Fig. 2

CREST Haploinsufficiency Leads to Upregulation of Inflammatory Responses in the Central Nervous System and the Denervation of Neuromuscular Junctions in TA Muscles. a Representative immunohistochemistry images of Iba1-positive microglia (highlighted by arrows, green, upper panels) and their skeletonized appearance (lower panels) in the lumbar spinal cords of 6-month-old Crest ^+/− mice (n = 3) and WT littermates (n = 3). Scale bar, 20μm. (b-e) Analysis and quantification of microglial morphological parameters including soma area (b) and roundness (c) of the projection of Iba1-positive cell bodies (each symbol indicating one cell), and branch length (d) and branch number (e) per cell (each symbol indicating one image, about 6 images per mouse) in the lumbar spinal cords of 6-month-old Crest ^+/− mice (n = 3) and WT littermates (n = 3). f Representative immunohistochemistry images of Iba1-positive microglia (highlighted by arrows, green, upper panels) and their skeletonized appearance (lower panels) in the cerebral motor cortices of 6-month-old Crest ^+/− mice (n = 3) and WT littermates (n = 3). Scale bar, 20μm. (G-J) Analysis and quantification of microglial morphological parameters including soma area (g) and roundness (h) of the projection of Iba1-positive cell bodies (each symbol indicating one cell), and branch length (i) and branch number (j) per cell (each symbol indicating one image, about 6 images per mouse) in the cerebral motor cortices of 6-month-old Crest ^+/− mice (n = 3) and WT littermates (n = 3). k mRNA levels of proinflammatory genes, such as IL-1β, TNF-α and COX-2, in frontal cortices from 18-month-old Crest ^+/− mice (n = 9) and WT littermates (n = 3) measured by quantitative RT-PCR. (l and m) Representative immunohistochemistry images (l) and quantification (m) of ChAT-positive (red) motor neurons (each symbol indicating one image, 4 images per mouse) in ventral horn of the lumbar spinal cords of 6-month-old Crest ^+/− mice (n = 3) and WT littermates (n = 3). Dashed lines indicate grey matter of lumbar spinal cord. Scale bar, 100μm. (n and o) Representative immunohistochemistry images (n) and quantification (o) of the innervation of neuromuscular junctions in tibialis anterior (TA) muscles of 6-month-old Crest ^+/− mice (n = 3) and WT littermates (n = 3). Each symbol indicates one innervation ratio of about 100 neuromuscular junctions). Acetylcholine receptors were labeled with α-bungarotoxin (BTX) CF488A conjugate (green), motor neuron axons were labeled with anti-neurofilament-L (NF) antibody (red), and synapses were labeled with anti-synapsin-1 (Syn) antibody (red). Scale bar, 100μm. Error bars represent SEM. *p < 0.05, **p < 0.01, Student’s t test