Fig. 4

Repeated dural IS stimulation induced gliosis and neuronal loss in the cerebral cortex and hippocampus, but no obvious white matter damage. (a-d) Representative immunofluorescence analysis showed an increase in Iba-1 positive microglia and GFAP positive astrocytes in the cortex and hippocampus following dural IS stimulation, indicating microgliosis and astrogliosis. Magnification × 40. Insets show a higher magnification view. Scale bar, 20 μm. (e–f) Representative immunofluorescence images showed a decrease in MAP2-positive areas (an indicator of neuronal loss) in the cerebral cortex and hippocampus, but no significant change in MBP-positive areas (an indicator of white matter integrity) after repeated dural IS stimulation. Magnification × 20. Scale bar, 20 μm. (g-h) Representative crystal violet images and quantification showed the loss of Nissl-positive neurons in the cortical and hippocampal CA3 regions after repeated dural IS stimulation. Magnification × 40. Scale bar, 20 μm. (i-j) Representative Luxol fast blue stained images and quantification showed no obvious white matter lesion in the corpus callosum (medial), corpus callosum (paramedian), caudoputamen, internal capsule, and optic tract after repeated dural IS stimulation. The severity of white-matter disruption was graded accordingly: Grade 0 = no disruption; Grade 1 = disarrangement of nerve fibers; Grade 2 = formation of marked vacuoles; Grade 3 = disappearance of myelinated fibers. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 as compared to sham mice. Datasets in (j) were conducted with Mann–Whitney U test; the other datasets were analyzed using Student’s t test. Data are represented as mean ± SEM. Abbreviations: IS, inflammatory soup; Iba-1, ionized calcium-binding adaptor molecule-1; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; MBP, myelin basic protein