Fig. 7
Chronic neuroinflammation and neurotransmitter changes in the pons and midbrain. The lateral parabrachial nuclei (LPBN; A; scp: superior cerebellar peduncle) displayed significantly increased mean IRL of co-stained Iba-1 (B, D)/TNFα (B1, D1) and iNOS (E1, G)/GFAP (E, G) in SCI tissues, compared to control tissues (Iba-1/TNFα: C; iNOS/GFAP: F; n = 4/group, *p < 0.05, Student’s t test). IHC images showed morphological features of aggregation of activated microglia and astrocytes in LPBN after SCI, corroborating with increased detection of Iba-1, TNFα and iNOS, and GFAP in these cells (B, B1, E1, and E, relative to controls in C and F). T10 compression heightened the activity of NeuN+ (H)/CGRP+ (I) neurons in the LPBN compared to the control group (J; confocal analysis in K/SCI and K1/control), which was demonstrated by significantly higher numbers of cFos+ cells co-expressing CGRP in SCI LPBN (statistics in L; n = 4/group, *p < 0.05, Student’s t test). The ventral lateral periaqueductal gray (VLPAG: Bregma −7.80 mm; M) in the midbrain of the SCI group showed higher mean IRL of GFAP and Iba1/TNFα co-staining (N and P1/P2/P3, respectively) than the control group (N1/GFAP; P4/Iba1 and TNFα); the difference was statistically significant (O/GFAP, n = 4/group, ***p < 0.001; Student’s t test; Q/Iba1 and TNFα, *p < 0.05; Mann–Whitney U test; n = 4/group). Under higher magnification, SCI VLPAG manifested extensive reactive gliosis (e.g., morphology of hypertrophic astrocytes: N inset), relative to the control tissue (N1 inset). Activated microglia (i.e., swollen ramified morphology and expressions of Iba1/P1 inset and TNFα/P2 inset (merged images: P3 inset) densely populated VLPAG of SCI animals only (relative to laminectomy controls: P4 and inset). Importantly, discernably elevated GAD67 expression scale existed in the VLPAG of the SCI group (R1) than the control group (R2). Confocal z-stack imaging with orthogonal optical slicing depicted a typical profile of GABAergic neurons with GAD67 immunostaining in the neuronal somas and neurites (R3). The mean IRL of GAD67 in the SCI VLPAG was significantly higher than that of the control group (S; n = 5/group, **p < 0.01; Student’s t test; scale bars: 100 µm/N–R2)