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Neurofilament Protein as a Potential Biomarker of Axonal Degeneration in Experimental Autoimmune Encephalomyelitis
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.280651
Keywords: Autoimmune, axonal degeneration, experimental allergic encephalomyelitis, neurofilament proteinKey Messages: The biomarker of NF-L can reflect the acute axonal damage mediated by inflammatory mechanisms and can imply prognostic value in early evaluation of MS.
Multiple sclerosis (MS) is a demyelinating disease mediated by an immune response against self-antigens from the central nervous system (CNS). This immune response triggers an initial inflammation in brain and spinal cord that is then followed by demyelination, axonal damage, and scar formation [1] Experimental autoimmune encephalomyelitis (EAE) is an induced inflammatory demyelinating disease of the CNS that is largely employed as a model to study MS.[2],[3] EAE is primarily used as an animal model of autoimmune inflammatory diseases of the CNS, and it resembles MS, the prototypical such disease, in many respects.[4],[5],[6] Biomarkers of axonal degeneration have the potentials to improve our capacity to predict and monitor neurological outcome in MS patients. Neurofilament proteins, one of the major proteins expressed within neurons and axons, have been detected in cerebrospinal fluid and blood samples from MS patients and are now being actively investigated for their utility as prognostic indicators of disease progression in MS.[7] So, in this paper we want to express that the detection of such components would provide a convenient means to assess the presence and degree of axonal degeneration in MS, and this information could be useful for predicting and monitoring the progression of the disease, and for assessing the efficacy of therapeutic strategies that are aimed at preventing axonal loss.
EAE induction Mice were maintained under conditions of strict confinement, which included automatic control of temperature (21°C) and photoperiod (12 h light/12 h dark). EAE was induced in 10- to 12-week-old female mice with an emulsion made of 2 mg/ml myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (Tocris, Bristol, UK) in phosphate buffered saline, pH 7.2, mixed with complete Freund's adjuvant (CFA, Difco, Detroit, MI) containing 10 mg/ml mycobacterium tuberculosis (H37RA, Difco) at a 1:1 ratio. The mice received a total of 200 μl of the emulsion subcutaneously, distributed in four injection sites (base of the tail and hind legs). Each mouse also received 400 ng of pertussis toxin (Tocris) applied intraperitoneally in 200 μl on day 0 and day 2 after the first injection. The mice were monitored daily for clinical signs of EAE and scored according to the following scale: 0, no clinical signs; 1, loss of tail tone; 2, flaccid tail; 3, incomplete paralysis of one or two hind legs; 4, complete hind limb paralysis; 5, moribund (animals that do not move, do not consume water or food, that loss weight greater than 20%, or have respiratory problems, were euthanized); 6, death. Cumulative clinical score was calculated as the sum of daily clinical scores. Three independent experiments were performed for clinical course tracking. The cerebrum were quickly dissected in different time such as 1d, 3d, 7d, 14d, and 28d and divided into cerebrum, spinal cord, and were stored at −80°C until analysis. The serum was obtained by centrifugation. All animals' care was in accordance with institutional guidelines. Preparation of 10% nerve tissue homogenate Cerebrum samples were homogenized in 0.9% saline (1/9, tissue/saline, w/v) at 4°C, using a homogenizer (10,000-15,000 rev./min, 10 s). The tubes with homogenates were kept in ice water for 30 min and centrifuged at 4°C (2,500 rev/min, 10 min) according to the commercial assay kits. The supernatants were separated and stored at −80°C. Protein content was determined using BCA™ protein assay kits. Statistical analysis The data was expressed as mean ± SD. Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by LSD's post-hoc tests, which was provided by SPSS 23.0 statistical software (Chicago, USA). A P value of less than 0.05 (P < 0.05) was considered as statistically significant.
Detection of the neurofilament proteins (NF-L, NF-M and NF-H) in nerve tissues. Time-dependent changes of NF-L in the cerebrum and spinal cord are shown in [Figure 1]. Representative immunoblots of NF-L are also shown below the graph.
Compared with the level of day 0, the cerebrum NF-L levels in the supernatant were decreased (P < 0.01) on day 1, 3, and 7, respectively, but were increased on day 14 and 28. While in the pellet, NF-L levels were also decreased on day 1, 3, and 7, respectively. As in the spinal cord, NF-L levels in the supernatant were increased (P < 0.01) on day 1, 3, 7, 14, and 28, respectively, especially on day 28. The time course of NF-M levels following EAE were firstly decreased on day 3 and then increased on day 7, 14, and 28 in the supernatant in the cerebrum as shown in [Figure 2]. NF-M levels were decreased on day 3 and 7, and then increased on day 14 and 28 (P < 0.01) in the pellet.
In the spinal cord, NF-M levels in the supernatant were firstly decreased on day 3 and 7, and then increased on day 14 and 28. As shown in [Figure 3], compared with the level of day 0, the concentration of NF-H in the supernanant of cerebrum after EAE were decreased on day 7, and then increased on day 28. While in the spinal cord, the NF-H levels were increased on day 14 and 28.
The axonal cytoskeleton is a finely organized system, essential for maintaining the integrity of the axon. Axonal degeneration is implicated in the pathogenesis of unremitting disability of MS. Biomarkers of axonal degeneration have the potential to improve our capacity to predict and monitor neurological outcome in MS patients. Neurofilament proteins, one of the major proteins expressed within neurons and axons, have been detected in cerebrospinal fluid and blood samples from MS patients and are now being actively investigated for their utilities as prognostic indicators of disease progression in MS. The potential use of the NF protein subunits as surrogate markers of axonal degeneration in MS was first explored by Lycke JN in 1998.[8] NF-L provides direct means to measure tissue damage and is a useful addition to our methods for evaluation of MS. Some report shows it can remain elevated for several weeks in MS patients.[9],[10] In our reports, the results showed that the concentration of NF-L in the supernanant of the cerebrum after EAE were decreased by 23%, 42%, 36% on day 1, 3, and 7, respectively, increased by 66%, 13% on day 14 and 28, while the concentration of NF-L in the pellet were decreased by 22%, 16%, 15%, and then increased by 28% and 128% on day 14 and 28. So from the experiment, we can see the elevation in the late stage in the EAE, and it is in accord with the conference. NF-M is another biomarker of axonal damage in the experiment. Compared with the level of day 0, the concentration of NF-M in the supernanant of cerebrum after EAE were decreased by 6%, increased by 31%, 30%, 33% on day 7, 14, and 28, while the concentration of NF-M in the pellet of cerebrum after EAE were changed by 12%, 4%, 21%, 6%, 148% on day 1, 3, 7, 14,28 respectively. It means that the concentration of NF-M was significantly increased on day 28 in supernanant and pellet. These results are related with reference.[11] Compared with the level of day 0, the concentration of NF-H in the supernanant of cerebrum after EAE were increased by 14% on day 1, and then decreased by 1%, 31%, 22%, 6% on day 3, 7, 14, 28 respectively, while the concentration of NF-H in the pellet were decreased by 5% and 8%, but then increased by 14%, 44%, and 52%. In this study, the average concentration of NF-L levels were much higher in supernanant and pellet in cerebrum of rats than spinal cord with all subtypes of EAE relative to samples taken from controls.[12] NF-M and NF-H changed in the later course of the disease. The biomarker of NF-L can reflect the acute axonal damage mediated by inflammatory mechanisms and can imply prognostic value for prognostic evaluation of MS, while NF-M and NF-H may be more related with nerve regeneration after the acute inflammation. Possibly due to the small sample size, this result did not reach statistical significance. As an indicator of disease activity and potentially therapeutic efficacy, the detection of NF subunits holds considerable promise as a means to monitor axonal loss in MS patients. Work is being focused on the development of yet more specific and sensitive assays for NF proteins of utility both in cerebrum and spinal cord.[13] Future studies should therefore aim to develop more sensitive methods for measuring the various NF subunits in CSF, plasma, or serum to ascertain whether these biomarkers will be useful in the clinic for predicting MS onset, and for monitoring MS progression and response to therapy. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3]
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