; $$$, p < 0.001 compared to D6 (Wilcoxon matched pairs signed rank test). Abbreviations: AUC, area under the curve; D2, day 2 post-TBI; D6, day 6, D14, day 14; NF-L, neurofilament light chain; ROC, receiver operating characteristics; TBI, traumatic brain injury.

Figure 5

Plasma NF-L and cognitive impairment after TBI. (A) Optimal cut-point analysis was performed in the EPITARGET animal cohort to identify parameters that differentiate cognitively impaired (CI+) and non-impaired (CI−) rats. The cut-point latency of 19.2 s (i.e., latency to reach the platform in the Morris water maze (MWM) on the 3rd day of testing, i.e., on D37, dashed line) differentiated TBI (n = 118) and sham-operated rats (n = 23) with AUC 0.94 (p < 0.001) and was set as a limit for cognitive impairment. (B) No correlation was detected between plasma NF-L levels on D2 and MWM latency on D37 (Spearman correlation rho (ρ) = 0.270, p > 0.05, n = 26). (C) Accordingly, ROC analysis indicated that plasma NF-L levels did not distinguish CI+ (latency > 19.2 s) from CI− (<19.2 s) rats (AUC 0.58, p > 0.05, Mann-Whitney U test). Abbreviations: AUC, area under the curve; D2, day 2 post-TBI; ROC, receiver operating characteristics; TBI, traumatic brain injury.

Figure 6

Plasma NF-L and epileptogenesis after TBI. (A) Box and whisker plots (whiskers: minimum and maximum; box: interquartile range; line: median) showing plasma NF-L levels (y-axis) in different groups and time-points (x-axis). Plasma NF-L levels on D2, D9, or D176 did not differ between rats that did (TBI+, n = 13) or did not develop epilepsy (TBI−, n = 13, p > 0.05). (B) Plasma NF-L levels on D2, D9, or D176 did not distinguish TBI+ and TBI− rats in the ROC analysis (p > 0.05, results for D2 shown in the figure). Statistical significance: ns, not significant (Mann-Whitney U test). Abbreviations: AUC, area under the curve; BL, baseline; D2, day 2 post-TBI; D9, day 9; D176, day 176; NF-L, neurofilament light chain; ROC, receiver operating characteristics.

Figure 7

Study design. (A) The first 34 rats [8 sham-operated experimental controls, and 26 with traumatic brain injury (TBI)] completing the 6-month follow-up in the EPITARGET animal cohort of 137 animals (23 sham, and 114 TBI) were included in the present analysis [22,37]. Within the TBI group, 13 rats exhibited no unprovoked seizures in video-encephalogram (vEEG) (TBI−). In 13 rats, we found at least 1 unprovoked seizure during the 6th month vEEG (TBI+). The number of animals was based on a power calculation. That is, we expected the plasma NF-L to separate the TBI− and TBI+ groups with AUC 0.800 (MedCalc software). (B) Timing of the tests included in the present analysis (for a complete study design, see [22]). During the 6-month follow-up, rats underwent blood sampling via the tail vein, behavioral tests (neuroscore and Morris water maze), in vivo quantitative T₂ magnetic resonance imaging (MRI), and 1-month continuous vEEG monitoring. Plasma levels of neurofilament light chain were assessed using a single molecule array (SIMOA). Abbreviations: D, day.

Figure 8

Cortical T₂ magnetic resonance imaging (MRI) analysis in the sham-operated experimental control group. (A) T₂ images of the sham animals were registered to a template brain. Then, the ipsilateral (left, blue outline) and the contralateral (right, orange outline) cortex was manually outlined in each image slice (thickness 0.5 mm). In each slice, a cortical profile of the T₂ relaxation time was measured, starting at the rhinal fissure and continuing dorsally towards the brain midline as indicated by the white arrow (bottom slice). (B) The cortical T₂ profiles of different slices were combined, filtered using a 2-dimensional isotropic Gaussian filter with a standard deviation of 0.5 mm, and interpolated on a grid with a 0.5 × 0.5 mm² resolution. We found an ipsilateral signal increase on D2, D7, and D21 in areas close to the rhinal fissure at the rostrocaudal level (-3.5 mm from the bregma) as the median of the ipsilateral T₂ area was increased compared with that contralaterally. We also found a parasagittal signal decrease on D7. Color coding of the heatmap: red indicates a positive ipsilateral vs. contralateral difference in T₂ (higher T₂ ipsilaterally), and blue indicates a negative difference (higher T₂ contralaterally). An asterisk (*) at a given grid point indicates a statistically significant difference for the median T₂ (Mann-Whitney U-test, after correcting for multiple comparisons using the Benjamini-Hochberg procedure with an average false discovery rate of 0.05).