Microglial‐Mediated PVN Inflammation Precedes Sympathoexcitation but not Hypertension in the Development of Gαi2 Protein‐Dependent Salt Sensitive Hypertension

Abstract

AimPrevious work in our laboratory has established that hypothalamic paraventricular nucleus (PVN) Gαi2 proteins mediate sympathoinhibitory responses to high salt (HS) diets. The inability to upregulate these proteins in response to a HS diet results in salt sensitive hypertension (ssHTN). Additionally, we have demonstrated that microglial‐mediated PVN inflammation correlates with Gαi2 protein dependent ssHTN. We hypothesize that microglial‐mediated PVN inflammation precedes sympathoexcitation in the development of Gαi2 protein‐dependent ssHTN.MethodsThree‐month‐old male Sprague Dawley rats were implanted with ICV cannulas fitted to osmotic minipumps to deliver an infusion of either targeted Gαi2, which downregulate CNS Gαi2 proteins by ~85%, or scrambled (SCR) oligodeoxynucleotides (ODN) (25μg/5μl/day) and were then put on a 24‐hour or 7‐day normal (NS; 0.6% NaCl) or high (HS; 4% NaCl) salt diet (n=5 per group). Rats were transcardially perfused and brains were extracted and fixed in paraformaldehyde and sucrose. Immunohistochemistry (IHC) was performed to assess PVN microglial activation and for protein levels of pro‐inflammatory cytokines (PIC) IL‐1β, and IL‐6. We ran additional groups in the same manner, previously fitted with radiotelemeters to assess MAP, and sacrificed these groups on day 1 and 7 to obtain whole blood for ELISA plasma norepinephrine (NE) analysis as a measure of sympathetic tone.ResultsIn SCR ODN infused rats, HS diet evoked no change in MAP or microglial reactivity compared to NS control. In rats lacking central Gαi2 proteins, a 24‐hour HS diet evoked significant increases in blood pressure (MAP, mmHg; Gαi2 ODN HS 115 ± 2 vs Gαi2 ODN NS 103 ± 1 p<0.05) and percent activated PVN microglia (% active per sample region; Gαi2 ODN HS 25.1% ± 3 vs Gαi2 ODN NS 16.8 ± 3.7 p<0.05) compared to Gαi2 ODN NS control and SCR ODN rats. Additionally, immunofluorescence detected qualitative increases in IL‐6 and IL‐1β expression in rats lacking central Gαi2 proteins compared to SCR ODN. Plasma NE values were significantly lower in SCR ODN infused animals on HS (plasma NE, nmol/L; SCR ODN HS 43 ± 4.6 vs. SCR ODN NS 65 ± 5.0, p<0.05) than NS control. Gαi2 ODN and HS infused animals on day 1 showed no change in plasma NE levels compared to Gαi2 NS control, but showed significantly lower NE levels than Gαi2 HS on day 7 (plasma NE, nmol/L; Gαi2 ODN day 1 56 ± 6.0 vs. Gαi2 ODN day 7 91 ± 3.2, p<0.05).ConclusionsWhile SCR ODN infused animals inhibit sympathetic activity after 1 day HS and remain normotensive, Gαi2 ODN infused animals fail to do so. Gαi2 ODN infused animals do not, however, exhibit sympathoexcitation on day 1, yet demonstrate increases in MAP, PVN microglial activation, and PVN inflammation. This suggests inflammation may precede sympathoexcitation in Gαi2 protein‐dependent ssHTN.Support or Funding InformationR01 HL139867, R01 HL141406, R56 AG057687, T32 HL007224 MAP, inflammation, and sympathetic characteristics of rats with and without central Gαi2 proteins Gαi2 ODN, 0.6% NaCl diet baseline Gαi2 ODN, 4% NaCl diet day 1 Gαi2 ODN, 4% NaCl diet day 7 SCR ODN, 0.6% NaCl diet baseline SCR ODN, 4% NaCl diet day 1 SCR ODN, 4% NaCl diet day 7 Mean Arterial Pressure (mmHg) 103 ± 1 115 ± 2 * 128 ± 2 * # 102 ± 1 103 ± 2 103 ± 1 Plasma NE (nmol/L) 67 ± 6.0 56 ± 6.0 91 ± 3.2 * # 65 ± 5.0 43 ± 4.6 * 37 ± 2.4 * # Microglia in 200 μm × 200 μm sample area in PVN 22.0 ± 3.1 22.6 ± 2.1 35.33 ± 3.1 * # 26.6 ± 4.2 17.4 ± 2.0 20.5 ± 2.7 % Microglia activated in 200 μm × 200 μm sample area in PVN 16.8 ± 3.7 25.1 ± 3.0 * 29.2 ± 2.5 * 18.0 ± 2.4 11.4 ± 3.5 15.6 ± 2.5 Data presented as mean ± SD, *p<0.05 vs NS baseline within ODN group; #p<0.05 vs. HS day 1 within ODN group