Centrally administered lipopolysaccharide elicits sympathetic excitation via NAD(P)H oxidase-dependent mitogen-activated protein kinase signaling

Abstract

Objective: The mechanisms by which inflammation activates sympathetic drive in heart failure and hypertension remain ill-defined. In this study, an intracerebroventricular injection of lipopolysaccharide (LPS) was used to induce the expression of cytokines and other inflammatory mediators in the brain, in the absence of other excitatory mediators, and the downstream signaling pathways leading to sympathetic activation were examined using intracerebroventricular injections of blocking or inhibiting agents.

Methods and results: In anesthetized rats, intracerebroventricular injection of LPS (5 microg) increased (P < 0.05) renal sympathetic nerve activity, blood pressure and heart rate. LPS increased (P < 0.05) hypothalamic mRNA for NAD(P)H oxidase subunits p47 and gp91, NAD(P)H oxidase-dependent superoxide generation, hypothalamic mRNA for tumor necrosis factor-alpha, cyclooxygenase-2 and cerebrospinal fluid levels of tumor necrosis factor-alpha and prostaglandin E2. In the paraventricular nucleus of hypothalamus, dihydroethidium staining for superoxide expression and c-Fos activity (indicating neuronal excitation) increased. The superoxide scavenger tempol significantly (P < 0.05) diminished the expression of inflammatory mediators, as well as superoxide expression and neuronal excitation in paraventricular nucleus. SB203580 (p38 mitogen-activated protein kinase inhibitor) also reduced the expression of inflammatory mediators in hypothalamus and cerebrospinal fluid. Tempol, apocynin [NAD(P)H oxidase inhibitor], SB203580 and NS398 (cyclooxygenase-2 inhibitor) all reduced cerebrospinal fluid prostaglandin E2 and the sympathoexcitatory response to LPS. LPS also increased angiotensin II type 1 receptor mRNA, a response blocked by apocynin and tempol but not by SB203580.

Conclusion: These findings suggest that central inflammation in pathophysiological conditions activates the sympathetic nervous system via NAD(P)H oxidase and p38 mitogen-activated protein kinase-dependent synthesis of prostaglandin E2.

Keywords: inflammation; lipopolysaccharide; oxidative stress; paraventricular nucleus of hypothalamus; prostaglandins.

Fig. 1

Representative recordings (A) and grouped data (B) showing LPS-induced increases in heart rate (HR), renal sympathetic nerve activity (RSNA) and blood pressure (BP) or mean BP (MBP) were prevented by continuous intracerebroventricular (ICV) infusion of tempol and diminished by apocynin infusion. Arrows indicate the LPS injection. Lines represent the continuous ICV infusion of tempol or apocynin. Each recording section is 200 seconds in duration with interval of 60 min. Values were expressed as mean ± SE. *P<0.05, versus baseline, †P<0.05 versus ICV LPS alone.

Fig. 2

Quantitative mRNA expression of representative subunits of NAD(P)H oxidase p47^phox (A) and gp91^phox (B), and superoxide production (C) in the hypothalamus of rats treated with ICV aCSF or ICV LPS. D: Representative confocal images of PVN sections showing that dihydroethidium (DHE) fluorescence was abundant in the PVN of ICV LPS-treated rats compared with ICV aCSF-treated rats. Rats treated with ICV tempol in addition to LPS had less DHE fluorescence in the PVN than rats treated with ICV LPS alone. E: Quantitative comparison of DHE immunofluorescence in the PVN for each group. Values were expressed as mean ± SE. *P<0.05 versus ICV aCSF, †P<0.05, ICV Tempol + ICV LPS versus ICV LPS alone.

Fig. 3

Expression of c-Fos activity in the PVN of hypothalamus. A: Representative sections showing ICV LPS treatment increased c-Fos immunoreactivity in the PVN neurons. ICV tempol treatment reduced LPS-induced c-Fos immunoreactivity in the PVN region. Dark dots indicate individual activated neurons. B: Quantification of c-Fos positive neurons in the dorsal pavocellular (dpPVN), medial parvocellular (mpPVN), ventrolateral pavocellular (vlpPVN), and posterior magnocellular (pmPVN) PVN. Values were expressed as mean ± SE. *P<0.05 versus ICV aCSF, †P<0.05, ICV Tempol + ICV LPS versus ICV LPS alone.

Fig. 4

ICV administration of LPS increased hypothalamic tissue TNF-α (A) and COX-2 (B) mRNA expression, and both were significantly reduced by continuous ICV infusion of tempol and SB203580. ICV LPS also significantly increased cerebrospinal fluid (CSF) level of TNF-α (C) and PGE₂ (D). ICV infusion of tempol and SB203580 reduced the LPS-induced increases in CSF TNF-α and PGE₂; the COX-2 inhibitor NS398 reduced the LPS-induced increase in CSF PGE₂. *P<0.05 versus ICV aCSF, †P<0.05, ICV Treatment + ICV LPS versus ICV LPS alone.

Fig. 5

Representative recordings (A) and grouped data (B) showing that central LPS induced sympatho-excitatory responses (increases in HR, RSNA and BP or MBP) can be diminished by continuous ICV infusion of the specific p38 MAPK inhibitor SB203580, and prevented by pretreatment with the specific COX-2 inhibitor NS398. Arrows indicate the ICV LPS injection. Line represents the continuous ICV infusion of SB203580. Each recording section is 200 seconds in duration at 60 min intervals. Values were expressed as mean ± SE. *P<0.05 versus ICV aCSF, †P<0.05, ICV Treatment + ICV LPS versus ICV LPS alone.

Fig. 6

Real-time PCR analysis showing increased hypothalamic tissue AT1R (A), but not ACE (B) mRNA expression 4 hours after ICV administration of LPS. The LPS-induced increase of AT1R mRNA expression was significantly diminished by continuous ICV infusion of tempol and apocynin, but not by SB203580. *P<0.05 versus ICV aCSF; †P<0.05, ICV Treatment + ICV LPS versus ICV LPS alone.

Fig. 7

Schematic illustrating putative molecular pathways mediating the effects of centrally administered LPS on sympathetic drive. LPS induces the production of pro-inflammatory cytokines, such as TNF-α, with subsequent induction of NAD(P)H oxidase dependent superoxide production. LPS also induces p38 MAPK, which is redox dependent. Downstream gene products of p38 MAPK include COX-2, the enzyme that generates PGE2. PGE2 activates E class prostanoid receptors (EP-R) to activate the sympathetic nervous system. LPS also induces expression of mRNA for the angiotensin II type 1 receptor (AT1R). The LPS induced sympatho-excitatory response can be blocked by inhibiting the LPS-induced NAD(P)H dependent p38 MAPK pathway with blockers or inhibitors at the levels indicated (see text for details). The increase in AT1R mRNA is blocked by agents that reduce superoxide production, but not by the p38 MAPK inhibitor, suggesting an alternative mechanism for that effect. LPS activation of the p44/42 MAPK pathway, which is also redox dependent, might account for this effect.