Antibodies to Gliomedin Cause Peripheral Demyelinating Neuropathy and the Dismantling of the Nodes of Ranvier
2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2012.06.034
ISSN1525-2191
Autores Tópico(s)Nerve injury and regeneration
ResumoGuillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP. Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP. Guillain-Barré syndrome (GBS) is a group of inflammatory neuropathies that affect peripheral nerves. In Europe, acute inflammatory demyelinating polyneuropathy (AIDP) is the most common form of GBS. Autopsy and biopsy studies indicated that both humoral and cellular immune reaction against Schwann cell or axonal antigens are implicated in GBS etiology.1Hughes R.A. Cornblath D.R. Guillain-Barré syndrome.Lancet. 2005; 366: 1653-1666Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar Early investigations have found that conduction defects closely correlate with myelin retraction and macrophage invasion in many patients.2Prineas J.W. 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Nodal proteins are target antigens in Guillain-Barré syndrome.J Peripher Nerv Syst. 2012; 17: 62-71Crossref PubMed Scopus (143) Google Scholar Notably, cell adhesion molecules (CAMs) at nodes or paranodes (gliomedin, neurofascin, and contactin) were recognized by IgG antibodies in patients with GBS or CIDP.12Devaux J.J. Odaka M. Yuki N. Nodal proteins are target antigens in Guillain-Barré syndrome.J Peripher Nerv Syst. 2012; 17: 62-71Crossref PubMed Scopus (143) Google Scholar, 13Pruss H. Schwab J.M. Derst C. Gortzen A. Veh R.W. Neurofascin as target of autoantibodies in Guillain-Barré syndrome.Brain. 2011; 134: 173Crossref Scopus (38) Google Scholar Autoantibodies against neurofascin and gliomedin were also detected in a rat model of AIDP and correlated with important conduction defects.14Lonigro A. Devaux J.J. Disruption of neurofascin and gliomedin at nodes of Ranvier precedes demyelination in experimental allergic neuritis.Brain. 2009; 132: 260-273Crossref PubMed Scopus (101) Google Scholar This finding suggested that antibodies to nodal CAMs may participate to the pathogenesis of AIDP and CIDP. However, the exact mechanisms by which these humoral factors mediate demyelination and conduction defects are still elusive. Several CAMs are implicated in node formation and are responsible for the enrichment of voltage-gated sodium (Nav) channels at the nodes of Ranvier.15Susuki K. Rasband M.N. Molecular mechanisms of node of Ranvier formation.Curr Opin Cell Biol. 2008; 20: 616-623Crossref PubMed Scopus (102) Google Scholar At peripheral, nodes gliomedin and NrCAM are secreted into the nodal gap lumen and interact with neurofascin-186 (NF186) expressed at nodal axolemma.16Eshed Y. Feinberg K. Carey D.J. Peles E. Secreted gliomedin is a perinodal matrix component of peripheral nerves.J Cell Biol. 2007; 177: 551-562Crossref PubMed Scopus (86) Google Scholar, 17Maertens B. Hopkins D. Franzke C.W. Keene D.R. Bruckner-Tuderman L. Greenspan D.S. Koch M. Cleavage and oligomerization of gliomedin, a transmembrane collagen required for node of Ranvier formation.J Biol Chem. 2007; 282: 10647-10659Crossref PubMed Scopus (76) Google Scholar, 18Davis J.Q. Lambert S. Bennett V. Molecular composition of the node of Ranvier: identification of ankyrin-binding cell adhesion molecules neurofascin (mucin+ third FNIII domain-) and NrCAM at nodal axon segments.J Cell Biol. 1996; 135: 1355-1367Crossref PubMed Scopus (306) Google Scholar, 19Feinberg K. Eshed-Eisenbach Y. Frechter S. Amor V. Salomon D. Sabanay H. Dupree J.L. Grumet M. Brophy P.J. Shrager P. Peles E. A glial signal consisting of gliomedin and NrCAM clusters axonal Na+ channels during the formation of nodes of Ranvier.Neuron. 2010; 65: 490-502Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar This interaction is crucial for Nav channel aggregation at nodes.19Feinberg K. Eshed-Eisenbach Y. Frechter S. Amor V. Salomon D. Sabanay H. Dupree J.L. Grumet M. Brophy P.J. Shrager P. Peles E. A glial signal consisting of gliomedin and NrCAM clusters axonal Na+ channels during the formation of nodes of Ranvier.Neuron. 2010; 65: 490-502Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Labasque M. Devaux J.J. Leveque C. Faivre-Sarrailh C. Fibronectin type III-like domains of neurofascin-186 protein mediate gliomedin binding and its clustering at the developing nodes of Ranvier.J Biol Chem. 2011; 286: 42426-42434Crossref PubMed Scopus (32) Google Scholar, 21Sherman D.L. Tait S. Melrose S. Johnson R. Zonta B. Court F.A. Macklin W.B. Meek S. Smith A.J. Cottrell D.F. Brophy P.J. Neurofascins are required to establish axonal domains for saltatory conduction.Neuron. 2005; 48: 737-742Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar In addition, the paranodal axoglial junctions are made by the association of contactin and contactin-associated protein (Caspr) with neurofascin-155 (NF155), a variant expressed in glia.22Charles P. Tait S. Faivre-Sarrailh C. Barbin G. Gunn-Moore F. Denisenko-Nehrbass N. Guennoc A.M. Girault J.A. Brophy P.J. Lubetzki C. Neurofascin is a glial receptor for the paranodin/Caspr-contactin axonal complex at the axoglial junction.Curr Biol. 2002; 12: 217-220Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar This adhesive junction forms a barrier to the lateral diffusion of nodal channels.19Feinberg K. Eshed-Eisenbach Y. Frechter S. Amor V. Salomon D. Sabanay H. Dupree J.L. Grumet M. Brophy P.J. Shrager P. Peles E. A glial signal consisting of gliomedin and NrCAM clusters axonal Na+ channels during the formation of nodes of Ranvier.Neuron. 2010; 65: 490-502Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 21Sherman D.L. Tait S. Melrose S. Johnson R. Zonta B. Court F.A. Macklin W.B. Meek S. Smith A.J. Cottrell D.F. Brophy P.J. Neurofascins are required to establish axonal domains for saltatory conduction.Neuron. 2005; 48: 737-742Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 23Zonta B. Tait S. Melrose S. Anderson H. Harroch S. Higginson J. Sherman D.L. Brophy P.J. Glial and neuronal isoforms of Neurofascin have distinct roles in the assembly of nodes of Ranvier in the central nervous system.J Cell Biol. 2008; 181: 1169-1177Crossref PubMed Scopus (149) Google Scholar In a rat model of AIDP, we found that the loss of NF186 and gliomedin at nodes preceded paranodal demyelination and the diffusion of Nav channels in demyelinated segments.14Lonigro A. Devaux J.J. Disruption of neurofascin and gliomedin at nodes of Ranvier precedes demyelination in experimental allergic neuritis.Brain. 2009; 132: 260-273Crossref PubMed Scopus (101) Google Scholar This finding indicated that antibodies to nodal CAMs may participate to conduction defects by dismantling axoglial attachment at nodes and paranodes. We investigated whether immunity toward gliomedin and NF186 can trigger peripheral neuropathies and be responsible for demyelination in GBS patients. We found that immunization against gliomedin induced a biphasic condition associated with conduction loss and demyelination. Passive transfer of antibodies to gliomedin exacerbated the clinical signs of EAN and resulted in the disorganization of the nodes of Ranvier. Altogether, these results demonstrate that humoral immune response directed against nodal CAMs participates in conduction abnormalities in peripheral nerves and in the etiology of GBS and CIDP. The extracellular domain of NF186 fused to human IgG Fc (NF186-Fc) and gliomedin fused to human IgG Fc (Gldn-Fc) were obtained as described previously22Charles P. Tait S. Faivre-Sarrailh C. Barbin G. Gunn-Moore F. Denisenko-Nehrbass N. Guennoc A.M. Girault J.A. Brophy P.J. Lubetzki C. Neurofascin is a glial receptor for the paranodin/Caspr-contactin axonal complex at the axoglial junction.Curr Biol. 2002; 12: 217-220Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 24Eshed Y. Feinberg K. Poliak S. Sabanay H. Sarig-Nadir O. Spiegel I. Bermingham J.R. Peles E. Gliomedin mediates Schwann cell-axon interaction and the molecular assembly of the nodes of Ranvier.Neuron. 2005; 47: 215-229Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar and stored at −80°C. The human IgG Fc fragment was purchased from Bethyl Laboratories (Montgomery, TX). Male inbred adult Lewis rats (6 to 7 weeks old; Elevage Janvier, Le Genest St Isle, France) were sensitized by subcutaneous injection at the base of the tail with 200 μL of an antigen emulsion. The antigens were emulsified with an equal volume of complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO). Final doses in the inoculum were 100 μg of H37RA Mycobacterium tuberculosis and 50 μg of Fc fusion proteins. Animals received intraperitoneal injections of 200 ng of pertussis toxin in PBS on the day of immunization and 48 hours after immunization. Animals were weighed and observed daily. Clinical signs were graded as follows: 0, no illness; 1, tail tip hanging; 2, limp tail; 3, tail paralysis; 4, gait ataxia; 5, mild paraparesis; 6, severe paraparesis; 7, paraplegia; 8, tetraparesis; 9, moribund; and 10, death. All of the experiments were in lines with the European Community's guiding principles on the care and use of animals (86/609/CEE). Blood was collected by cardiac puncture at disease peaks (30 days after immunization) from six animals immunized with Gldn-Fc or control Fc. IgG was purified from serum samples by affinity chromatography with protein G sepharose according to the manufacturer protocol (Sigma-Aldrich). The synthetic peptide of bovine P2 myelin protein (amino acids 53 to 78)25Uyemura K. Suzuki M. Kitamura K. Horie K. Ogawa Y. Matsuyama H. Nozaki S. Muramatsu I. Neuritogenic determinant of bovine P2 protein in peripheral nerve myelin.J Neurochem. 1982; 39: 895-898Crossref PubMed Scopus (38) Google Scholar was purchased from Bachem (Bubendorf, Switzerland) and dissolved in saline (2 mg/mL). Lewis rats were sensitized with 50 μg of P2 antigen (EAN-P2) in 100 μL of saline emulsified with 100 μL of complete Freund's adjuvant. At the onset of disease (12 days after immunization), rats received i.p. injections of 500 μg of purified anti-gliomedin IgG or control rat IgG. In parallel, naive Lewis rats received i.p. injections of 500 μg of purified anti-gliomedin IgG. Animals were weighed and examined daily for clinical signs. L6 spinal nerves from immunized Lewis rats and sciatic nerves from adult C57BL/6J mice were dissected and fixed in 2% paraformaldehyde in PBS for 1 hour at 4°C, then rinsed in PBS. Axons were gently teased, dried on glass slides, and stored at −20°C. In some experiments, unfixed L6 spinal roots were rapidly teased, dried, and frozen. Alternatively, fixed spinal nerves were cryoprotected in 30% sucrose in 0.1 mol/L PBS overnight at 4°C, then cut into 5- to 10-μm–thick cryosections. Frozen sections and teased fibers were permeabilized by immersion in −20°C acetone for 10 minutes, blocked at room temperature for 1 hour with 5% fish skin gelatin containing 0.1% Triton X-100 in PBS, and incubated overnight at 4°C with various combinations of primary antibodies or sera: rabbit anti-sera against gliomedin (1/500),24Eshed Y. Feinberg K. Poliak S. Sabanay H. Sarig-Nadir O. Spiegel I. Bermingham J.R. Peles E. Gliomedin mediates Schwann cell-axon interaction and the molecular assembly of the nodes of Ranvier.Neuron. 2005; 47: 215-229Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar NF186 (1/500),26Southwood C. He C. Garbern J. Kamholz J. Arroyo E. Gow A. CNS myelin paranodes require Nkx6-2 homeoprotein transcriptional activity for normal structure.J Neurosci. 2004; 24: 11215-11225Crossref PubMed Scopus (70) Google Scholar or Caspr (1/1000)27Menegoz M. Gaspar P. Le Bert M. Galvez T. Burgaya F. Palfrey C. Ezan P. Arnos F. Girault J.A. Paranodin, a glycoprotein of neuronal paranodal membranes.Neuron. 1997; 19: 319-331Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar; mouse monoclonal antibodies against PanNav channels (K58/35; 1:500; Sigma-Aldrich), Nav1.6 (1/100; University of California, Davis, National Institute of Neurological Disorders and Stroke, National Institute of Mental Health, NeuroMab Facility, Davis, CA), ED1 (1/200; AbD Serotec, Oxford, UK), CD3 (1/200; AbD Serotec), or C5b-9 (1/50; DakoCytomation, Glostrup, Denmark); goat antibody against contactin (1/200; R&D Systems, Minneapolis, MM) or rat complement C3 (Nordic Immunological Laboratories, Tilburg, The Netherlands); or rat sera diluted 1/200 to 1/2000. The slides were then washed several times and incubated with the appropriate Alexa-conjugated secondary antibodies (1/500; Invitrogen, Paisley, UK). Slides were mounted with Mowiol plus 2% 1,4-diazabicyclo[2.2.2]octane and examined using an ApoTome fluorescence microscope (ApoTome, AxioObserver and AxioCam MRm, Carl Zeiss MicroImaging GmbH, Jena, Germany). Digital images were manipulated into figures with Adobe Photoshop (Adobe Systems Inc, San Jose, CA) and CorelDraw (Corel Corporation, Ottawa, ON, Canada). Teased fibers from five animals were analyzed for each group (∼500 axons counted in total). The lengths of individual Caspr-positive paranodes and intercalated nodes were measured using ImageJ software version 1.43u (NIH, Bethesda, MD). For the quantification of demyelinated axons, teased fibers were stained for Caspr, and intercalated nodes larger than 5 μm were counted as demyelinated. For histopathologic analysis, L6 spinal nerves were fixed in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L PBS overnight at 4°C and postfixed in 1% OsO4 in 0.1 mol/L PBS for 1 hour. Nerves were dehydrated and embedded in epoxy resin. Transverse semithin sections were stained with toluidine blue and examined by light microscopy. The number of degenerated myelinated axons was measured in five animals for each group (all of the fibers in the roots were counted; ∼1000 axons per animals). Schwann tubes, >2 μm in diameter and typically containing myelin debris but no recognizable axon, were considered to be degenerating myelinated axons. Blood was collected from the lateral tail vein before immunization (preimmune), and at 7, 14, 21, 30, 45, and 80 days after immunization. Blood was allowed to clot for 30 minutes at room temperature, then blood was centrifuged for 10 minutes at 3000 × g, and serum samples were collected. Human embryonic kidney (HEK) cells were transiently transfected with rat NF186 (NM_001160314.1; HA tagged) or rat gliomedin (NM_181382.2; Myc tagged) using JetPEI (Polyplus-transfection, Illkirch, France). One day after transfection, cells were trypsinated, suspended in serum free Opti-MEM medium (Invitrogen), and plated onto poly-l-lysine coated glass coverslips in 24-well plates at a density of 100,000 cells per well. One day after, living cells were incubated for 20 minutes with 50 μL of serum diluted at 1/50 to 1/10,000 in blocking solution (5% fish skin gelatin in PBS). Serum samples were preincubated for 30 minutes at room temperature with fluorescein isothiocyanate–conjugated anti-rat IgM (1/50) or tetramethyl rhodamine isothiocyanate–conjugated anti-rat IgG (1/50). Cells were washed three times in PBS, fixed with 2% paraformaldehyde in 0.1 mol/L PBS for 20 minutes, rinsed in PBS, and blocked for 30 minutes. Cells were then incubated for 1 hour with primary antibodies: rat monoclonal antibodies against HA (1/200; Roche, Basel, Switzerland) or mouse monoclonal antibodies against Myc (1/500; Roche). The cells were then washed and revealed with the appropriate Alexa-conjugated secondary antibodies (1/500; Invitrogen). Cells were stained with DAPI and mounted with Mowiol plus 2% 1,4-diazabicyclo[2.2.2]octane. Recordings were performed at different stages of the disease. After euthanizing, the caudal equine were quickly dissected and transferred into artificial cerebrospinal fluid equilibrated with 95% O2−5% CO2, which contained 126 mmol/L NaCl, 3 mmol/L KCl, 2 mmol/L CaCl2, 2 mmol/L MgSO4, 1.25 mmol/L NaH2PO4, 26 mmol/L NaHCO3, and 10 mmol/L dextrose, pH 7.4 to 7.5. The L6 ventral spinal roots were cut into 2-cm segments, and recordings of nerve compound action potentials (CAPs) were made at 36°C in a three-compartment recording chamber as previously described.14Lonigro A. Devaux J.J. Disruption of neurofascin and gliomedin at nodes of Ranvier precedes demyelination in experimental allergic neuritis.Brain. 2009; 132: 260-273Crossref PubMed Scopus (101) Google Scholar Nerves were stimulated at a single site. The delay and duration of the CAPs were calculated at half the maximal amplitude. Conduction velocities were estimated from latencies. For recruitment analysis, nerves were stimulated at increasing intensities. For refractory period analysis, two stimuli were applied at different intervals, and the amplitude of the second CAP was measured and plotted as a function of the stimulus interval. To determine whether the immune reaction toward nodal proteins participates in the pathogenesis of AIDP, Lewis rats were immunized with the extracellular domains of NF186-Fc; 50 μg) and Gldn-Fc (50 μg). Animals were treated with pertussis toxin on days 0 and 2 to potentiate the immune response. NF186-Fc or Fc alone did not induce significant neurologic signs in Lewis rats. By contrast, animals immunized against Gldn-Fc developed progressive neurologic symptoms within 7 to 9 days (Figure 1A). The clinical course was biphasic, with a first peak at days 12 to 15 followed by a complete remission and a clinical worsening starting around 21 days after immunization (Figure 1, A and B). The course of the secondary phase was variable among individuals and lasted for several weeks. Some animals presented a relapse-remitting course, whereas other presented a secondary progressive rise to a plateau (Figure 1B). At disease peaks, animals exhibited a maximal clinical score of approximately 4 (see Supplemental Figure S1C at http://ajp.amjpathol.org) characterized by tail paralysis and gait abnormalities. Higher doses of immunogen did not enhance clinical signs or reveal symptoms in the case of NF186-Fc. Electrophysiologic examinations at disease peaks (between 30 and 45 days after immunization) demonstrated that gliomedin-sensitized animals exhibited important conduction deficits in L6 ventral spinal nerves with a marked decrease in CAP amplitude and in conduction velocity (Figure 1 and Table 1). These alterations were associated with a significant increase in the refractory period and a displacement of the recruitment curve toward higher voltages (P < 0.01; see Supplemental Figure S1A at http://ajp.amjpathol.org), which are hallmarks of demyelinating conditions. However, sciatic nerves, L5 ventral spinal roots, or dorsal roots were less affected (data not shown). Altogether, these results indicated that immunity to gliomedin induces a predominantly motor demyelinating neuropathy.Table 1Characteristics of CAPs from Ventral Spinal Roots of Animals Immunized Against GliomedinCharacteristicLewis rats immunized againstFcGldn-FcAmplitude (mV)17.2 ± 6.87.3 ± 5.1⁎Significantly different P < 0.01 with two-tailed t-tests for two samples of equal variance.Area (mV · ms)4041 ± 20142247 ± 1395⁎Significantly different P < 0.01 with two-tailed t-tests for two samples of equal variance.Duration (ms)0.43 ± 0.090.54 ± 0.27CVV½ (m · s−1)40.2 ± 3.831.8 ± 3.5⁎Significantly different P < 0.01 with two-tailed t-tests for two samples of equal variance.CVVmax (m · s−1)27.5 ± 3.321.7 ± 4.5⁎Significantly different P < 0.01 with two-tailed t-tests for two samples of equal variance.No.†No. represents the number of nerves tested. The number of animals tested is indicated in parentheses.8 (4)10 (6)Days of analysis after immunization3030–45The data were recorded at the peaks of severity from L6 ventral roots of animals immunized against human Fc or Gldn-Fc. Data are presented as mean ± SD.CVV½, conduction velocity at half the maximal amplitude; CVVmax, conduction velocity at peak amplitude. Significantly different P < 0.01 with two-tailed t-tests for two samples of equal variance.† No. represents the number of nerves tested. The number of animals tested is indicated in parentheses. Open table in a new tab The data were recorded at the peaks of severity from L6 ventral roots of animals immunized against human Fc or Gldn-Fc. Data are presented as mean ± SD. CVV½, conduction velocity at half the maximal amplitude; CVVmax, conduction velocity at peak amplitude. To examine the organization of the nodes of Ranvier, L6 ventral spinal roots were immunostained for Caspr or contactin. Caspr and contactin showed the expected distinct paranodal distribution (Figure 2). However, many axons showed signs of nodal elongation or paranodal demyelination (Figure 2, C and E). The length of unstained nodal gap was determined from nine different animals and was found to be significantly widened in gliomedin-sensitized animals (Figure 2, F and G). More than 5% of the nodes were >5 μm in length and were undergoing paranodal demyelination. No signs of demyelination were observed in animals immunized against NF186-Fc or control Fc. Next, I investigated the possibility that nodal organization was affected in gliomedin-sensitized animals. In most axons, NF186, gliomedin, and Nav channels were properly clustered at normal-appearing nodes (Figure 2 and Table 2). However, NF186, gliomedin, and Nav channels were missing or diffusely localized in most demyelinated segments (Figure 2E and Table 2). Only a few demyelinated segments showed NF186, gliomedin, or Nav channel clusters at heminodes that flanked the paranodes (Figure 2C). These results indicated that node elongation, demyelination, and node disruption might account for conduction defects and neurologic signs in these animals.Table 2Percentage of Nodal Cluster Disruption in Animals Immunized Against GliomedinFcGldn-FcRabbit α-gliomedin Normal nodes (%)99.192.2 Disrupted nodes (%)0.97.8 No.802805Rabbit α-NF186 Normal nodes (%)99.290.8 Disrupted nodes (%)0.89.2 No.602692Mouse α-PanNav Normal nodes (%)99.296.1 Disrupted nodes (%)0.83.9 No.10541144Days of analysis after immunization3030–45Teased fibers were prepared at the peaks of severity from L6 ventral roots of five different animals immunized against human Fc or Gldn-Fc. The teased fibers were stained with the indicated antibodies and a goat antiserum anti-contactin. Nodes and heminodes with bright and focal gliomedin, NF186, or PanNav staining were considered normal nodes. Nodes and heminodes with diffuse and weak staining were considered disrupted. No. represents the number of nodes examined. Open table in a new tab Teased fibers were prepared at the peaks of severity from L6 ventral roots of five different animals immunized against human Fc or Gldn-Fc. The teased fibers were stained with the indicated antibodies and a goat antiserum anti-contactin. Nodes and heminodes with bright and focal gliomedin, NF186, or PanNav staining were considered normal nodes. Nodes and heminodes with diffuse and weak staining were considered disrupted. No. represents the number of nodes examined. To understand the immunopathologic mechanisms underlying EAN, spinal nerves were examined for immune cell infiltration or IgG deposits at disease peaks (30 to 45 days after immunization). Semithin transverse sections confirmed the presence of demyelination. Only a few degenerated axons were visible at disease peaks and may be secondary to demyelination or inflammation (Figure 2G). Worth noting, spinal nerves appeared devoid of immune cell infiltration. Consistently, no signs of CD3-positive T cells were found in spinal nerves (data not shown), and only a few ED1-positive macrophages were detected in gliomedin-sensitized animals (see Supplemental Figure S2 at http://ajp.amjpathol.org). Instead, a strong IgG deposit was detected at most nodes (57%; n = 4 animals) in L6 ventral spinal nerves (Figure 3, F and G). This finding was associated with a faint deposition of the terminal complement complex (C5b-9) in 22% of the nodes (n = 4 animals; Figure 3, H and I). Worth noting, IgG deposits were mostly found at in
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