Objective To investigate the morphological features of chronic inflammatory demyelinating polyneuropathy (CIDP) with autoantibodies directed against paranodal junctional molecules, particularly focusing on the fine structures of the paranodes.
Methods We assessed sural nerve biopsy specimens obtained from 9 patients with CIDP with anti-neurofascin-155 antibodies and 1 patient with anti-contactin-1 antibodies. 13 patients with CIDP without these antibodies were also examined to compare pathological findings.
Results Characteristic light and electron microscopy findings in transverse sections from patients with anti-neurofascin-155 and anti-contactin-1 antibodies indicated a slight reduction in myelinated fibre density, with scattered myelin ovoids, and the absence of macrophage-mediated demyelination or onion bulbs. Teased-fibre preparations revealed that segmental demyelination tended to be found in patients with relatively higher frequencies of axonal degeneration and was tandemly found at consecutive nodes of Ranvier in a single fibre. Assessment of longitudinal sections by electron microscopy revealed that detachment of terminal myelin loops from the axolemma was frequently found at the paranode in patients with anti-neurofascin-155 and anti-contactin-1 antibody-positive CIDP compared with patients with antibody-negative CIDP. Patients with anti-neurofascin-155 antibodies showed a positive correlation between the frequencies of axo–glial detachment at the paranode and axonal degeneration, as assessed by teased-fibre preparations (p<0.05).
Conclusions Paranodal dissection without classical macrophage-mediated demyelination is the characteristic feature of patients with CIDP with autoantibodies to paranodal axo–glial junctional molecules.
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Chronic inflammatory demyelinating polyneuropathy (CIDP) is an immune-mediated neuropathy characterised by demyelination.1–8 The typical CIDP is defined as neuropathy manifesting progressive, stepwise or recurrent symmetrical proximal and distal weakness and sensory impairment in all four limbs, that develops over 2 months.9 Although occurring at a lower proportion than that of so-called typical CIDP, atypical forms, such as multifocal acquired demyelinating sensory and motor (MADSAM), distal acquired demyelinating symmetric (DADS), pure sensory, pure motor and focal, are also considered to be subtypes of CIDP.9 ,10 Immune-modulating therapies, such as intravenous immunoglobulin (IVIg), corticosteroids and plasma exchange, are the first-line treatments for CIDP;5–9 however, some patients do not respond to these therapies.11 ,12 The diversity of clinical features and responses to treatment indicate that the pathogenesis of CIDP is heterogeneous. Although cell-mediated and humoral immunities have been implicated in the pathogenesis of CIDP,6 recent studies have suggested an association between autoantibodies directed against paranodal junctional molecules and certain subpopulations of CIDP.13 Among these autoantibodies, anti-neurofascin-155 and anti-contactin-1 antibodies have been found in a subpopulation of patients with characteristic clinical features.14–21 Patients with these antibodies are characterised by sensory ataxia, tremor and poor response to IVIg treatment.16 ,17 ,19–21 As pathological assessments of patients with these antibodies have been performed in only a small number of patients,18 ,20 ,21 the morphological abnormalities leading to the observed damage to the peripheral nervous system have not been clarified.
In this study, we assessed sural nerve biopsy specimens obtained from 10 CIDP cases that had either anti-neurofascin-155 or anti-contactin-1 antibodies, particularly focusing on the fine structures of the paranodes.
Patients and methods
We examined electron microscopy findings in 10 patients with CIDP with anti-neurofascin-155 or anti-contactin-1 antibodies who were referred to Nagoya University Graduate School of Medicine for diagnostic sural nerve biopsy. Nine patients (Patients 1–9), consisting of seven male patients and two female patients aged 15–57 (35.7±16.7, mean±SD) years, were positive for anti-neurofascin-155 antibodies (table 1). The duration from the onset of neuropathy to biopsy in these patients ranged from 4 to 14 (7.7±3.5) months. Another 72-year-old male patient (Patient 10) was positive for anti-contactin-1 antibodies. The duration of neuropathy in this patient was 4 years. CIDP was diagnosed based on the diagnostic criteria of the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS).9 According to these criteria, the study patients consisted of four typical CIDP and six (including the patient with anti-contactin-1 antibodies) DADS neuropathy cases. All patients fulfilled definite electrodiagnostic criteria. The pathological features of these patients have not been reported elsewhere, whereas information on their clinical features was incorporated into another study analysing a larger series of patients.22 Administration of corticosteroids had been initiated in Patients 4 and 8, and IVIg had been given to Patient 7, prior to sural nerve biopsy.
Sural nerve biopsy specimens obtained from 13 CIDP patients fulfilling the EFNS/PNS criteria, but with no neurofascin-155 or contactin-1 antibodies, were also examined to compare pathological findings, particularly those of the paranode (table 1). These patients (Patients 11–23) consisted of five male patients and eight female patients aged 15–73 (51.3±17.1 mean±SD) years and included five cases of typical CIDP, three of MADSAM neuropathy, three of acquired DADS neuropathy, one of pure motor neuropathy and one of pure sensory neuropathy. Antibodies were examined using sera obtained at the time of biopsy in all of these patients. All patients except for Patients 17 and 19 fulfilled definite EFNS/PNS electrodiagnostic criteria. Diagnostic category of Patients 17 and 19 was definite because they had at least two supportive criteria. Although the biopsy was performed at 1 month from the onset of neuropathy in Patients 14 and 15, neuropathy in these patients progressed more than 1 month thereafter. None of the patients had monoclonal gammopathy.
All patients provided written informed consent. The study was approved by the ethics committees of Nagoya University Graduate School of Medicine, National Defense Medical College and Kyushu University.
Determination of anti-neurofascin-155 and anti-contactin-1 antibodies
Antibodies were examined using sera obtained from 131 patients with CIDP who fulfilled the EFNS/PNS criteria (see online supplementary data 1). Sera were obtained at the same time as the sural nerve biopsies, except for Patient 9, whose antibodies were examined 1 year after biopsy.
For the determination of the anti-neurofascin-155 and anti-contactin-1 antibodies, we performed ELISA using recombinant human neurofascin protein (R&D Systems, Minneapolis, USA) and recombinant human contactin-1 protein (Sino Biological, Beijing, China) as antigens, as detailed elsewhere (see online supplementary data 1).22 Briefly, patient sera diluted at 1:200 with 1% bovine serum albumin in phosphate-buffered saline were used as primary antibodies. Horseradish peroxidase-conjugated anti-human IgG-Fc antibody (MP Biomedicals, Solon, USA) diluted at 1:500 with 1% bovine serum albumin in phosphate-buffered saline was used as the secondary antibody, and then a chromogenic reaction with O-phenylenediamine in phosphate–citrate buffer was developed. The optical density values were measured at 490 nm. Immunohistochemical studies were done to confirm the results using longitudinal frozen sections of sciatic nerves dissected from adult Sprague-Dawley rats as described elsewhere.22
To further confirm positive results for anti-neurofascin-155 antibodies, we performed western blotting on ELISA-positive sera, as detailed elsewhere (see online supplementary data 1).22 Briefly, patient sera diluted at 1:200 with blocking buffer were used as primary antibodies, and horseradish peroxidase-conjugated anti-human IgG-Fc antibody diluted at 1:5000 with blocking buffer was used as the secondary antibody. The results were also verified by cell-based assay as previously described (see online supplementary data 1).15 ,20
Sural nerve biopsy and light microscopy observation
A sural nerve biopsy was performed as previously described.23 ,24 Each specimen was divided into two portions. The first portion was fixed in 2.5% glutaraldehyde in 0.125 M cacodylate buffer (pH 7.4) and embedded in epoxy resin. The density of myelinated fibres was evaluated in transverse sections using light microscopy. Toluidine-blue-stained semithin sections were assessed using a computer-assisted image analyser (Luzex FS; Nikon, Tokyo, Japan).24 ,25 A fraction of the glutaraldehyde-fixed sample was processed for a teased-fibre study, and pathological conditions were assessed according to the previously described criteria.26 Teased myelinated fibres with condition F were considered as those with segmental remyelination. The second portion was fixed in a 10% formalin solution and embedded in paraffin. Sections were cut by routine methods and stained with H&E and Masson's trichrome. Normal control values were obtained from 10 autopsy cases in which the patients died of non-neurological diseases (36.8±19.1 years of age, mean±SD; three men, seven women).
To evaluate the deposition of autoantibodies and complements, immunofluorescent studies were done using longitudinal fresh-frozen sections from Patients 7 and 10 as described previously.27 ,28 As the IgG subclass of autoantibodies in our patients was predominantly IgG4,22 we used sheep polyclonal antibody to IgG4 (The Binding Site Group, Birmingham, UK) and rabbit polyclonal antibody to contactin-associated protein (Caspr) (Abcam, Cambridge, UK) as primary antibodies to detect the deposition of autoantibodies. Rabbit monoclonal antibody to complement component 3d (C3d) (Abcam, Cambridge, UK) and sheep monoclonal antibody to IgG4 were used as primary antibodies to evaluate the deposition of complements. The methods for immunofluorescent studies are described in online supplementary data 2.
Electron microscopic study
For electron microscopy, the epoxy-resin-embedded specimens were cut into 70 nm thick ultrathin sections and stained with uranyl acetate and lead citrate. To assess the density of unmyelinated fibres, electron micrographs were captured at 4000× magnification in a random manner that covered the area of the transverse sections, as described previously.23 ,29 Unmyelinated axons in Schwann cell subunits that previously contained myelinated axons (ie, bands of Büngner) were not counted because they were considered regenerating myelinated fibres.29 ,30 Disproportionately large unmyelinated axons over 3 μm in diameter were not counted because these fibres were considered to be originally myelinated and to have formed as a consequence of demyelination.29
To assess the morphology of nodes of Ranvier and paranodes, longitudinal sections were used. As anti-neurofascin-155 or anti-contactin-1 antibodies are located at the paranode to connect axons and terminal loops of Schwann cells,31 we observed this portion at a final magnification of 100 000×. At least 30 nodes of Ranvier and their associated paranodes were evaluated for each case. Paranodal alterations were quantified as the frequency of nodes of Ranvier with corresponding paranodal findings in the total nodes of Ranvier assessed on longitudinal sections.
Quantitative data are presented as the mean±SD. Statistical analyses were performed using the χ2 test, Mann-Whitney U test, or Spearman's rank correlation analysis as appropriate. A p value of <0.05 was considered statistically significant.
Light microscopy findings
In the anti-neurofascin-155 antibody-positive group, moderate endoneurial oedema was observed in all cases. Onion bulb formation was not observed in any cases. Epineurial and endoneurial inflammatory cellular infiltration was not conspicuous, and no signs of vasculitis were found in any of the cases. The densities of myelinated fibres were reduced compared with those of normal controls (6594±985 fibres/mm2 vs 8887±1330 fibres/mm2, p<0.01; table 2). Myelin ovoids were occasionally found (figure 1A). Axonal sprouts, indicative of regenerating fibres, were not conspicuous in any case.
As for the assessment of teased-fibre preparations, patients with anti-neurofascin-155 antibodies showed axonal degeneration, except for one patient (Patient 6) with a short disease duration (4 months from the onset of neuropathy) at the time of examination. Axonal degeneration was more frequently found in anti-neurofascin-155 antibody-positive patients than in normal controls (3.6±4.2% vs 0.6±0.7%, p<0.05). Seven of the antibody-negative patients showed fibres with the classical feature of long segmental demyelination indicative of CIDP,1 ,7 while only three patients (Patients 2, 8 and 9) in the anti-neurofascin-155 antibody-positive group had apparent segmental demyelination. These three patients had a relatively high frequency of axonal degeneration compared with the others in the anti-neurofascin-155 antibody-positive group (p<0.05). In these patients, a widening of the nodes of Ranvier tended to be observed tandemly at consecutive nodes of Ranvier in a single fibre. The frequency of segmental remyelination was not increased compared with that of normal controls (3.6±2.6% vs 4.3±4.5%).
Similar findings were obtained from the patient with anti-contactin-1 antibodies as those with anti-neurofascin-155 antibodies in terms of a slight reduction in myelinated fibre density with occasional myelin ovoids and the absence of inflammatory cells or onion bulbs. The extent of endoneurial oedema was mild in this case.
Immunofluorescent studies revealed colocalisation of IgG4 and Caspr, suggesting the deposition of IgG4 at paranodes in anti-neurofascin-155 and anti-contactin-1 antibody-positive cases (see online supplementary data 2). On the contrary, the deposition of C3d was not detected in either case.
Electron microscopy findings in transverse sections
In accordance with the light microscopy findings, the absence of onion bulbs, defined as flattened Schwann cell processes surrounding myelinated fibres, and scattered myelin ovoids were common among the anti-neurofascin-155 and anti-contactin-1 antibody-positive patients. In the patients with antibody-negative CIDP, classical features of demyelination reported in CIDP, namely, the degradation of the myelin sheath by macrophages that surround the myelinated axons and contain myelin debris in their cytoplasm,1 ,5 ,7 ,32 ,33 were observed in three of the four typical patients with CIDP (Patients 14, 15 and 18) and one of the three patients with DADS neuropathy (Patient 21). In contrast, such macrophage-mediated demyelination was not found in any of the antibody-positive patients. Uncompacted myelin lamellae were observed in three of the anti-neurofascin-155 antibody-positive patients (Patients 3, 6 and 9) and six of the antibody-negative patients (Patients 12, 13, 15, 16, 19 and 23). Onion bulbs were obviously observed in five patients with antibody-negative CIDP (Patients 13, 17, 18, 19, 21). In the antibody-positive group, one patient with anti-neurofascin-155 antibodies (Patient 2) showed only one myelinated fibre, which was incompletely surrounded by one layer of flattened Schwann cell processes.
The density of unmyelinated fibres in the anti-neurofascin-155 antibody-positive patients was not significantly reduced compared with that of normal controls (30 298 ±4794 fibres/mm2 vs 30 586 ±4376 fibres/mm2). In addition, the percentage of non-myelinating Schwann cell subunits that contained unmyelinated axons out of the total number of non-myelinating Schwann cell subunits in these patients was not decreased (86.9±6.6% vs 82.1±10.6%).29 ,34 Ballooning of unmyelinated axons suggestive of axonal degeneration was also not apparent. Hence, unmyelinated fibres were relatively well preserved as opposed to myelinated fibres in these patients (figure 1B).
Morphology of nodes of Ranvier and paranodes in longitudinal sections
Demyelinated fibres and remyelinating fibres with thin myelin relative to neighbouring myelinated fibres were occasionally found in patients with antibody-negative CIDP, and a macrophage degrading myelin at a paranode was found (figure 2A). However, paranodal structures tended to be preserved in these patients. Myelin lamellae usually terminate consecutively at paranodes as they reach the axolemma of small myelinated fibres (figure 2B, C). Each major dense line opens just prior to contacting the axolemma to enclose a small pocket of cytoplasm, called the terminal loop.35 Terminal loops are normally apposed closely to the axolemma.35 Even though there were spaces between the terminal loops and axolemma, the spaces were usually <20 nm and occupied by electron-dense regions, suggesting that the terminal loops and axolemma were tightly connected to each other. In large myelinated fibres, some of the myelin lamellae may terminate before reaching the axolemma (figure 2D, E). However, myelin lamellae that terminate on axolemma were closely apposed to the axolemma as those of the small myelinated fibres. Although clear spaces devoid of electron-dense regions between paranodal terminal loops and axolemma were found in 9 of 13 patients in the antibody-negative group, the frequency was low (3.4±3.7%), and the extent of the widening was mild. These spaces tended to be present at paranodes of remyelinating internodes (see online supplementary data 3). However, they were found irrespective of the presence or absence of demyelination, as defined by widening of the node of Ranvier.
In contrast, a clear space between Schwann cell terminal loops and axolemma was frequently found in small and large myelinated fibres in patients with CIDP with anti-neurofascin-155 antibodies (figure 3A–C). This finding (ie, axo–glial detachment) was observed in association with 53.5±25.5% of nodes of Ranvier, and such axo–glial detachment at the paranodes was more frequent (p<0.001) compared with that of patients with antibody-negative CIDP. The detachment tended to be observed at the portion of the paranode close to the node of Ranvier. Large spaces were sometimes observed between the terminal loops and the axolemma (figure 3B). However, the extent of detachment was relatively mild at the terminal loops in the outermost layers of the myelin sheath of such paranodes (figure 3C). Hence, the extreme dislocation of terminal loops leading to the peeling off and exposure of axons (ie, demyelination), which has been reported in neuropathy with anti-myelin-associated glycoprotein (MAG) antibodies,27 was not observed. Patients with anti-neurofascin-155 antibodies showed a positive correlation between the frequency of axo–glial detachment at the paranode and axonal degeneration, as assessed by teased-fibre preparations (p<0.05). Whorling of myelin was frequently found at the paranodes, particularly in large myelinated fibres. Similar axo–glial detachment was also found in the case with anti-contactin-1 antibodies (figure 4A, B), particularly at the terminal loops neighbouring nodes of Ranvier.
Although the frequency was low, the widening of nodes of Ranvier was observed in antibody-positive patients, particularly in two patients (Patients 8 and 9) with a high frequently of axonal degeneration in teased-fibre preparations (figure 3D). However, the extent of the widening of the nodes of Ranvier was usually mild in antibody-positive patients, and associated macrophages were not found. The widening of nodes of Ranvier was found in nodes with and without axo–glial detachment.
In the present study, we assessed the morphological features of sural nerve biopsy specimens from CIDP patients with and without autoantibodies to paranodal axo–glial junctional molecules. Characteristic light and electron microscopy findings in transverse sections from patients positive for these antibodies revealed a slight reduction in myelinated fibre density with scattered myelin ovoids and the absence of macrophage-mediated demyelination or onion bulbs. As for the light microscopy observations of the transverse sections of the sural nerve biopsy specimens, similar findings have been reported previously in the assessment of two patients with anti-neurofascin-155 antibodies.20 Photographs taken from two other patients in a subsequent report also revealed similar features, although the presence of demyelination was also mentioned.21 Three sural nerve biopsy specimens taken from patients with CIDP with anti-contactin-1 antibodies also seemed to show similar pathological findings.18 These studies, in addition to ours, indicate that the classical macrophage-mediated demyelinating process so far reported in CIDP1 ,5 ,7 ,32 ,33 does not participate in the pathogenesis of the neuropathies with these antibodies.
Myelinated fibres of the peripheral nervous system are divided into four structurally essential domains along the axon: nodes of Ranvier, paranodes, juxtaparanodes and internodes.36 The node of Ranvier is the site where action potentials are generated to sustain saltatory conduction, while the paranode acts as an insulator.13 ,31 Neurofascin-155 is among the paranodal molecules that are expressed on the myelin side and is tightly connected to axonal contactin-1.31 These structural and molecular distributions around the node of Ranvier are essential for saltatory conduction, which is invariably impaired in CIDP, and are important for the axon–Schwann cell interactions that maintain axons.36 In this context, assessment of paranodal structures using longitudinal sections, which facilitate observation of the relationships between the four domains, is needed to clarify if the initial lesion results from autoantibodies directed against paranodal axo–glial junctional molecules such as neurofascin-155 and contactin-1.
The most impressive finding in our study was the paranodal dissection at the axo–glial junctions in patients with anti-neurofascin-155 or anti-contactin-1 antibodies, as shown by ultrastructural assessments of the longitudinal sections. In previous studies of neurofascin-155 and contactin-1-deficient mice, loss of electron-dense regions between myelin terminal loops and axolemma despite their close apposition was reported.37 ,38 These results suggest that abnormalities of paranodal axo–glial junctional molecules affect tight connection between myelin terminal loops and axolemma. As the space between myelin terminal loops and axolemma became wide in our cases, it may induce nerve conduction abnormalities that fulfil the electrodiagnostic criteria for CIDP. As IgG subclass of anti-neurofascin-155 and anti-contactin-1 antibodies was predominantly IgG4 that does not activate complement,22 it may therefore not initiate an inflammatory response. The absence of macrophage-mediated demyelination was the characteristic feature in patients with anti-neurofascin-155 and anti-contactin-1 antibodies. IgG4-subclass antibodies to neurofascin-155 and contactin-1 may affect adhesion between myelin terminal loops and axolemma without inducing inflammatory cascades at paranode. A correlation between anti-neurofascin-155 antibody titres and the frequency of axo–glial detachment was not found in this study. This result may be attributed to small sample size and initiation of immunotherapies before antibody determination in some of the cases.
Although segmental demyelination, detected as the widening of nodes of Ranvier, was observed in teased-fibre preparations from 3 of the 10 patients in the present study, such widening was not necessarily related to paranodal dissection on electron microscopic examination. In a previous study of neuropathy with anti-MAG antibodies, the detachment and dislocation of terminal loops adjacent to a node of Ranvier led to demyelination at the paranode.27 In contrast, a significant dislocation of the terminal loops close to the node of Ranvier, which peel off the axons, was not observed in our patients, although abnormal spaces were frequently observed at that portion. In polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes (POEMS) syndrome, in which uncompacted myelin lamellae frequently start at the junction of the paranodes and juxtaparanodes, the widening of nodes of Ranvier was observed despite the preservation of paranodal junctions adjacent to nodes of Ranvier.28 Although uncompacted myelin lamellae were seen in some of our cases, they were observed regardless of the presence or absence of anti-paranodal axo–glial junctional autoantibodies. The frequency of axonal degeneration in cases with a high frequency of segmental demyelination was relatively high compared with that of the other cases in the anti-neurofascin-155 antibody-positive group. The widening of consecutive nodes of Ranvier in a single fibre may also result from axonal atrophy or degeneration.24 ,39 Hence, the demyelination found in our patients with anti-neurofascin-155 antibodies may be, at least partly, a secondary change related to the disturbances in axon–Schwann cell interactions resulted from axo–glial detachment. Interestingly, typical axo–glial detachment, as shown in figure 3, is not found in anti-MAG neuropathy and POEMS syndrome even though axonal degeneration is extensive.27 ,28 Although the extent was much smaller than the antibody-positive group, clear spaces between terminal loops and axolemma were also found in the antibody-negative group. Slightly widened axo–glial junction of a patient with CIDP has also been shown in a previous literature.40 Further studies are needed to clarify the significance of such small spaces.
In conclusion, we clarified the morphological changes of peripheral nerves in patients with CIDP with anti-paranodal axo–glial junctional molecules. Paranodal dissection and the absence of classical macrophage-mediated demyelination were the characteristic features in these patients. Impairment of saltatory conduction and axonal damage may occur as a result of axo–glial detachment in these patients.
Correction notice Since this paper was first published online the terms anticontactin and antineurofascin now read anti-contactin and anti-neurofascin.
Contributors HK and GS developed the hypotheses and conceived the study. HK, SI, YK, MI, DK, NM, MK and GS compiled and analysed the clinical data. MK and KK determined anti-neurofascin-155 and anti-contactin-1 antibodies; HO, RY and JK verified the results. HK, SI, YK, MI and GS performed the pathological analysis. GS supervised the study. HK wrote the first draft and all authors critically evaluated the manuscript.
Funding This work was supported by grants from the Ministry of Health, Labour and Welfare (Research on rare and intractable diseases, H26-057) and the Ministry of Education, Culture, Sports, Science and Technology (25461276) of Japan.
Competing interests None declared.
Patient consent Obtained.
Ethics approval Ethics Committees of Nagoya University Graduate School of Medicine, National Defense Medical College and Kyushu University.
Provenance and peer review Not commissioned; externally peer reviewed.