Prolonged Cerebrospinal Fluid Neurofilament Light Chain Increase in Patients with Post-Traumatic Disorders of Consciousness

Abstract

The mechanisms involved in secondary brain injury after the acute phase of severe traumatic brain injury (TBI) are largely unknown. Ongoing axonal degeneration, consequent to the initial trauma, may lead to secondary brain injury. To test this hypothesis, we evaluated the cerebrospinal fluid (CSF) level of neurofilament light chain (NF-L), a proposed marker of axonal degeneration, in 10 patients who developed a severe disorder of consciousness after a TBI, including 7 in a minimally conscious state and 3 with unresponsive wakefulness syndrome (time since brain injury, 309 ± 169 days). CSF NF-L level was measured with a commercially available NF-L enzyme-linked immunosorbent assay. CSF NF-L level was very high in all 10 patients, ranging from 2.4- to 60.5-fold the upper normal limit (median value, 4458 pg/mL; range, 695–23,000). Moreover, NF-L level was significantly higher after a severe TBI than in a reference group of 9 patients with probable Alzheimer's disease, a population with elevated levels of CSF NF-L attributed to neuronal degeneration (median value, 1173 pg/mL; range, 670–3643; p < 0.01). CSF NF-L level was correlated with time post-TBI (p = 0.04). These results demonstrate prolonged secondary brain injury, suggesting that patients exhibit ongoing axonal degeneration up to 19 months after a severe TBI.

Introduction

Diffuse axonal injury is a common neuropathology in patients who are left with severe disabilities after a traumatic brain injury (TBI). It impairs axonal transport, resulting in protein accumulation in axonal swellings within hours of trauma.¹ Although damage to axons may play a role in the immediate loss of consciousness and cognitive dysfunctions characteristic of TBI, the contributions of persistent axonal degeneration to prolonged disorders of consciousness or severe cognitive dysfunctions have yet to be determined. Notably, several studies suggested that TBI may trigger persistent neurodegenerative processes and TBI has been proposed as a risk factor for the development of Alzheimer's disease (AD).²

Neurofilaments (NFs) are the dominant intermediate filaments of the neural cytoskeleton.³ NFs are obligate heteropolymers, comprised of light (NF-L), medium, and heavy molecular weight proteins. NFs play an important role in maintaining axon diameter and morphology, which are critical for proper axonal conductance.⁴ Because NFs are released into the extracellular fluid subsequent to axonal injury or degeneration, NF-L in cerebrospinal fluid (CSF) is a proposed biomarker of axonal injury.⁵ CSF levels of NF-L within a few days of trauma have been related to injury severity and outcome in patients with TBI.⁶ However, CSF NF-L has not yet been evaluated as a biomarker of prolonged secondary axonal injury in the months after severe TBI.

In this study, we evaluated CSF NF-L levels in a convenience sample of patients with severe and prolonged disorders of consciousness caused by TBI, including unresponsive wakefulness syndrome (UWS) and minimally conscious state (MCS). We hypothesized that we would detect high levels of NF-L in CSF samples from these patients, thereby revealing evidence of long-term axonal degeneration as a component of secondary brain injury after severe TBI. Moreover, we compared CSF NF-L levels in patients with post-traumatic disorders of consciousness with those obtained from a group of patients with probable AD, expected to have high NF-L levels because of their neurodegenerative disease.

Methods

Protocol approval and consent to participate in the study

The Regional Ethical Review Board (Palermo 2 Ethical Committee, Palermo, Italy) approved this study (approval number 042004/2016). Patients' legal guardians gave their written informed consent to all procedures.

Patients

We recruited 10 patients admitted to our Unit for Severe Acquired Brain Injuries for intensive rehabilitation after a severe TBI, including 3 with UWS and 7 in a MCS after a coma (Table 1). All patients underwent a standard neurological examination and assessment with the Coma Recovery Scale Revised (CRS-R).⁷ The CRS-R provides criteria for a diagnosis of UWS, MCS, and emergence from MCS. It has been identified as the most reliable tool with which to assess patients with disorders of consciousness after a coma in both clinical practice and research.⁸ The CRS-R consists of 29 hierarchically organized items grouped into six subscales addressing auditory, visual, motor, oromotor/verbal, communication, and arousal functions. Each subscale provides a range of scores that allow discrimination among UWS, MCS, and emergence from MCS through clinical diagnostic criteria.⁷ We recruited patients who underwent to a lumbar puncture during their hospitalization as a standard diagnostic or therapeutic procedure, specifically to test the efficacy of intrathecal administration of baclofen in 4 cases, to conduct a CSF tap test in 4 cases, and to exclude CNS infections in 2 cases. The inclusion criteria were: 1) diagnosis of UWS or MCS after a TBI at the time of lumbar puncture execution and 2) a TBI-to-lumbar puncture interval greater than 90 days. Patients were not included if a CSF tap test indicated an improvement in their level of consciousness or if their CSF analysis revealed a current central nervous system (CNS) infection. Patients with a history of prior neurological diseases were not included.

Table 1.

Patients' Demographic, Clinical, and NF-L Characteristics

Pt	Age (years)	Cause of brain injury	Time post-TBI (days)	CT/MRI data at CSF NF-L sampling	Reason for lumbar puncture	Disorder of consciousness	CRS-R score (subscores)	CSF NF-L (pg/mL)^a	NF-L increase^b
1	41.8	Motor vehicle accident	95	Diffuse axonal injury	To exclude suspected CNS infection	MCS	20 (Au4, V5, M5, O3, C1, Ar2)	9402	11.3 times
2	22.5	Motor vehicle accident	465	Hydrocephalus, large ischemic lesion in the right hemisphere	CSF tap test for predicting shunt responsiveness	MCS	19 (Au3, V4, M5, O3, C1, Ar3)	5120	17.7 times
3	33.3	Motor vehicle accident	159	Diffuse axonal injury	Intrathecal baclofen test	MCS	10 (Au2, V3, M2, O1, C0, Ar2)	23,000^a	60.5 times
4	50.9	Fall	130	Hydrocephalus	CSF tap test for predicting shunt responsiveness	MCS	11 (Au2, V3, M3, O1, C0, Ar2)	22,500^a	27.1 times
5	38	Motor vehicle accident	581	Diffuse axonal injury	Intrathecal baclofen test	MCS	13 (Au2, V3, M3, O2, C1, Ar2)	2290	6 times
6	18.2	Motor vehicle accident	305	Ischemic lesions in both hemispheres, basal ganglia, and mesencephalon, hydrocephalus	To exclude suspected CNS infection	UWS	4 (Au0, V0, M2, O0, C0, Ar2)	2675	9.2 times
7	40.9	Motor vehicle accident	272	Diffuse axonal injury	Intrathecal baclofen test	UWS	5 (Au1, V0, M1, O1, C0, Ar2)	3796	10 times
8	19	Motor vehicle accident	186	Left frontal ischemic lesion, hydrocephalus	CSF tap test for predicting shunt responsiveness	UWS	6 (Au1, V1, M1, O1, C0, Ar2)	2097	7.2 times
9	56.8	Motor vehicle accident	492	Diffuse axonal injury, right temporal ischemic lesion	Intrathecal baclofen test	MCS	7 (Au0, V3, M2, O0, C0, Ar2)	5235	6.3 times
10	25.2	Motor vehicle accident	402	Diffuse axonal injury, hydrocephalus	CSF tap test for predicting shunt responsiveness	MCS	9 (Au1, V3, M2, O1, C0, Ar2)	695	2.4 times

Samples that yielded results >10,000 pg/mL were re-tested (see Methods for details).

Relative to age-matched upper normal limits indicated by the NF-L ELISA kit manufacturer.

CNS, central nervous system; CRS-R, Coma Recovery Scale Revised (CRS-R subscale abbreviations: Au, auditory; V, visual; M, motor; O, oro-motor/verbal; C, communication; Ar, arousal); CSF, cerebrospinal fluid; CT, computed tomography; ELISA, enzyme-linked immunosorbent assay; MCS, minimally conscious state; MRI, magnetic resonance imaging; NF-L, neurofilament light chain; Pt, patient; TBI, traumatic brain injury; UWS, unresponsive wakefulness syndrome.

CSF NF-L levels were compared with those obtained from 9 patients without a history of TBI who were labeled diagnostically as probable AD according to the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer's Disease and Related Disorders Association criteria. All of these patients were referred to our center for AD for their first diagnosis of dementia, which was made by neurologists trained in the evaluation of patients with dementia. The Clinical Dementia Rating score was 0.5 (very mild dementia) in 1 patient, 1 (mild dementia) in 6, and 3 (severe dementia) in 2.

Cerebrospinal fluid neurofilament light chain measurement

CSF was obtained by lumbar puncture, and CSF NF-L level was measured with a commercially available NF-L enzyme-linked immunosorbent assay (ELISA) kit (Uman Diagnostics, Umeå, Sweden), according to the manufacturer's instructions. The sensitivity of the NF-L assay is 32 pg/mL. The upper limits of normal NF-L ranges are: 290 (<30 years), 380 (30–39 years), and 830 pg/mL (40–59 years). Samples for which ELISAs indicated levels >10,000 pg/mL were subjected to further testing; they were diluted 1:100 in dilution buffer and reassayed with the final result factoring in the dilution.

Statistical analysis

Demographic and clinical data are expressed as mean ± standard deviation; NF-L levels are expressed as median, lowest, and highest values. We used the Mann-Whitney U test to compare demographic characteristics and NF-L levels between patients with disorders of consciousness post-TBI and the reference group with probable AD. We used the Spearman's rank-correlation test to examine whether NF-L percentage increase, with respect to age-matched upper normal limit, correlates with time post-TBI.

Results

The subjects of this study were 10 consecutive patients with disorders of consciousness post-TBI (all males; mean age, 34.6 ± 13.7 years; range, 18–56; mean interval post-TBI, 309 ± 169 days; range, 95–581). In the reference AD group, the mean time between the first symptoms of dementia and the diagnosis was 3.9 ± 2.8 years. Because CSF NF-L levels are higher in men than in women,⁹ patients with probable AD were sex-matched to TBI patients (all males; mean age, 72 ± 7.7 years).

Patients with disorders of consciousness were younger than the AD reference group (U = 1; p < 0.01). All of the patients with disorders of consciousness post-TBI were found to have very high CSF NF-L levels (median value, 4458 pg/mL; range, 695–23,000), ranging from 2.4- to 60.5-fold of the upper age-matched normal limit (Table 1). As shown in Figure 1, the CSF NF-L levels in patients with TBI were higher than those in the AD reference group (median value, 1173 pg/mL; range, 670–3643; U = 12; p < 0.01). Finally, CSF NF-L percentage increase correlated with time post-TBI (Spearman ρ = −0.65; p = 0.04).

FIG. 1.

NF-L levels in patients with TBI and controls. (A) CSF NF-L levels in patients with TBI exceeded that in patients with probable AD markedly. The upper and lower extremes of the whiskers indicate the highest and lowest NF-L levels observed. The 25th and 75th percentile and median value are shown as the lower and upper limits of the boxes and the line within each box, respectively (see Methods for age-matched NF-L normal limits). (B) Percent NF-L increase in patients with TBI correlated with time post-TBI. Value increases were determined in reference to age-matched upper normal limit for each patient. AD, Alzheimer's disease; CSF, cerebrospinal fluid; NF-L, neurofilament light chain; TBI, traumatic brain injury.

Discussion

The main finding of this study was that CSF NF-L levels were tremendously elevated in the present group of patients with disorders of consciousness after a TBI, relative to normal range values. The CSF NF-L levels in these patients were far greater than those with probable AD, despite the fact that the AD reference group had two characteristics that would predispose them to high CSF NF-L levels, namely more advanced age and a progressive neurodegenerative disease.⁹ The present data, obtained from samples collected more than 10 months post-TBI, reveal that a severe TBI may initiate persistent axonal degeneration.

After axons have suffered mechanical stretching, multiple mechanisms converge to produce acute axonal degeneration, including calcium-mediated cytotoxicity, mitochondrial dysfunction, and an inflammatory response, which involves the release of cytokines and chemokines.¹⁰ These mechanisms may account for CSF NF-L increases immediately after a TBI. A few studies have suggested that subsequent to the acute phase, TBI leads to persistent inflammatory mechanisms that can lead to axonal degeneration that lasts for months or even years.^11,12 In concordance, here, we found highly elevated CSF NF-L levels up to 19 months post-TBI, with a significant correlation between NF-L level and time post-injury. Together, these data suggest that TBI can result in long-term ongoing axonal degeneration, possibly mediated by ongoing inflammatory responses. The progressive decrease of NF-L levels suggests that the mechanisms leading to axonal degeneration tend to attenuate over time. However, these data should be considered with caution, given that it could be attributed to the small sample size. Indeed, the small sample size, variability in brain injuries, and lack of NF-L normative values obtained from healthy subjects are the main limitations of this study. This topic warrants further consideration in a larger number of patients with more homogeneous brain lesions, ideally evaluated in a study with a longitudinal design.

The CSF NF-L levels observed among the present group of patients with disorders of consciousness after severe TBI were highly variable. Although the biological significance of this difference has not been determined, we suppose that it may reflect differing degrees of axonal degeneration in different patients. Recovery from disorders of consciousness depends on the brain's ability to recover neural circuitry and functions involved in conscious behaviors.^13,14 Persistent axonal degeneration impairs the brain's ability to restore connectivity and synaptic transmission. Hence, it is reasonable to postulate that the magnitude of axonal degeneration caused by TBI affects the clinical outcomes of patients with severe disorders of consciousness.

Although AD is characterized primarily by gray matter loss, patients with AD also exhibit significant white matter changes,¹⁵ which likely account for the high CSF NF-L levels.⁹ TBI represents a major environmental risk factor for dementia, and epidemiological evidence indicates that both moderate and severe TBIs are associated with increased risk of developing AD.² After a single severe TBI, we found a persistent CSF NF-L increase, significantly higher than in patients with AD without a history of TBI. These data suggest that the axonal degeneration that develops after a single TBI can be more severe than that in patients with AD.

Experimental data show that some drugs, such as FK506, may attenuate NF compaction and axonal degeneration selectively post-TBI.¹⁶ The present evidence indicating that NF-L release into CSF can persist for up to 19 months post-TBI may be useful for planning the timing of interventions aimed at preventing secondary axonal degeneration.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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