Figure 2.
Inhibition of CaM or PKA Reduces Spinal Cord Water Content, AQP4 Translocation to the BSCB, and AQP4 Expression after DC Crush Injury In Vivo
(A) Water content of the thoracic spinal cord 3 days after dorsal column (DC) crush and treatment with CaM or PKA inhibitors (CaMi or PKAi, respectively). Sham, laminectomy only; DC+vehicle, T8 DC crush +PBS; DC+CaMi, DC crush + intra-lesion injection of 41 mM TFP; DC+PKAi, DC crush + intra-lesion injection of 10 μM H89; n = 4 rats per treatment group, except DC+vehicle 3 dpi (n = 8) and DC+vehicle 28 dpi (n = 3).
(B) Water content of the thoracic spinal cord 3 days after DC crush to investigate the mode of action of TFP. DC+CaMi, DC crush + intra-lesion injection of 41 mM TFP or 164 mM W-7; DC+A1Ri, DC crush + intra-lesion injection of 53 mM α1 adrenergic receptor antagonist terazosin; DC+D2Rii, DC crush + intra-lesion injection of 6.6 mM dopamine D2 receptor antagonist L-741,626; DC+PKCi, DC crush + intra-lesion injection of 9.94 μM PKC inhibitor Gö 6983. n = 3–12 rats per treatment group, normalized to sham controls across multiple experiments.
(C–F) Representative images used to quantify data in (G) and (H) (scale bar, 50 μm). Sham tissue (C) was compared with tissue from animals 3 days after DC crush (3 dpi; D), for which an increase in total AQP4 expression and translocation to the blood-spinal cord barrier (BSCB; white arrows identify astrocyte endfeet) is observed, which is ablated by treatment with TFP (E) or treatment with H89 (F). Two representative magnified images are shown for each panel. The contrast in the insets was manually adjusted to aid the eye (scale bars, 10 μm).
(G) Quantification of at least 24 images (a minimum of 4 images per animal, n = 6) shows changes in AQP4 BSCB localization.
(H) Quantification of at least 18 images (a minimum of 3 images per animal, n = 6) shows changes in total AQP4 expression. For all comparisons, ANOVA was followed by post hoc Bonferroni-corrected t tests. ∗p < 0.05, ns represents p > 0.05 (Table S2).