Fig. 6
Chronic neuroinflammation, neuronal hyperactivity, and serotonin reduction in the brainstem. Neurons in the gracile nucleus (GrN; A) of SCI rats had significantly higher group mean IRL of Iba-1 (B, D) and TNFα (B1, D1), compared to those of control animals (C). IHC double stains revealed significantly elevated mean IRL of GFAP (E, G1) and iNOS (E1, G) in the SCI group relative to the control group (F; n = 4/group; p < 0.05, Student’s t test). The number of neurons expressing c-Fos, a neuronal activity marker in the GrN of SCI animals was significantly higher (H versus I) than that of controls (K, n = 4/group; p < 0.05, Student’s t test). Under the orthogonal slicing, co-localization of cFos and NeuN, a highly specific marker of neuronal nuclei (J) and DAPI, a standard nuclear counterstain (J1; scale bars: 100 µm/C and I; 40 µm/J) was confirmed. In the nucleus raphe magnus (RMg or NRM; L), a serotonergic neuronal center (L1, M), significantly increased group average IRL of GFAP was observed in the SCI group, compared to the control group (N1 versus N2). GFAP was mainly produced by hypertrophic astrocytes in injured spinal cords (inset image in N1 versus that of N2). In contrast, SCI NRM had discernibly lower group mean IRL of 5-HT expression (O) than that of the control NRM (O1; statistics in P; p < 0.001/GFAP and 0.01/5HT; Student’s t test; n = 5/group). Confocal z-stack imaging with orthogonal optical slicing captured typical morphology of serotonergic neurons (i.e., 5-HT presence in beaded axons and cell soma: O2; scale bars: 100 µm/M–O1)