Quantification of Axonal Damage in Traumatic Brain Injury

Abstract : Diffuse axonal injury is a primary feature of head trauma and is one of the most frequent causes of mortality and morbidity. Diffuse axonal injury is microscopic in nature and difficult or impossible to detect with imaging techniques. The objective of the present study was to determine whether axonal injury in head trauma patients could be quantified by measuring levels of CSF tau proteins. Tau proteins are structural microtubule binding proteins primarily localized in the axonal compartment of neurons. Monoclonal antibodies recognizing the form of tau found in the CSF of head trauma patients were developed by differential CSF hybridoma screening using CSF from head trauma and control patients. Clones positive for head trauma CSF tau proteins were used to characterize this form of tau and for ELISA development. Using the developed ELISA, CSF tau levels were elevated >1,000-fold in head trauma patients (mean, 1,519 ng/ml of CSF) when compared with patients with multiple sclerosis (mean, 0.014 ng/ml of CSF ; p < 0.001), normal pressure hydrocephalus (nondetectable CSF tau), neurologic controls (mean, 0.031 ng/ml of CSF ; p < 0.001), or nonneurologic controls (nondetectable CSF tau ; p < 0.001). In head trauma, a relationship between clinical improvement and decreased CSF tau levels was observed. These data suggest that CSF tau levels may prove a clinically useful assay for quantifying the axonal injury associated with head trauma and monitoring efficacy of neuroprotective agents. Affinity purification of CSF tau from head trauma patients indicated a uniform cleavage of ~ 18 kDa from all six tau isoforms, reducing their apparent molecular sizes to 30-50 kDa. These cleaved forms of CSF tau consisted of the interior portion of the tau sequence, including the microtubule binding domain, as judged by cyanogen bromide digestion. Consistent with these data, CSF cleaved tau bound taxolpolymerized microtubules, indicating a functionally intact microtubule binding domain. Furthermore, epitope mapping studies suggested that CSF cleaved tau proteins consist of the interior portion of the tau sequence with cleavage at both N and C terminals.     

Novel cerebrospinal fluid and serum autoantibody targets for clinically isolated syndrome

Limited information is available on the identity of antigens targeted by antibodies present in cerebrospinal fluid (CSF) of patients with clinically isolated syndrome (CIS). The aim of this study was to identify novel antigens for CIS and investigate their prognostic potential to predict conversion to multiple sclerosis (MS). We applied serological antigen selection (SAS) to identify antigens interacting with antibodies present in the pooled CSF from four CIS patients, who developed MS. Antibody reactivity towards CIS antigens identified by SAS was tested in CSF and serum from patients with CIS (n = 123/n = 108), MS (n = 65/n = 44), and other (inflammatory) neurological diseases (n = 75/n = 38) as well as in healthy control sera (n = 44). Using SAS, a panel of six novel CIS candidate antigens was identified. CSF antibody reactivity was detected in both CIS and relapsing‐remitting (RR) MS. Serum reactivity was significantly increased in CIS and RR‐MS as compared with controls (p = 0.03). For two antigens, the frequency of antibody‐positive patients was higher in CIS patients who converted to MS as compared with CIS patients without conversion. We identified novel CIS antigens to which antibody reactivity was primarily detected in CIS and RR‐MS as compared to controls. Possible prognostic potential could be demonstrated for two antigens.     

Glucopeptides derived from myelin-relevant proteins and hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin cross-react with multiple sclerosis specific antibodies: A step forward in the identification of native autoantigens in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory and autoimmune disorder, in which an antibody-mediated demyelination mechanism plays a critical role. We prepared two glucosylated peptides derived from the human myelin proteins, that is, oligodendrocyte-myelin glycoprotein (OMGp) and reticulon-4 receptor (RTN4R), selected by a bioinformatic approach for their conformational homology with CSF114(Glc), a designed β-turn antigenic probe derived from myelin oligodendrocyte glycoprotein (MOG), a glycoprotein present in the CNS. This synthetic antigen is specifically recognized by antibodies in sera of MS patients. We report herein the antigenic properties of these peptides, showing, on the one hand, that MS patient antibodies recognize the two glucosylated peptides and, on the other hand, that these antibodies cross-react with CSF114(Glc) and with the previously described hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin protein HMW1ct(Glc). These observations point to an immunological association between human and bacterial protein antigens, underpinning the hypothesis that molecular mimicry triggers the breakdown of self-tolerance in MS and suggesting that RTN4R and OMGp can be considered as autoantigens.     

Axonal pathology in myelin disorders

Myelination provides extrinsic trophic signals that influence normal maturation and long-term survival of axons. The extent of axonal involvement in diseases affecting myelin or myelin forming cells has traditionally been underestimated. There are, however, many examples of axon damage as a consequence of dysmyelinating or demyelinating disorders. More than a century ago, Charcot described the pathology of multiple sclerosis (MS) in terms of demyelination and relative sparing of axons. Recent reports demonstrate a strong correlation between inflammatory demyelination in MS lesions and axonal transection, indicating axonal loss at disease onset. Disruption of axons is also observed in experimental allergic encephalomyelitis and in Theiler's murine encephalomyelitis virus disease, two animal models of inflammatory demyelinating CNS disease. A number of dysmyelinating mouse mutants with axonal pathology have provided insights regarding cellular and molecular mechanisms of axon degeneration. For example, the myelin-associated glycoprotein and proteolipid protein have been shown to be essential for mediating myelin-derived trophic signals to axons. Patients with the inherited peripheral neuropathy Charcot-Marie Tooth disease type 1 develop symptomatic progressive axonal loss due to abnormal Schwann cell expression of peripheral myelin protein 22. The data summarized in this review indicate that axonal damage is an integral part of myelin disease, and that loss of axons contributes to the irreversible functional impairment observed in affected individuals. Early neuroprotection should be considered as an additional therapeutic option for these patients.     

Comparison of Antibodies Hydrolyzing Myelin Basic Protein from the Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

It was found that antibodies (Abs) against myelin basic protein (MBP) are the major components of the antibody response in multiple sclerosis (MS) patients. We have recently shown that IgGs from sera of MS patients are active in the hydrolysis of MBP. However, in literature there are no available data concerning possible MBP-hydrolyzing Abs in cerebrospinal fluid (CSF) of MS patients. We have shown that the average content of IgGs in their sera is about 195-fold higher than that in their CSF. Here we have compared, for the first time, the average content of lambda- and kappa-IgGs as well as IgGs of four different subclasses (IgG1-IgG4) in CSF and sera of MS patients. The average relative content of lambda-IgGs and kappa –IgGs in the case of CSFs (8.0 and 92.0%) and sera (12.3 and 87.7%) are comparable, while IgG1, IgG2, IgG3, and IgG4: CSF - 40.4, 49.0, 8.2, and 2.5% of total IgGs, respectively and the sera - 53.6, 36.0, 5.6, and 4.8%, decreased in different order. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We present first evidence showing that IgGs from CSF efficiently hydrolyze MBP and that their average specific catalytic activity is unpredictably ∼54-fold higher than that of Abs from sera of the same MS patients. Some possible reasons of these findings are discussed. We suggest that anti-MBP abzymes of CSF may promote important neuropathologic mechanisms in this chronic inflammatory disorder and in MS pathogenesis development.     

Comparison of DNA-Hydrolyzing Antibodies from the Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

It was found that high-affinity anti-DNA antibodies were one of the major components of the intrathecal IgG response in multiple sclerosis (MS) patients [Williamson et al., PNAS, 2001]. Recently we have shown that IgGs from the sera of MS patients are active in the hydrolysis of DNA. Here we have shown, for the first time, that average concentration of total proteins (132-fold), total IgGs (194-fold) and anti-DNA antibodies (200-fold) in the sera is significantly higher than that in the cerebrospinal fluid (CSF) of fifteen MS patients. The relative activities of total protein from sera and CSFs varied remarkably from patient to patient. It was surprising that the specific DNase activity of the total protein of CSF reparations were 198-fold higher than the serum ones. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We present first evidence showing that IgGs from CSF not only bind but efficiently hydrolyze DNA and that average specific DNase activity of homogeneous antibodies from CSF is unpredictably ∼49-fold higher than that from the sera of the same MS patients. Some possible reasons of these findings are discussed. We suggest that DNase IgGs of CSF may promote important neuropathologic mechanisms in this chronic inflammatory disorder and MS pathogenesis development.     

Axonal conduction and injury in multiple sclerosis: the role of sodium channels

Multiple sclerosis (MS) is the most common cause of neurological disability in young adults. Recent studies have implicated specific sodium channel isoforms as having an important role in several aspects of the pathophysiology of MS, including the restoration of impulse conduction after demyelination, axonal degeneration and the mistuning of Purkinje neurons that leads to cerebellar dysfunction. By manipulating the activity of these channels or their expression, it might be possible to develop new therapeutic approaches that will prevent or limit disability in MS.     

Axonal defects in mouse models of motoneuron disease

Human motoneuron disease is characterized by loss of motor endplates, axonal degeneration, and cell death of motoneurons. The identification of the underlying gene defects for familial ALS, spinal muscular atrophy (SMA), and spinal muscular atrophy with respiratory distress (SMARD) has pointed to distinct pathophysiological mechanisms that are responsible for the various forms of the disease. Accumulating evidence from mouse models suggests that enhanced vulnerability and sensitivity to proapoptotic stimuli is only responsible for some but not all forms of motoneuron disease. Mechanisms that modulate microtubule assembly and the axonal transport machinery are defective in several spontaneous and ENU (ethylnitrososurea) mutagenized mouse models but also in patients with mutations in the p150 subunit of dynactin. Recent evidence suggests that axonal growth defects contribute significantly to the pathophysiology of spinal muscular atrophy. Reduced levels of the survival motoneuron protein that are responsible for SMA lead to disturbed RNA processing in motoneurons. This could also affect axonal transport of mRNAs for beta-actin and other proteins that play an essential role in axon growth and synaptic function. The local translation of specific proteins might be affected, because developing motoneurons contain ribosome-like structures in distal axons and growth cones. Altogether, the evidence from these mouse models and the new genetic data from patients suggest that axon growth and maintenance involves a variety of mechanisms, including microtubule assembly and axonal transport of proteins and ribonucleoproteins (RNPs). Thus, defects in axon maintenance could play a leading role in the development of several forms of human motoneuron disease.     

Effect of Porphyromonas gingivalis outer membrane vesicles on gingipain-mediated detachment of cultured oral epithelial cells and immune responses

Porphyromonas gingivalis is a major etiological agent of periodontal diseases and the outer membrane vesicles (OMVs) contain virulence factors such as LPS and gingipains. In this study, we investigated the potential role of the OMVs in host immune response and tissue destruction during P. gingivalis infection. Firstly, we found that sera from periodontitis patients had significantly stronger reactivity against an OMV-producing wild type strain than the isogenic OMV-depleted strain. OMVs were found to be highly antigenic, as absorption of patient sera with OMVs greatly reduced reactivity with whole cells of P. gingivalis. LC-MS/MS analysis of OMVs revealed multiple forms of gingipains and several gingipain-related proteins. Western blots of OMVs using patient sera revealed a conserved immunoreactive antigen profile resembling the profile of OMV antigens that were recognized by gingipain antiserum, suggesting a potential role of OMV-associated gingipains in triggering antibody-mediated immune responses to P. gingivalis infection. When OMVs were added to a monolayer of an oral squamous epithelial cell line, OMVs caused cell detachment, which was inhibited by preincubating OMVs with anti-gingipain antiserum. These data suggest that gingipain-laden OMVs may contribute to tissue destruction in periodontal diseases by serving as a vehicle for the antigens and active proteases.     

Biomarkers of Inflammation and Axonal Degeneration/Damage in Patients with Newly Diagnosed Multiple Sclerosis: Contributions of the Soluble CD163 CSF/Serum Ratio to a Biomarker Panel

Background

Expression of soluble CD163 (sCD163), a macrophage/microglia biomarker, is increased in inflammatory conditions, and sCD163 levels in the cerebrospinal fluid (CSF) have recently been shown to be elevated in patients with multiple sclerosis (MS): the sCD163 CSF/serum ratio was elevated in patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and clinically isolated syndrome (CIS) compared with symptomatic controls.

Objective

To investigate the contributions of the sCD163 CSF/serum ratio to a biomarker panel focusing on inflammation and axonal degeneration in newly diagnosed MS; thus optimising a diagnostic biomarker panel for MS.

Methods

After a full MS diagnostic work-up, including collection of paired samples of CSF and serum, 125 patients were included in this study. Patients were divided into groups based on their diagnosis, and patients with normal clinical and paraclinical findings were defined as symptomatic controls. Serum and CSF levels, ratios, and indices of sCD163, CXCL13, osteopontin, neopterin, and CSF levels of neurofilament light polypeptide were determined by enzyme-linked immunosorbent assays (ELISAs). For sCD163 the results constitute a post-hoc analysis of already published data.

Results

All tested biomarkers, notably the sCD163 ratio, the CXCL13 ratio, the NEO ratio, the CSF level of NfL, the IgG index, and the serum level of OPN, were significantly correlated to RRMS, PPMS, and/or CIS. The individual biomarkers in single tests had a lower performance than the IgG index, however, their combined receiver operating characteristic (ROC) curve demonstrated excellent diagnostic discriminatory power.

Conclusion

The biomarker panel showed distinct profiles for each patient group and could be a valuable tool for clinical differentiation of MS subgroups. The combined ROC analysis showed that sCD163 contributes positively as a diagnostic marker to a panel of established MS biomarkers. Patients with PPMS were demonstrated to have significantly elevated levels of both inflammatory and degenerative markers.     

Triosephosphate isomerase- and glyceraldehyde-3-phosphate dehydrogenase-reactive autoantibodies in the cerebrospinal fluid of patients with multiple sclerosis

Our previous results revealed that Igs in lesions and single chain variable fragment Abs (scFv-Abs) generated from clonal B cells in the cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) bind to axons in MS brains. To study the axonal Ags involved in MS, we identified the glycolytic enzymes, triosephosphate isomerase (TPI) and GAPDH, using Igs from the CSF and scFv-Abs generated from clonal B cells in the CSF and in lesions from MS patients. Elevated levels of CSF-Abs to TPI were observed in patients with MS (46%), clinically isolated syndrome (CIS) suggestive of MS (40%), other inflammatory neurological diseases (OIND; 29%), and other noninflammatory neurological diseases (ONIND; 31%). Levels of GAPDH-reactive Abs were elevated in MS patients (60%), in patients with CIS (10%), OIND (14%), and ONIND (8%). The coexistence of both autoantibodies was detected in 10 MS patients (29%), and 1 CIS patient (3%), but not in patients with OIND/ONIND. Two scFv-Abs generated from the CSF and from lesions of a MS brain showed immunoreactivity to TPI and GAPDH, respectively. The findings suggest that TPI and GAPDH may be candidate Ags for an autoimmune response to neurons and axons in MS.     

Neutralizing Antibodies Inhibit Axonal Spread of Herpes Simplex Virus Type 1 to Epidermal Cells In Vitro

The ability of antibodies to interfere with anterograde transmission of herpes simplex virus (HSV) from neuronal axons to the epidermis was investigated in an in vitro model consisting of human fetal dorsal root ganglia innervating autologous skin explants in a dual-chamber tissue culture system. The number and size of viral cytopathic plaques in epidermal cells after axonal transmission from HSV type 1 (HSV-1)-infected dorsal root ganglionic neurons were significantly reduced by addition to the outer chamber of neutralizing polyclonal human sera to HSV-1, of a human recombinant monoclonal group Ib antibody to glycoprotein D (gD), and of rabbit sera to HSV-1 gB and gD but not by rabbit anti-gE or anti-gG. A similar pattern of inhibition of direct infection of epidermal cells by these antibodies was observed. High concentrations of the monoclonal anti-gD reduced transmission by 90%. Rabbit anti-gB was not taken up into neurons, and human anti-gD did not influence spread of HSV in the dorsal root ganglia or axonal transport of HSV antigens when applied to individual dissociated neurons. These results suggest that anti-gD and -gB antibodies interfere with axonal spread of HSV-1, possibly by neutralizing HSV during transmission across an intercellular gap between axonal termini and epidermal cells, and thus contribute to control of HSV spread and shedding. Therefore, selected human monoclonal antibodies to protective epitopes might even be effective in preventing epidermis-to-neuron transmission during primary HSV infection, especially neonatal infection.     

Axonal degeneration as a therapeutic target in the CNS

Degeneration of the axon is an important step in the pathomechanism of traumatic, inflammatory and degenerative neurological diseases. Increasing evidence suggests that axonal degeneration occurs early in the course of these diseases and therefore represents a promising target for future therapeutic strategies. We review the evidence for axonal destruction from pathological findings and animal models with particular emphasis on neurodegenerative and neurotraumatic disorders. We discuss the basic morphological and temporal modalities of axonal degeneration (acute, chronic and focal axonal degeneration and Wallerian degeneration). Based on the mechanistic concepts, we then delineate in detail the major molecular mechanisms that underlie the degenerative cascade, such as calcium influx, axonal transport, protein aggregation and autophagy. We finally concentrate on putative therapeutic targets based on the mechanistic prerequisites.     

Simultaneous Measurement of 2':3' Cyclic-Nucleotide 3' Phosphodiesterase and RNase Activities in Sera and Spinal Fluids of Multiple Sclerosis Patients

Abstract: The enzymes 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and RNase were simultaneously measured in the sera and CSF of multiple sclerosis (MS) and non-MS patients. No evidence of increased activity for these enzymes could be found regardless of pathology in either fluid source. Discrepancies between the present results and those from two previous studies that reported significant increases in CNPase activity in the CSF of patients with MS were carefully analyzed. It was concluded that the apparent increased CNPase activity correlated with MS in both previous studies was most probably the result of methodological and computational difficulties.     

Cerebrospinal fluid α-synuclein levels are elevated in multiple sclerosis and neuromyelitis optica patients during replase

The concept that the immune system plays a central role in the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica (NMO) was indisputable. However, neurodegenerative pathological features including loss of axons and neurons were also found in the lesions of these diseases. α-Synuclein is one of the most abundant proteins in pre-synaptic terminals. Recently, many research show α-synuclein level in CSF may reflect the progression of synaptic dysfunction and neuronal apoptosis. Whether the levels of CSF α-synuclein are changed in MS and NMO patients remain unknown. In this study, CSF α-synuclein was measured by an enzyme-linked immunosorbent assay (ELISA) in MS (n = 18) patients, NMO (n = 22) patients, Parkinson's disease patients (n = 9), and the controls (n = 11). We found concentration of α-synuclein in MS and NMO patients were significantly higher than Parkinson's disease subgroup and the controls. Both MS and NMO revealed an increased disease disability with increased CSF α-synuclein. Thus, CSF α-synuclein may be reflect injure axons and neurons in inflammatory demyelinating diseases.     

Increased intrathecal high-avidity anti-tau antibodies in patients with multiple sclerosis

Background

Antibodies against tau protein indicate an interaction between the immune system and the neurocytoskeleton and therefore may reflect axonal injury in multiple sclerosis (MS).

Methodology/Principal Findings

The levels and avidities of anti-tau IgG antibodies were measured using ELISA in paired cerebrospinal fluid (CSF) and serum samples obtained from 49 MS patients and 47 controls. Anti-tau antibodies were significantly elevated intrathecally (p<0.0001) in the MS group. The CSF anti-tau antibody levels were lower in MS patients receiving therapy than those without treatment (p<0.05). The avidities of anti-tau antibodies were higher in the CSF than in the serum (MS group p<0.0001; controls p<0.005). Anti-tau avidities in the CSF were elevated in MS patients in comparison with controls (p<0.05), but not in serum.

Conclusions

MS patients have higher levels of intrathecal anti-tau antibodies. Anti-tau antibodies have different avidities in different compartments with the highest values in the CSF of MS patients.     

Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway

Cerebrospinal fluid (CSF) includes conserved factors whose function is largely unexplored. To assess the role of CSF during embryonic development, CSF was repeatedly drained from embryonic zebrafish brain ventricles soon after their inflation. Removal of CSF increased cell death in the diencephalon, indicating a survival function. Factors within the CSF are required for neuroepithelial cell survival as injected mouse CSF but not artificial CSF could prevent cell death after CSF depletion. Mass spectrometry analysis of the CSF identified retinol binding protein 4 (Rbp4), which transports retinol, the precursor to retinoic acid (RA). Consistent with a role for Rbp4 in cell survival, inhibition of Rbp4 or RA synthesis increased neuroepithelial cell death. Conversely, ventricle injection of exogenous human RBP4 plus retinol, or RA alone prevented cell death after CSF depletion. Zebrafish rbp4 is highly expressed in the yolk syncytial layer, suggesting Rbp4 protein and retinol/RA precursors can be transported into the CSF from the yolk. In accord with this suggestion, injection of human RBP4 protein into the yolk prevents neuroepithelial cell death in rbp4 loss‐of‐function embryos. Together, these data support the model that Rbp4 and RA precursors are present within the CSF and used for synthesis of RA, which promotes embryonic neuroepithelial survival. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 75–92, 2016     

Label‐free analysis of human cerebrospinal fluid addressing various normalization strategies and revealing protein groups affected by multiple sclerosis

The aims of the study were to: (i) identify differentially regulated proteins in cerebrospinal fluid (CSF) between multiple sclerosis (MS) patients and non‐MS controls; (ii) examine the effect of matching the CSF samples on either total protein amount or volume, and compare four protein normalization strategies for CSF protein quantification. CSF from MS patients (n = 37) and controls (n = 64), consisting of other noninflammatory neurological diseases (n = 50) and non neurological spinal anesthetic subjects (n = 14), were analyzed using label‐free proteomics, quantifying almost 800 proteins. In total, 122 proteins were significantly regulated (p < 0.05), where 77 proteins had p‐value <0.01 or AUC value >0.75. Hierarchical clustering indicated that there were two main groups of MS patients, those with increased levels of inflammatory response proteins and decreased levels of proteins involved in neuronal tissue development (n = 30), and those with normal protein levels for both of these protein groups (n = 7). The main subgroup of controls clustering with the MS patients showing increased inflammation and decreased neuronal tissue development were patients suffering from chronic fatigue. Our data indicate that the preferable way to quantify proteins in CSF is to first match the samples on total protein amount and then normalize the data based on the median intensities, preferably from the CNS‐enriched proteins.     

Physical and Serologic Properties of an Antigen Prepared from Erythrocytes of Horses with Acute Babesiosis*

SYNOPSIS. By means of precipitation with protamine sulfate, a soluble antigen (PS) was obtained from erythrocytes of horses with acute babesiosis due to Babesia caballi and B. equi. This antigen reacted in gel diffusion tests with sera from horses recovered from acute babesiosis. The PS antigen was found to be muco-protein, susceptible to destruction by trypsin and taka-diastase. Analysis of the antigen by paper electrophoresis revealed 2 components which were not present in similar preparations made from erythrocytes of Babesia-free horses. When the PS antigen was heated in boiling water for 30 minutes, a serologically inactive precipitate was formed; however, the supernate remained serologically active and was termed boiled PS (BPS) antigen. This antigen was polysaccharide in nature; its serologic activity was destroyed by taka-diastase. In gel diffusion tests with sera of recovered horses, the PS antigen formed 2 lines of precipitation which coalesced in a single line formed between these sera and the BPS antigen. Both PS and BPS antigens reacted with sera of horses recovered from acute babesiosis in the gel-diffusion test, but not with sera of dogs and rats recovered from acute infection with Babesia canis and Babesia rodhaini, respectively. The serologic specificity of these antigens suggests that they might have application in the serodiagnosis of inapparent Babesia infections of equine animals.     

Time-Dependent Progression of Demyelination and Axonal Pathology in MP4-Induced Experimental Autoimmune Encephalomyelitis

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal pathology. Myelin basic protein/proteolipid protein (MBP-PLP) fusion protein MP4 is capable of inducing chronic experimental autoimmune encephalomyelitis (EAE) in susceptible mouse strains mirroring diverse histopathological and immunological hallmarks of MS. Limited availability of human tissue underscores the importance of animal models to study the pathology of MS.MethodsTwenty-two female C57BL/6 (B6) mice were immunized with MP4 and the clinical development of experimental autoimmune encephalomyelitis (EAE) was observed. Methylene blue-stained semi-thin and ultra-thin sections of the lumbar spinal cord were assessed at the peak of acute EAE, three months (chronic EAE) and six months after onset of EAE (long-term EAE). The extent of lesional area and inflammation were analyzed in semi-thin sections on a light microscopic level. The magnitude of demyelination and axonal damage were determined using electron microscopy. Emphasis was put on the ventrolateral tract (VLT) of the spinal cord.ResultsB6 mice demonstrated increasing demyelination and severe axonal pathology in the course of MP4-induced EAE. In addition, mitochondrial swelling and a decrease in the nearest neighbor neurofilament distance (NNND) as early signs of axonal damage were evident with the onset of EAE. In semi-thin sections we observed the maximum of lesional area in the chronic state of EAE while inflammation was found to a similar extent in acute and chronic EAE. In contrast to the well-established myelin oligodendrocyte glycoprotein (MOG) model, disease stages of MP4-induced EAE could not be distinguished by assessing the extent of parenchymal edema or the grade of inflammation.ConclusionsOur results complement our previous ultrastructural studies of B6 EAE models and suggest that B6 mice immunized with different antigens constitute useful instruments to study the diverse histopathological aspects of MS.