The protein kinase C activator phorbol myristate acetate decreases brain edema by aquaporin 4 downregulation after middle cerebral artery occlusion in the rat

Abstract

The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) is known to interact with aquaporin 4 (AQP 4), a water-selective transporting protein that is abundant in astrocytes, and has experimentally been found to decrease osmotically-induced cell swelling. The purpose of this study was to examine whether PMA reduces brain edema following focal ischemia induced by middle cerebral artery (MCA) occlusion by modulation of AQP4 expression. Male Sprague-Dawley rats were randomly assigned to either sham surgery (n = 6), or a continuous intravenous infusion of vehicle (1% dimethylsulfoxide), followed by MCA occlusion (n = 18), and administration of PMA at 50 microg/kg (n = 6) or at 200 microg/kg (n = 6) starting 60 min before or 30 min (200 microg/kg; n = 6) or 60 min (200 microg/kg; n = 6) after MCA occlusion. Cerebral blood flow was monitored with laser Doppler over the MCA territory, and confirmed a 70% reduction during occlusion. After a 2-h period of ischemia and 2 h of reperfusion, the animals were sacrificed for assessment of brain water content and sodium and potassium concentration. AQP4 expression was assessed by immunoblotting and quantified by densitometry (n = 24). Statistical analysis was performed by ANOVA followed by Tukey's post-hoc test. PMA treatment at 200 microg/kg significantly reduced brain water concentration in the infarcted area when started 60 min before or 30 min after occlusion (p < 0.001 and p = 0.022, respectively), and prevented the subsequent sodium shift (p < 0.05). PMA normalized the AQP4 upregulation in ischemia (p = 0.021). A downregulation of AQP4 in the ischemic area paralleling the reduction in brain edema formation following PMA treatment suggests that the effect was mediated by AQP4 modulation.

FIG. 1.

Tissue processing. (A) Dorsal view and (B) coronal view of the rat brain showing the brain tissues sampled. Coronal sections 4 mm thick were cut through the middle cerebral artery core territory for water and electrolyte assessment. Representative coronal sections 7 mm posterior to the frontal pole were assessed for ischemic injury. Phorbol 12-myristate 13-acetate (PMA) treatment reduced brain edema in a dose-dependent manner. Brains from animals receiving (C) vehicle alone, (D) 50 μg/kg PMA, and (E) 200 μg/kg PMA (* = cerebral cortex; † = caudate putamen; LA, left anterior; LP, left posterior; RA, right anterior; RP, right posterior; MCA, middle cerebral artery).

FIG. 2.

Changes in regional cerebral blood flow (CBF) during and after middle cerebral artery occlusion in sham controls (open circles), vehicle-alone (open triangles), and animals receiving 50 μg/kg PMA (solid circles), and 200 μg/kg PMA (open squares). Data are expressed as percentage of baseline and are means ± SD; n = 6 rats in each group (SD, standard deviation; PMA, phorbol 12-myristate 13-acetate; MCAO, middle cerebral artery occlusion).

FIG. 3.

Effect of different doses of PMA on brain water content after MCA occlusion. PMA administration by continuous intravenous infusion significantly reduced brain water content in both the right anterior and posterior coronal sections in a dose-dependent manner. Data are means ± SD; n = 6 rats/group; *p < 0.0001; **p < 0.02 compared with the vehicle-alone group; †p < 0.04 compared with the 200 μg/kg PMA-treated group; SD, standard deviation; PMA, phorbol 12-myristate 13-acetate; MCA, middle cerebral artery; LA, left anterior; LP, left posterior; RA, right anterior; RP, right posterior).

FIG. 4.

Effect of PMA given at different time points before or after MCA occlusion on brain edema and electrolyte content. When started 60 min before or 30 min after MCAO, intravenous PMA infusion significantly reduced ischemia-induced brain edema. Data are expressed as the difference of percentage of water content (A), sodium concentration (B), and potassium concentration (C), between the ischemic and the non-ischemic side (means ± SEM; PMA, phorbol 12-myristate 13-acetate; SEM, standard error of the mean).

FIG. 5.

Effect of PMA on AQP4 expression in the ischemic hemisphere after MCA occlusion. (A) Intravenous PMA infusion started 1 h before MCA occlusion (2 h ischemia, then 2 h reperfusion) reduced the ischemia-induced AQP4 upregulation seen in vehicle-infused animals. (B) AQP4 expression as assessed by immunoblotting and quantified by densitometry revealed significant differences (means ± SEM; AQP4, aquaporin 4; PMA, phorbol 12-myristate 13-acetate; SEM, standard error of the mean; MCA, middle cerebral artery; Post., posterior; Ant., anterior).