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. 2021 Jun 4;8(5):e1028. doi: 10.1212/NXI.0000000000001028

Figure 2. Assessment of Pain-Like Behaviors and Neuronal Activation in Passive Transfer Mice 24 Hours After Injection.

Figure 2

(A) Mechanical pain hypersensitivity was assessed by calibrated von Frey filaments (0.04, 0.07, 0.16, 0.40, and 0.60 g) 24 hours after injection. Mice treated with purified IgG from patients 1, 2, and 3 showed significantly higher reaction rates to each stimulation strength (0.07, 0.16, 0.40, and 0.60 g filaments) than HC IgG-treated mice (1-way ANOVA with Dunnett test; *p < 0.01). No significant differences in reaction rates were seen with IgG from 2 inflammatory disease controls (1 patient with NBD and 1 patient with NPSLE) and patients 1, 2, and 3 after preadsorption with rhPlexin D1 compared with control IgG (1-way ANOVA with Dunnett test). (B) Indirect IFA of mouse L5 DRG sections revealed that IgG (green) from the patient with SFN (patient 3) with plexin D1-IgG but not control IgG bound to small DRG neurons. IgG from patient 3 preabsorbed with rhPlexin D1 (2 µg/mL) showed no significant immunoreactivity to mouse DRG. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (blue). Immunostaining of pERK, a marker of primary afferent neuron activation, in L5 DRG of mice treated with purified IgG from patient 3 and control 24 hours after injection. Most of the pERK-labeled neurons in mice treated with purified IgG from patient 3 are small DRG neurons (≤25 µm in diameter). Few neurons are labeled for pERK in mice treated with control IgG and IgG from patient 3 after preabsorption with rhPlexin D1. Sections were counterstained with hematoxylin. (C) Distribution of pERK immunoreactivity among different-sized neurons was analyzed. DRG neurons were stratified as small (≤25 mm in diameter) and large (>25 mm in diameter) neurons. The pERK-labeled neurons in L5 DRG of mice treated with purified IgG from patients 1, 2, and 3 were predominantly small neurons of less than 25 μm in diameter. Scale bars = 50 µm and (inset) = 25 µm. ANOVA = analysis of variance; DRG = dorsal root ganglia; HC = healthy control; IFA = immunofluorescence assay; IgG = immunoglobulin G; NBD = neuro-Behçet disease; NPSLE = neuropsychiatric systemic lupus erythematosus; pERK = phosphorylated extracellular signal-regulated protein kinase; Pre-Ab = preabsorbed; rhPlexin D1 = recombinant human plexin D1.