Inhibition of Microgliosis with Minocycline Attenuates Central Inflammation Driving Gαi2 Protein Dependent Sympathetically Mediated Salt Sensitive Hypertension

Abstract

AimPrevious work in our laboratory has established that hypothalamic PVNGαi2 proteins mediate sympathoinhibitory responses to high dietary sodium. Downregulation of these proteins is implicated in salt sensitive hypertension, and central inflammation may play a role in the pathogenesis of salt sensitive hypertension by increasing sympathetic outflow. We hypothesize that microglial‐mediatedPVN inflammation contributes to salt sensitive hypertension (HTN) that develops following central Gαi2 protein down regulation.Methods3‐month‐old male Sprague Dawley rats were implanted with I.C.V. cannulas fitted to osmotic minipumps to deliver an infusion of either Gαi2 or scrambled oligodeoxynucleotides (ODN) (25μg/5μl/day) and were then put on a 7‐day time course of either high (HS; 4% NaCl) or normal (NS; 0.6% NaCl) salt diets (n=6 per group), and instrumented on day 7 with a femoral cannula to measure blood pressure (BP) and obtain arterial blood for plasma norepinephrine (NE) analysis by ELISA. Rats were then sacrificed via transcardial perfusion and brains were extracted and fixed in 4% paraformaldehyde and 30% sucrose. Immunohistochemistry (IHC) was performed for markers of microglial activation and astrocyte activation, assessed via ImageJ analysis, and mRNA levels of pro‐inflammatory cytokines (PIC) TNFα, IL‐1β, and IL‐6 and anti‐inflammatory cytokine (AIC) IL‐10 in the PVN were analyzed. We additionally co‐infused a separate group of animals with minocycline, an inhibitor of microglial activation, and ODNs (n=6 per group), to abolish inflammatory responses and ran the same assessment for PIC upregulation, BP, and plasma NE.ResultsDownregulation of PVN Gαi2 proteins in combination with a HS diet resulted in significant increases in blood pressure, as well as significant increases in plasma NE. In our control, scrambled ODN infused animals on HS, there was a suppression of plasma NE as well as an increase in AICs. In contrast, PIC mRNA was significantly upregulated, while AIC mRNA was downregulated in response to Gαi2 ODN downregulation on HS diet. Microgliosis, but not astrogliosis, was seen in response to Gαi2 downregulation and HS diet, but neither were seen in scrambled ODN animals on HS or NS. Finally, minocycline co‐infusion caused complete restoration of PIC and AIC mRNA levels to baseline in Gαi2 ODN infused animals on HS diets, complete restoration of plasma NE content to baseline, and an approximately 50% reduction in the magnitude of hypertension.ConclusionsWe conclude that microgliosis, but not astrogliosis, mediates central inflammation driving Gαi2 protein mediated salt sensitive hypertension that lead to increases in global NE and systemic BP.Support or Funding InformationFunding Source: R01HL139867, R01 HL141406, K02 HL112718, R01 HL107330, R56 AG057687 0.6% NaCl SCR ODN 4% NaCl SCR ODN 4% NaCl SCR ODN + Mino 0.6% NaCl Gαi2 ODN 4% NaCl Gαi2 ODN 4% NaCl Gαi2 ODN + Mino MAP (mmHg) 123 ± 4 121 ± 3 119 ± 3 118 ± 4 144 ± 3 * 130 ± 4*# Plasma NE (nmol/L) 62 ± 7 38 ± 4* 42 ± 5* 59 ± 5 92 ± 7* 62 ± 6# IL‐1β mRNA fold increase 0.9 ± 0.2 0.8 ± 0.1 1.1 ± 0.2 1.1 ± 0.2 2.4 ± 0.4* 1.3 ± 0.3# TNFα mRNA fold increase 1.1 ± 0.1 1.0 ± 0.2 1.0 ± 0.1 1.3 ± 0.2 2.3 ± 0.5* 1.1 ± 0.2# IL‐6 mRNA fold increase 1.0 ± 0.2 0.9 ± 0.1 1.1 ± 0.1 1.0 ± 0.1 2.2 ± 0.3* 1.2 ± 0.2# IL‐10 mRNA fold increase 1.0 ± 0.1 1.3 ± 0.1 1.3 ± 0.2 1.1 ± 0.2 0.4 ± 0.1* 1.1 ± 0.2# Microglial Density in PVN (number/.04mm2) 26.6 ± 1.88 20.5 ± 2.44 ND 22 ± 3.11 35.33 ± 2.54 * ND Percent Activation Microglia 18.3 ± 0.024 13.4 ± 0.021 ND 16.8 ± 0.015 29.24 ± 0.01 * ND Astrocyte Density in PVN (% of tissue) 30.2 ± 0.0324 27.3 ± 0.021 ND 32.9 ± 0.034 26.6 ± 0.026 ND p<0.05 vs. respective normal salt (0.6% NaCl) group; p<0.05 vs. respective high salt (4% NaCl) group (N=4–6/group ± SEM). Mino = Minocycline. ND = not determined This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.