Brain perivascular macrophages and the sympathetic response to inflammation in rats after myocardial infarction

Abstract

Inflammation is associated with increased sympathetic drive in cardiovascular diseases. Blood-borne proinflammatory cytokines, markers of inflammation, induce cyclooxygenase 2 (COX-2) activity in perivascular macrophages of the blood-brain barrier. COX-2 generates prostaglandin E(2), which may enter the brain and increase sympathetic nerve activity. We examined the contribution of this mechanism to augmented sympathetic drive in rats after myocardial infarction (MI). Approximately 24 hours after acute MI, rats received an intracerebroventricular injection (1 microL/min over 40 minutes) of clodronate liposomes (MI+CLOD) to eliminate brain perivascular macrophages, liposomes alone, or artificial cerebrospinal fluid. A week later, COX-2 immunoreactivity in perivascular macrophages and COX-2 mRNA and protein had increased in hypothalamic paraventricular nucleus of MI rats treated with artificial cerebrospinal fluid or liposomes alone compared with sham-operated rats. In MI+CLOD rats, neither perivascular macrophages nor COX-2 immunoreactivity was seen in the paraventricular nucleus, and COX-2 mRNA and protein levels were similar to those in sham-operated rats. Prostaglandin E(2) in cerebrospinal fluid, paraventricular nucleus neuronal excitation, and plasma norepinephrine were less in MI+CLOD rats than in MI rats treated with artificial cerebrospinal fluid or liposomes alone but more than in sham-operated rats. Intracerebroventricular CLOD had no effect on interleukin 1beta and tumor necrosis factor-alpha mRNA and protein in the paraventricular nucleus or plasma interleukin-1beta and tumor necrosis factor-alpha, which were increased in MI compared with sham-operated rats. In normal rats, pretreatment with intracerebroventricular CLOD reduced (P<0.05) the renal sympathetic, blood pressure, and heart rate responses to intracarotid artery injection of tumor necrosis factor-alpha (0.5 microg/kg); intracerebroventricular liposomes had no effect. The results suggest that proinflammatory cytokines stimulate sympathetic excitation after MI by inducing COX-2 activity and prostaglandin E(2) production in perivascular macrophages of the blood-brain barrier.

Figure 1

Representative laser confocal images from PVN of SHAM and MI rats treated with artificial cerebrospinal fluid (aCSF), clodronate liposomes (CLOD) and liposomes alone (LIPO). (A) triple immunostaining for the perivascular macrophage marker ED2 (top, bright red), COX-2 (middle, bright green) and combined image (bottom) with nuclear staining (blue). The arrows point to ED2 positive cells expressing COX-2, as indicated by the yellow staining in the merge image. Neither perivascular macrophages nor COX-2 immunoreactivity were found in the MI rats treated with CLOD. (B) OX42 positive microglial cells (bright green). The arrows indicate cells positive for OX42. The microglial cells in the PVN were not affected by CLOD treatment. Scale bar, 20 μm.

Figure 2

Quantitative comparison of mRNA expression (A) and protein levels (B) for COX-2, TNF-α and IL-1β from the PVN and adjacent regions of hypothalamus of each treatment group. Representative Western blots of COX-2, TNF-α, IL-1β and β-actin are shown in figure B. Values were expressed as mean ± SEM (n=5 to 8 for each group). *P<0.05 vs SHAM in same region, †P<0.05, MI+treatment vs MI+aCSF in same region.

Figure 3

Plasma TNF-α (A) and IL-1β (B), CSF PGE₂ (C) and plasma NE (D) levels in each group. Values were expressed as mean ± SEM (n=6 to 8 for each group). *P<0.05 vs SHAM, †P<0.05, MI+treatment vs MI+aCSF.

Figure 4

Expression of Fra-LI activity in the PVN. (A) Representative sections from each group showing Fra-LI immunoreactivity in PVN neurons. Dark dots indicate Fra-LI positive neurons. Squares (dotted lines) indicate the regions in panel A from which the data in panel B are derived. Scale bar=200 μm. (B) Quantification of Fra-LI positive neurons in 4 different regions of the PVN. Values are expressed as mean ± SEM (n=4 for each group). *P<0.05 vs SHAM. †P<0.05, MI+treatment vs MI+aCSF. pm indicates posterior magnocellular; mp, medial parvocellular; vlp, ventrolateral parvocellular; dp, dorsal parvocellular.

Figure 5

Effects of bolus ICA injection of TNF–α on COX-2 mRNA (A) and protein (B) expression in PVN, and PGE₂ level in CSF (C) in normal rats pre-treated a week earlier with ICV CLOD, LIPO or aCSF. aCSF-treated rats not receiving TNF–α injection were used as control. Representative Western blots are aligned with the matching grouped data (B). Values were expressed as mean ± SEM (n=5 to 7 for each group). *P<0.05 vs aCSF (no TNF-α); †P<0.05, Treatment+TNF–α vs aCSF+TNF–α.

Figure 6

Effects of CLOD on cardiovascular and sympathetic responses to intracarotid artery (ICA) injection of TNF-α. Heart rate (HR), renal sympathetic nerve activity (RSNA, shown both as integrated nerve activity and as windowed spike activity), and arterial pressure (AP) responses to ICA bolus injection of TNF–α in normal rats pre-treated a week earlier with (A) intracerebroventricular (ICV) aCSF, (B) ICV CLOD, or (C) ICV LIPO. (D) Grouped data showing peak changes from baseline in mean blood pressure (MBP), HR and RSNA in each group. Values were expressed as mean ± SEM (n=6 to 7 for each group). *P<0.05 vs baseline; †P<0.05 vs aCSF+TNF–α.