Fig. 4
ARNT2 expression is decreased during peak disease in neuronal populations. a A relative loss of NeuN+ cells is observed in EAE mice compared to healthy sham-immunized controls examined in five to seven levels from three to four mice each. b Expression levels of ARNT2 were normalized to the staining of ependymal cells and categorized as high (mean intensities greater than 2× the background) or moderate (greater than 1.5× the background) as shown in representative images. c The % of NeuN+ cells expressing moderate or greater ARNT2 intensity was reduced by almost 30% in animals at peak disease. These differences were even greater in analyses of the dorsal horn (DH) regions in each section. Bars represent the mean intensity of 4–6 levels pooled from 7 healthy/CFA immunized animals (n = 39) compared to levels pooled from 5 EAE animals (n = 29) where whiskers indicate the SEM. p ≤ 0.05*, p ≤ 0.01**, p ≤ 0.001***, Mann-Whitney T test. d GFAP+ astrocytes were enumerated per mm2 of gray matter in each of the same levels analyzed in c. e A comparison of healthy to EAE animals at the L5/L6 level showed a marked increase in the frequency of GFAP+ cells in the gray matter of EAE mice; notably, these cells are largely positive for ARNT2 (arrows). f Each infiltrate was scored and pooled per each of 5 levels from 5 EAE mice. This infiltration score from 25 levels of EAE cord was compared to the mean intensity of ARNT2 in NeuN+ cells in the GM. g Serial sections from the same levels examined in C were stained for MBP and SMI-32. SMI32 positivity was not detected in sham-immunized animals, nor was a decrease in MBP staining. Associated with regions of infiltrates in EAE animals (inset), SMI32+ axons and spheroids were more frequent and in the lower areas of cord tended to be associated with regions of demyelination. In higher thoracic areas of the cord, SMI32 positivity was still present but demyelination was less prevalent. h The no. of SMI32+ events/mm2 and i % demyelination as calculated by masking regions devoid of MBP staining were correlated with ARNT2 mean intensity as outlined in f