Microglial‐Mediated PVN Inflammation Precedes Sympathoexcitation but not Hypertension in the Development of Gαi 2 Protein‐Dependent Salt Sensitive Hypertension

Aim Previous work in our laboratory has established that hypothalamic paraventricular nucleus (PVN) Gαi 2 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 Gαi 2 protein dependent ssHTN. We hypothesize that microglial‐mediated PVN inflammation precedes sympathoexcitation in the development of Gαi 2 protein‐dependent ssHTN. Methods Three‐month‐old male Sprague Dawley rats were implanted with ICV cannulas fitted to osmotic minipumps to deliver an infusion of either targeted Gαi 2 , which downregulate CNS Gαi 2 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. Results In SCR ODN infused rats, HS diet evoked no change in MAP or microglial reactivity compared to NS control. In rats lacking central Gαi 2 proteins, a 24‐hour HS diet evoked significant increases in blood pressure (MAP, mmHg; Gαi 2 ODN HS 115 ± 2 vs Gαi 2 ODN NS 103 ± 1 p<0.05) and percent activated PVN microglia (% active per sample region; Gαi 2 ODN HS 25.1% ± 3 vs Gαi 2 ODN NS 16.8 ± 3.7 p<0.05) compared to Gαi 2 ODN NS control and SCR ODN rats. Additionally, immunofluorescence detected qualitative increases in IL‐6 and IL‐1β expression in rats lacking central Gαi 2 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. Gαi 2 ODN and HS infused animals on day 1 showed no change in plasma NE levels compared to Gαi 2 NS control, but showed significantly lower NE levels than Gαi 2 HS on day 7 (plasma NE, nmol/L; Gαi 2 ODN day 1 56 ± 6.0 vs. Gαi 2 ODN day 7 91 ± 3.2, p<0.05). Conclusions While SCR ODN infused animals inhibit sympathetic activity after 1 day HS and remain normotensive, Gαi 2 ODN infused animals fail to do so. Gαi 2 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 Gαi 2 protein‐dependent ssHTN. Support or Funding Information R01 HL139867, R01 HL141406, R56 AG057687, T32 HL007224MAP, inflammation, and sympathetic characteristics of rats with and without central Gαi 2 proteinsGαi 2 ODN, 0.6% NaCl diet baseline Gαi 2 ODN, 4% NaCl diet day 1 Gαi 2 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 7Mean Arterial Pressure (mmHg) 103 ± 1 115 ± 2 * 128 ± 2 * # 102 ± 1 103 ± 2 103 ± 1Plasma 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 ± 2.5Data presented as mean ± SD, * p<0.05 vs NS baseline within ODN group; # p<0.05 vs. HS day 1 within ODN group