pNF-H Levels Research Articles

Abstract TMP72: L-carnitine Enhances Axonal Plasticity Via Akt/mammalian Target Of Rapamycin Pathway In A Rat Chronic Cerebral Hypoperfusion

Background and Purpose: Chronic cerebral hypoperfusion (CCH) induces white matter lesions with oligodendrocytic damage and causes cognitive impairment. Oxidative stress has been elucidated as a pivotal role of its pathophysiology. Damage of axon can be also involved in CCH, however, mechanisms that associated with axonal damage under CCH have not been fully studied. In the present study, we analyzed axonal plasticity and damage of oligodendrocytes in a rat CCH model using L-carnitine, antioxidant agent. Methods: Adult rats subjected to ligation of bilateral common carotid artery (LBCCA), were randomly divided into two groups: vehicle-treated rats; and L-carnitine-treated rats (600mg/kg/day), and were sacrificed at 7, 14, 21 and 28 days after LBCCA (n=7/group). Sham operated rats (n=7) were used as a control group. The Morris water maze was performed to evaluate spatial learning and memory tasks, and immunohistochemical and Western blot analysis were studied. Results: Rats subjected to LBCCA substantially increased escape latency on the Morris water maze in a time-dependent fashion. L-carnitine-treated rats significantly reduced escape latency compared to vehicle-treated rats at 28 days after LBCCA (P<0.05). Protein levels of phosphorylated neurofilament heavy protein (pNFH), a marker of axons, were reduced after LBCCA, however, L-carnitine significantly increased pNFH levels compared to levels in vehicle-treated rats at 28 days after LBCCA (P<0.01). Concurrently, increases of phosphorylated Akt (P<0.05) and mammalian target of rapamycin (mTOR) (P<0.05) were detected in L-carnitine-treated rats compared to vehicle-treated rats at 28 days after LBCCA. 4-hydroxy-2-nonenal (HNE)+ and 8-hydroxy-deoxyguanosine (8-OHdG)+, markers of oxidative stress, oligodendrocytes substantially increased until 28 days after LBCCA (P<0.001). L-carnitine-treated rats significantly reduced HNE+ and 8-OHdG+ oligodendrocytes at each time point after LBCCA (P<0.001). Conclusion: Our results indicated that L-carnitine increased axonal plasticity via Akt/mTOR pathway and ameliorated oxidative stress in oligodendorocytes damaged by LBCCA, and thereby resulting an improvement of cognitive impairment in a rat CCH model.

Expression of serum IL-6,TNF-α,ICAM-1,pNF-H and NSE in patients with acute brain injury

Objective To explore the expression and significance of ser(u)m IL-6,TNF-α,ICAM-1,pNF-H and NSE in patients with acute brain injury.Methods 100 patients with acute brain injury were selected and divided into different groups(mild injury,moderate injury,severe injury).The serum levels of IL-6,TNF-α,ICAM-1,pNF-H and NSE were detected.30 healthy persons were selected as healthy control group.Results The serum IL-6,TNF-α,ICAM-1,pNF-H and NSE levels in the patients were higher than those in the healthy control groups(all P＜0.05).The serum levels of them were increased steply with the order of severity(all P＜0.05).IL-6,TNF-α,ICAM-1 were positive correlated with pNF-H and NSE(all P＜0.05).Conclusion The serum levels of IL-6,TNF-α,ICAM-1,pNF-H and NSE were correlated with degree of injury severity.The measurement of them may contribute to predict the severity of brain injury. Key words: Acute brain injury; Inflammafory factors; pNF-H; NSE

Phosphorylated neurofilament heavy subunit (pNF-H) in peripheral blood and CSF as a potential prognostic biomarker in amyotrophic lateral sclerosis

BackgroundThe phosphorylated neurofilament heavy subunit (pNF-H), a major structural component of motor axons, is a promising putative biomarker in amyotrophic lateral sclerosis (ALS) but has been studied mainly in CSF....

PNfH is a promising biomarker for ALS

A diagnostic biomarker for ALS would permit early intervention with disease-modifying therapies while a biomarker for disease activity could accelerate the pace of drug discovery by facilitating shorter, and less costly, drug trials to be conducted with a smaller number of patients. Neurofilaments are the most abundant neuronal cytoskeletal protein. We set out to determine whether pNfH was a credible biomarker for ALS. pNfH levels were determined using an ELISA for 150 ALS subjects and 140 controls. We demonstrated a seven-fold elevation in the cerebrospinal fluid (CSF) levels of phosphorylated neurofilament heavy subunit (pNfH) in ALS (median = 2787 pg/ml, n = 150), compared to headache and other benign controls (394 pg/ml, n = 100, p = < 0.05). There was a 10-fold elevation of pNfH compared to ALS mimics (266 pg/ml, n = 20) and other neurodegenerative and inflammatory conditions (279 pg/ml for n = 20) which was also highly significant (p = < 0.05). pNfH achieved a diagnostic sensitivity of 90% and specificity of 87% in distinguishing ALS from all controls. We also detected an inverse correlation between CSF pNfH levels and disease duration (time from symptom onset to death, r2 = 0.1247, p = 0.001). In conclusion, pNfH represents a promising candidate for inclusion in a panel of diagnostic and prognostic biomarkers.

Serum neurofilament concentrations in children with prolonged febrile seizures

ObjectiveNeurofilament (NF) is a major cytoskeletal protein of neurons. Elevation of serum NF concentration suggests neuronal injury, especially damage to axons. We assessed neuronal damage in febrile seizure (FS) by using NF. MethodsWe used enzyme-linked immunosorbent assay to measure serum levels of the phosphorylated form of NF-heavy chain (pNF-H) in 42 children with FS and 28 controls. ResultsSerum pNF-H levels in children with prolonged febrile seizure (PFS) (n=29) were significantly higher than those in the controls (p<0.001). There were no significant differences in serum pNF-H levels between children with simple febrile seizure (n=9) and the controls. There was a significant correlation between seizure duration and serum pNF-H levels during the first week in children with FS (p=0.022, r=0.37). ConclusionOur results suggest that PFS could lead to some degree of neuronal damage even in the absence of abnormal clinical neurological findings during the short-term follow up period.

Elevated levels of S100B, tau and pNFH in cerebrospinal fluid are correlated with subtypes of Guillain–Barré syndrome

Guillain-Barré syndrome (GBS) is an immune-mediated inflammatory disease in the peripheral nervous system. Specific biomarkers for the two most common clinical subtypes of GBS, i.e., acute inflammatory demyelinating polyneuropathy (AIDP) and acute motor axonal neuropathy (AMAN) are still missing. The distinctive pathological features of AIDP and AMAN may lead to release of such specific biomarkers including glial markers (calcium-binding astroglial protein, S100B) and axonal damage markers [axoskeletal protein, phosphorylated neurofilament heavy protein (pNFH); cytoskeletal protein, tau], etc. To explore the potentials of biochemical markers for differential diagnosis and evaluation of prognosis of clinical subtypes in GBS, we used ELISA to measure the levels of S100B, tau and pNFH in serum and cerebrospinal fluid (CSF) from the patients with AIDP, AMAN, viral encephalitis and other non-inflammatory neurological disorders (OND), respectively. The values of albumin quotient and IgG index in CSF are significantly higher in AIDP and AMAN than in OND. The levels of S100B, tau and pNFH in serum and CSF are elevated in the patients with AIDP and AMAN compared to OND. The concentrations of these proteins are all higher in CSF than in serum. Increased levels of S100B in CSF at the acute phase are positively correlated with the GBS disability scale scores (GDSs) in AIDP, whereas enhanced levels of tau and pNFH in CSF are positively correlated with the GDSs in AMAN. Increased CSF levels of S100B, tau and pNFH at the acute phase may predict a poor prognosis and evaluate the severity of AIDP or AMAN at plateau and the recovery phase. Elevated levels of pNFH in CSF may be used for differentiating between AMAN and AIDP.

P.4.015 Gene expression profile associated with major depression and with antidepressant response

It has been proposed that depression is associated with dysfunction of hippocampal plasticity. Novel hypotheses suggest that antidepressants induce neuronal structural plasticity, although the underlying mechanisms still remain unclear. Therefore, the aim of this study was to investigate the effects of amitriptyline on levels of phosphorylated heavy neurofilament subunit (NF-H) in the hippocampus of mice exposed to acute and chronic behavioral despair paradigms. Immunoblotting experiments showed that animals exposed to the tail suspension test (TST) displayed diminished levels of pNF-H 24 h after testing. Repeated administration of amitriptyline (10 mg/kg i.p.) prevented this decreased hippocampal phosphorylation of NF-H. Conversely, administration of citalopram (10 mg/kg i.p.) left unchanged pNF-H levels. The expression of pNF-H was also analyzed by immunofluorescence in mice exposed to the unpredictable chronic mild stress paradigm (UCMS), an experimental model of depression. Mice that developed a depressive-like behavior showed a decreased pNF-H immunostaining selectively in the hippocampal CA3 region. Chronic administration of amitriptyline reversed the despaired behavior induced by exposure to UCMS paradigm and, fully recovered pNF-H labeling to control values. Our results indicate that the cytoskeletal remodeling induced by amitriptyline in the hippocampal CA3 region might underpin its behavioral efficacy. Hippocampal alterations of the NF appeared associated with the mechanism of this antidepressant drug and may contribute to its psychotherapeutic actions.

A Pilot Trial of Pioglitazone HCl and Tretinoin in ALS: Cerebrospinal Fluid Biomarkers to Monitor Drug Efficacy and Predict Rate of Disease Progression

Objectives. To determine if therapy with pioglitazone HCl and tretinoin could slow disease progression in patients with ALS. Levels of tau and pNFH in the cerebrospinal fluid were measured to see if they could serve as prognostic indicators. Methods. 27 subjects on stable doses of riluzole were enrolled. Subjects were randomized to receive pioglitazone 30 mg/d and tretinoin 10 mg/BID for six months or two matching placebos. ALSFRS-R scores were followed monthly. At baseline and at the final visit, lumbar punctures (LPs) were performed to measure cerebrospinal fluid (CSF) biomarker levels. Results. Subjects treated with tretinoin, pioglitazone, and riluzole had an average rate of decline on the ALSFRS-R scale of −1.02 points per month; subjects treated with placebo and riluzole had a rate of decline of −.86 (P = .18). Over six months of therapy, CSF tau levels decreased in subjects randomized to active treatment and increased in subjects on placebo. Further higher levels of pNF-H at baseline correlated with a faster rate of progression. Conclusion. ALS patients who were treated with tretinoin and pioglitazone demonstrated no slowing on their disease progression. Interestingly, the rate of disease progression was strongly correlated with levels of pNFH in the CSF at baseline.

Sigma-1 receptor ligand PRE-084 reduced infarct volume, neurological deficits, pro-inflammatory cytokines and enhanced anti-inflammatory cytokines after embolic stroke in rats

Sigma receptor agonists have been found to provide potent neuroprotection in rats and mice. This neuroprotection is thought to be mediated through anti-excitotoxic mechanisms. Neuroprotective and immune modulatory effects of sigma ligands have not been investigated in embolic stroke. In the present study, rats were subjected to embolic stroke or sham stroke and were treated with the sigma-1 receptor agonist PRE-084 (5 mg/kg i.p.) or saline vehicle 3 and 24 h after stroke. Infarct volume and behavioural tests were conducted, and cytokine levels (ILs-1α and β, IL-2, IL-4, IL-6, IL-10, GM-CSF and TNF-α) were determined in ischemic and non-ischemic cortices. Axonal damage was determined using the pNF-H ELISA assay. Treatment with PRE-084 afforded neuroprotection following embolic stroke as evidenced by significantly reduced infarct volume and improved behavioural outcome. Remarkably, treatment with PRE-084 reduced levels of pro-inflammatory cytokines and enhanced anti-inflammatory cytokines. Levels of pNF-H were lower in rats treated with PRE-084 suggesting reduced axonal damage but this finding did not reach statistical significance. The findings of the present study suggest that part of the neuroprotective effects of sigma-1 receptor agonists may be mediated through a dual effect on cytokine release following stroke.

Changes of protein oxidation, calpain and cytoskeletal proteins (alpha tubulin and pNF-H) levels in rat brain after nerve agent poisoning

Highly toxic organophosphorus (OP) nerve agents, sarin and soman act by inhibiting acetylcholinesterase (AChE) function at neuronal synapses and cause many toxic effects including death within minutes. The effect of nerve agents on protein oxidation, calpain, and cytoskeletal protein levels was not well known. In the present study we investigated these parameters after subcutaneous injection of sarin (120 μg/kg) and soman (80 μg/kg) in the rat brain. Results indicate that several rat brain proteins were intensely oxidized after nerve agent poisoning. Immunoreactivity levels of μ-calpain were significantly elevated in cerebral cortex and cerebellum regions of rat brain from 2.5 h to 30 days. Alpha tubulin levels reduced from 1 to 7 days in the supernatant and 1 to 3 days in the pellet fractions of cerebellum and cerebral cortex, where as phosphorylation of high molecular weight neurofilament (pNF-H) was increased significantly in nerve agent intoxicated rat brains as compared to control rats. AChE activity was inhibited up to 3 days after nerve agent exposure in plasma and brain. Results suggest that altered protein oxidation, calpain and cytoskeletal protein levels are due to multiple mechanisms of nerve agents actions and these changes might be involved in nerve agent induced complex neurotoxicity.

Combination of neurofilament heavy chain and complement C3 as CSF biomarkers for ALS

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease with an average survival of 3 years from symptom onset. Rapid and conclusive early diagnosis is essential if interventions with disease-modifying therapies are to be successful. Cytoskeletal modification and inflammation are known to occur during the pathogenesis of ALS. We measured levels of cytoskeletal proteins and inflammatory markers in the CSF of ALS, disease controls and healthy subjects. We determined threshold values for each protein that provided the optimal sensitivity and specificity for ALS within a training set, as determined by receiver operating characteristic analysis. Interestingly, the optimal assay was a ratio of the levels for phosphorylated neurofilament heavy chain and complement C3 (pNFH/C3). We next applied this assay to a separate test set of CSF samples to verify our results. Overall, the predictive pNFH/C3 ratio identified ALS with 87.3% sensitivity and 94.6% specificity in a total of 71 ALS subjects, 52 disease control subjects and 40 healthy subjects. In addition, the level of CSF pNFH correlated with survival of ALS patients. We also detected increased pNFH in the plasma of ALS patients and observed a correlation between CSF and plasma pNFH levels within the same subjects. These findings support large-scale prospective biomarker studies to determine the clinical utility of diagnostic and prognostic signatures in ALS.

Levels of phosphorylated axonal neurofilament subunit H (pNfH) are increased in acute ischemic stroke

For the study of stroke outcomes, there is the need for measurements of severity of stroke damage. Phosphorylated neurofilament heavy protein (pNfH) levels are elevated in axonal injury. We have measured levels of pNfH in stroke and correlated these levels with measures of stroke severity. Blood samples were collected from 54 ischaemic stroke patients at day 1, week 1 (days 7–10) and weeks 3–6, and an ELISA was used to measure pNfH levels in each patient at each time-point. Serum pNfH levels were significantly elevated in stroke patients compared to healthy controls. The levels were low at day 1, higher at day 7 and reached a peak at week 3, the latest day that we assessed. Significant associations were found between the pNfH levels at week 3 and early and stroke severity, size and outcome. Blood pNfH levels that reflect the severity of ischaemic stroke, are correlated with outcome and rise during the weeks after stroke. This may be a useful measure of tissue damage in stroke.

Hyperphosphorylated neurofilament NF-H as a predictor of mortality after brain injury in children

Objective: The aim of the study was to determine whether serum levels of hyperphosphorylated neurofilament NF-H correlate with severity of brain injury in children.Methods: Forty-nine patients with traumatic brain injury (TBI) were enrolled into the prospective study. Venous blood samples were taken after admission and every 24 h for a maximum of 6 consecutive days. Serum NF-H concentrations were quantified by enzyme-linked immunosorbent assay. The outcome was evaluated 6 months after TBI using Glasgow Outcome Scale (GOS) in all patients.Results: The quantitative level of pNF-H remained significantly higher in patients with poor outcome (GOS = 1) in comparison with the other patients for the 2nd–4th day (p = 0.027; p = 0.019; p = 0.01). Levels of pNF-H were significantly higher in patients with diffuse axonal injury on initial CT scan (p = 0.004). Normal levels pNF-H in the paediatric population are unknown. Objective ROC analysis was identification of optimal cut-offs of proteins for prediction of GOS = 1.Conclusions: Although further, prospective study is warranted, these findings suggest that levels of hyperphosphorylated neurofilament NF-H correlate with mortality and may be useful as predictors of outcome in children with TBI.

A Pilot Study of Novel Biomarkers in Neonates With Hypoxic-Ischemic Encephalopathy

Severe hypoxic-ischemic encephalopathy (HIE) is a devastating condition that can lead to mortality and long-term disabilities in term newborns. No rapid and reliable laboratory test exists to assess the degree of neuronal injury in these patients. We propose two possible biomarkers: 1) phosphorylated axonal neurofilament heavy chain (pNF-H) protein, one of the major subunits of neurofilaments, found only in axonal cytoskeleton of neurons and 2) Ubiquitin C-terminal hydrolase 1 (UCHL1 protein) that is heavily and specifically concentrated in neuronal perikarya and dendrites. High-serum pNF-H and UCHL1 levels are reported in subarachnoid hemorrhage and traumatic brain injury, suggesting that they are released into blood following neuronal injury. We hypothesized that serum pNF-H and UCHL1 were higher in neonates with moderate-to-severe HIE than in healthy neonates. A time-limited enrollment of 14 consecutive patients with HIE and 14 healthy controls was performed. UCHL1 and pNF-H were correlated with clinical data and brain MRI. UCHL1 and pNF-H serum levels were higher in HIE versus controls. UCHL1 showed correlation with the 10-min Apgar score, and pNF-H showed correlation with abnormal brain MRI. Our findings suggest that serum UCHL1 and pNF-H could be explored as diagnostic and prognostic tools in neonatal HIE.

Hyperphosphorylated neurofilament NF-H as a biomarker of the efficacy of minocycline therapy for spinal cord injury

An in vivo study in a rat model of acute spinal cord contusion. To assess the efficacy of novel therapies for acute spinal cord injury (SCI), methods to evaluate accurately the effects of these therapies should be developed. Although neurological examination is commonly used for this purpose, unstable clinical conditions and the spontaneous recovery of neurological function in the acute and subacute phases after injury make this measurement unreliable. Recent studies have reported that the phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), a new biomarker for axonal degeneration, can be measured in serum samples in experimental SCI animals. Therefore, we aimed to investigate the use of plasma pNF-H as an indicator of the efficacy of minocycline, a neuroprotective drug, for treating SCI. This study was carried out at Saitama, Japan. Spinal cord injured rats received either minocycline or saline intraperitoneally. The plasma pNF-H levels and functional hind limb score were determined after the injury. Minocycline treatment reduced plasma pNF-H levels at 3 and 4 days post-injury (dpi). Rats with lower plasma pNF-H levels at 3 dpi had higher hind limb motor score at 28 dpi. pNF-H levels may serve as a biomarker for evaluating the efficacy of therapies for SCI.

Identification and preliminary characterization of ubiquitin C terminal hydrolase 1 (UCHL1) as a biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage

By using two different approaches, ubiquitin C-terminal hydrolase 1 (UCHL1) was identified as a potential cerebrospinal fluid (CSF) biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage (ASAH) and presumably other CNS damage and disease states. Appropriate antibodies and a sensitive ELISA were generated, and the release of UCHL1 into CSF was compared with that of pNF-H and S100beta in a cohort of 30 ASAH patients. Both UCHL1 and pNF-H showed persistent release into CSF in almost all patients in the second week postaneurysmal rupture (AR), and S100beta levels rapidly declined to baseline levels in 23 of 30 patients. Seven of thirty patients showed persistently elevated S100beta levels over the first 5 days post-AR and also had relatively higher levels of pNF-H and UCHL1 higher compared with the rest. These patients proved to have very poor outcomes, with 6 of 7 expiring. Patients who did reduce S100beta levels tended to have a better outcome if pNF-H and UCHL1 levels were also lower, and elevated UCHL1 levels in the second week post-AR were particularly predictive of poor outcome. Acute coordinated releases of large amounts of UCHL1, pNF-H, and S100beta in 16 of 30 patients were observed, suggesting sudden loss of brain tissues associated with secondary events. We conclude that measurement of the CSF levels of these proteins reveals details of ASAH progression and recovery and predicts patient outcome.

Phosphorylated Neurofilament Heavy Chain Correlations to Visual Function, Optical Coherence Tomography, and Treatment

Objective. To correlate visual and neurologic clinical scores and treatment of optic neuritis and multiple sclerosis (MS) patients with assays of serum phosphorylated neurofilament heavy chain (pNF-H) and optical coherence tomography (OCT) measurements of axonal loss. Design/Methods. The Optic Neuritis Treatment Trial (ONTT) randomized 457 patients with acute optic neuritis to intravenous methylprednisolone (IVMP) followed by oral prednisone, oral prednisone or placebo treatment arms. We quantified serum pNF-H levels in 175 ONTT patients 5 years after study entry. We performed OCT measurements of macular volume and the retinal nerve fiber layer (RNFL) in a subset of 51 patients at year 15. Results. Elevated pNF-H levels at year 5 correlated to poorer visual function at study entry. Lower 15 year macular volumes and RNFL thickness correlated better with follow-up than with baseline visual function measures. With IVMP treatment, 15 year RNFL differences of the fellow eye (FE) minus the affected eye (SE) RNFLFEmSE correlated with five-year pNF-H levels. PNF-H was reduced by half with IVMP relative to placebo or by 40% relative to prednisone. Conclusions/Relevance. Acute optic neuritis patients who have more severe visual loss during initial presentation have a higher incidence of axonal loss that was slightly suppressed with IVMP treatment.

Immunoreactivity of the phosphorylated axonal neurofilament H subunit (pNF‐H) in blood of ALS model rodents and ALS patients: evaluation of blood pNF‐H as a potential ALS biomarker

Levels of neurofilament subunits, potential biomarkers of motor axon breakdown, are increased in amyotrophic lateral sclerosis (ALS) patient's CSF but data on blood are not available. We measured blood levels of the phosphorylated axonal form of neurofilament H (pNF-H) by ELISA in transgenic rodent models of superoxide dismutase 1 (SOD1) ALS, and in 20 ALS patients and 20 similar aged controls monthly for 4 months. All symptomatic rodent ALS models showed robust levels of blood pNF-H, while control rodents or mice transgenic for unmutated SOD1 showed no detectable blood pNF-H. Average pNF-H levels in the G93A SOD1 mouse progressively increased from day 74 through death (day approximately 130). Median blood pNF-H level in ALS patients was 2.8-fold higher than controls (p < 0.001). Median ALSFRS-R declined a median of 0.8 pt/month (p < 0.001); higher baseline pNF-H level appeared to be associated with faster ALSFRS-R decline over 4 months (p = 0.087). The median rate of decline in ALSFRS-R was 1.9 pt/month in patients with baseline pNF-H levels above the median pNF-H value of 0.53 ng/mL; ALSFRS-R declined at a median of 0.6 pt/month in patients below this level. The pNF-H levels were relatively stable month to month in individual patients, raising questions regarding the molecular pathogenesis of ALS. Baseline control human pNF-H levels were higher in men than women and increased minimally over time. These data suggest that blood pNF-H can be used to monitor axonal degeneration in ALS model rodents and support further study of this protein as a potential biomarker of disease prognosis in ALS patients.

The Phosphorylated Axonal Form of the Neurofilament Subunit NF-H (pNF-H) as a Blood Biomarker of Traumatic Brain Injury

The detection of neuron-specific proteins in blood might allow quantification of the degree of neuropathology in experimental and clinical contexts. We have been studying a novel blood biomarker of axonal injury, the heavily phosphorylated axonal form of the high molecular weight neurofilament subunit NF-H (pNF-H). We hypothesized that this protein would be released from damaged and degenerating neurons following experimental traumatic brain injury (TBI) in amounts large enough to allow its detection in blood and that the levels detected would reflect the degree of injury severity. An enzyme-linked immunosorbent assay (ELISA) capture assay capable of detecting nanogram amounts of pNF-H was used to test blood of rats subjected to experimental TBI using a controlled cortical impact (CCI) device. Animals were subjected to a mild (1.0 mm), moderate (1.5 mm), or severe (2.0 mm) cortical contusion, and blood samples were taken at defined times post-injury. The assay detected the presence of pNF-H as early as 6 h post-injury; levels peaked at 24-48 h, and then slowly decreased to baseline over several days post-injury. No signal above baseline was detectable in control animals. Analysis of variance (ANOVA) showed a significant effect of lesion severity, and post hoc analysis revealed that animals given a moderate and severe contusion showed higher levels of blood pNF-H than controls. In addition, the peak levels of pNF-H detected at both 24 and 48 h post-injury correlated with the degree of injury as determined by volumetric analysis of spared cortical tissue. Relative amounts of pNF-H were also determined in different areas of the central nervous system (CNS) and were found to be highest in regions containing large-diameter axons, including spinal cord and brainstem, and lowest in the cerebral cortex and hippocampus. These findings suggest that the measurement of blood levels of pNF-H is a convenient method for assessing neuropathology following TBI.

Validation of a novel biomarker for acute axonal injury in experimental autoimmune encephalomyelitis

In multiple sclerosis, inflammatory axonal injury is a key pathological mechanism responsible for the development of progressive neurological dysfunction. The injured axon represents a therapeutic target in this disease; however, therapeutic trials of neuroprotective candidates will initially require preclinical testing in an animal model of inflammatory axonal injury and subsequently the development of a reliable paraclinical measure of axonal degeneration in humans. In the present study, we demonstrate the validity of serum phosphorylated neurofilament H (pNF-H) as a marker of axonal injury in murine experimental autoimmune encephalomyelitis (EAE). At the time of maximum disease severity (EAE day 22), the average serum pNF-H level reached 5.7 ng/ml, correlating significantly with the EAE paraplegia score (r = 0.75, P < 0.001). On average, 40% of axons in the spinal cord were lost in EAE, and serum pNF-H levels were highly correlated with axon loss (r = 0.8, P < 0.001). Axonal injury was a severe and acute event, insofar as serum pNF-H levels were not significantly elevated at early (EAE day 12) or late (EAE days 35 and 50) disease time points. Our results demonstrate that acute inflammatory axonal injury is a pathological feature of murine MOG(35-55) EAE, indicating that this model may mirror the acute pathological events in active multiple sclerosis lesions. Furthermore, we have validated the serum pNF-H assay as an unbiased measurement of axonal injury in EAE, facilitating rapid screening of potential neuroprotective therapies in this model.