Brain microglial cytokines in neurogenic hypertension

Abstract

Accumulating evidence indicates a key role of inflammation in hypertension and cardiovascular disorders. However, the role of inflammatory processes in neurogenic hypertension remains to be determined. Thus, our objective in the present study was to test the hypothesis that activation of microglial cells and the generation of proinflammatory cytokines in the paraventricular nucleus (PVN) contribute to neurogenic hypertension. Intracerebroventricular infusion of minocycline, an anti-inflammatory antibiotic, caused a significant attenuation of mean arterial pressure, cardiac hypertrophy, and plasma norepinephrine induced by chronic angiotensin II infusion. This was associated with decreases in the numbers of activated microglia and mRNAs for interleukin (IL) 1beta, IL-6, and tumor necrosis factor-alpha, and an increase in the mRNA for IL-10 in the PVN. Overexpression of IL-10 induced by recombinant adenoassociated virus-mediated gene transfer in the PVN mimicked the antihypertensive effects of minocycline. Furthermore, acute application of a proinflammatory cytokine, IL-1beta, into the left ventricle or the PVN in normal rats resulted in a significant increase in mean arterial pressure. Collectively, this indicates that angiotensin II induced hypertension involves activation of microglia and increases in proinflammatory cytokines in the PVN. These data have significant implications on the development of innovative therapeutic strategies for the control of neurogenic hypertension.

Figure 1. ICV minocycline attenuates Ang II-induced cardiovascular effects

(a) Outline of experimental protocol. (b) Minocycline abolishes Ang II-induced hypertension. * P<0.05, † P<0.01 vs. the other three groups at the same time points. (c) Minocycline infusion attenuates cardiac hypertrophy induced by Ang II. Bar graphs are mean ± SEM of HW/BW ratio following each treatment (n=6 in each group). * P<0.05 vs. other three groups. Mino: minocycline. (d) Minocycline inhibits the increase in plasma NE induced by Ang II infusion. * P<0.05 vs. control; † P<0.05 vs. Ang II by one-way ANOVA followed by Bonferoni. (Control n=5; n=6 in the other three groups)

Figure 2. Effect of ICV minocycline on activation of microglia in the PVN

Microglia within the PVN under each treatment condition were identified via OX-42 immunostaining, and their activation was assessed as detailed in the Methods. (a) Representative micrographs showing the activation of PVN microglia under each treatment condition, 4 weeks after the start of Ang II infusions. The scale bar equals 10 μm. (b) Quantification of the length of processes of microglia in the PVN under each treatment condition. * P<0.001 vs. other groups by Two-way ANOVA, n=4 in each group. (c) Quantification of the numbers of activated microglial based on the morphological analysis. Mino: minocycline. * P<0.05 v.s. control by one-way ANOVA followed by Bonferoni.

Figure 3. Effect of ICV minocycline on PVN cytokine mRNA expression

Following euthanization of the rats used in the experiment in Figure 1, brains were removed and mRNA levels of cytokines in the PVN were assessed by Real-Time RT-PCR. * P<0.05 vs. corresponding control; † P<0.05 vs. Ang II by One-way ANOVA (n=6 in each group). Mino: minocycline.

Figure 4. Increased expression of IL-10 in the PVN blunts Ang II-induced hypertension

SD rats were microinjected with either AAV5-IL-10, AAV5-GFP or PBS into the PVN as detailed in the Methods. (a) Outline of experimental protocol. (b) Time dependent changes in MAP in each treatment group. Dashed lines indicated the treatment time points. * P<0.05, ** P<0.01, *** P<0.001 vs. GFP or PBS groups at the same time points. (c) Effect of IL-10 transduction on Ang II-induced cardiac hypertrophy. * P<0.05 vs. IL-10 + saline; † P<0.05 IL-10 + Ang II; ‡ P<0.05 vs. GFP or PBS plus Ang II infusion (n=4 in each group).

Figure 5. AAV5-IL-10 induced neuronal expression of IL-10 in the PVN

(a) IL-10 mRNA levels in the PVN at 28 days following microinjections of AAV5-IL-10, AAV5-GFP or PBS. * P<0.05 vs. GFP or PBS group. † P<0.05 vs. GFP or PBS groups. (b) Representative fluorescence micrographs showing IL10- and NeuN- immunoreactivity in the PVN following AAV5-CBA-IL10 microinjection. The right panel shows the merged images of IL10 and NeuN. Bar = 50 um.

Figure 6. IL-1β increases MAP in anesthetized rats

(a) Representative tracings showing the effects of ICV injection of IL-1β (10 ng/ml, 500 nl) on MAP in the absence or presence of the IL-1 receptor antagonist (IL-1RA: 100 ng/ml, 500 nl). (b) Representative tracing showing effect of PVN injection of IL-1β (10 ng/ml, 100 nl) on MAP.