Angiotensin II, blood-brain barrier permeability, and microglia interplay during the transition from pre-to hypertensive phase in spontaneously hypertensive rats

Abstract

Background: Hypertension is characterized by upregulation of the renin-angiotensin system, increased blood-brain barrier (BBB) permeability, microglia activation within autonomic nuclei, and an intense sympathoexcitation. There is no information on the interplay of these events during the development of neurogenic hypertension. We sought to identify the interaction and time-course changes of Ang II availability, barrier dysfunction, microglia activation, and autonomic imbalance within autonomic areas during the development of neurogenic hypertension.

Methods: Sequential changes of hemodynamic/autonomic parameters, BBB permeability, microglia structure/density (IBA-1), and angiotensin II (Ang II) immunofluorescence were evaluated within the paraventricular hypothalamic nucleus, nucleus of the solitary tract, and rostral ventrolateral medulla of Wistar and spontaneously hypertensive rats (SHRs) aged 4 weeks, 5 weeks, 6 weeks, 8 weeks, and 12 weeks. The somatosensory cortex and hypoglossal nucleus were also analyzed as non-autonomic control areas.

Results: Increased brain Ang II availability (4th-5th week) was the first observed change, followed by the incipient BBB leakage and increased microglia density (6th week). From the 5th-6th weeks on, BBB leakage, Ang II, and IBA-1 densities increased continuously, allowing a parallel increase in both Ang II-microglia colocalization and the transition of microglial cells from highly ramified in the basal surveillant condition (4th-5th week) to shorter process arbors, fewer endpoints, and enlarged soma in the disease-associate condition (6th week to the 12th week). Simultaneously with increased Ang II-microglia colocalization and microglia morphologic phenotypic changes, sympathetic activity and pressure variability increased, autonomic control deteriorated, and blood pressure increased. These responses were not specific for autonomic nuclei but also occurred at a lower magnitude in the somatosensory cortex and hypoglossal nucleus, indicating the predominance of hypertension-induced effects on autonomic areas. No changes were observed in age-matched controls where Ang II density did not change.

Conclusion: Brain Ang II density is the initial stimulus to drive coordinated changes in BBB permeability and microglial reactivity. Increased BBB dysfunction allows access of plasma Ang II and increases its local availability and the colocalization and activation of microglial cells. It is a potent stimulus to augments vasomotor sympathetic activity, autonomic imbalance, and pressure elevation during the establishment of hypertension.

Keywords: angiotensin II; autonomic control; blood–brain barrier; microglia; spontaneously hypertensive rats.

FIGURE 1

Hemodynamic and autonomic recordings in SHRs and Wistar rats from 4 weeks to 12 weeks of age. Baseline mean arterial pressure [MAP, (A)] and heart rate [HR, (B)], systolic arterial pressure [SAP, (C)], and pulse interval [PI, (F)] variabilities with their spectral components at low [LF-SAP, (D) and LF-PI, (G)] and high frequencies [HF-PI, (H)], and spontaneous baroreflex sensitivity (alpha HF, (E). n = 6–9 rats/group. Comparisons made by two-way factorial ANOVA. MAP: group F (1,61) = 26.18 P < 0.001, age F (4,61) = 23.33 P < 0.001, interaction F (4,61) = 6.45, P = 0.001; HR: group F (1,61) = 3.78 P = 0.050, age F (4,61) = 13.88 P < 0.001, interaction F (4,61) = 1.68 P = 0.166; SAP variability: group F (1,61) = 57.61 P < 0.001, age F (4,61) = 8.67 P < 0.001, interaction F (4,61) = 5.16 P = 0.001; PI variability: group F (1,61) = 19.10 P < 0.001, age F (4,61) = 4.79 P = 0.002, interaction F (4,61) = 1.80 P = 0.136; LF-SAP: group F (1,61) = 67.96 P < 0.001, age F (4,61) = 5.17 P = 0.001, interaction F (4,61) = 3.36 P = 0.014; LF-PI: group F (1,61) = 11.72 P = 0.001, age F (4,61) = 1.08 P = 0.373, interaction F (4,61) = 0.53 P = 0.717; HF-PI: group F (1,61) = 15.54 P < 0.001, age F (4,61) = 1.30 P = 0.277, interaction F (4,61) = 0.08 P = 0.987; Alpha HF: group F (1,61) = 38.43 P < 0.001, age F (4,61) = 0.82 P = 0.516, interaction F (4,61) = 0.55 P = 0.701; Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 2

BBB permeability changes within autonomic nuclei during the development of hypertension. Representative images of SHRs (PVN, NTS, and RVLM) and Wistar rats (PVN) aged 4 weeks, 6 weeks, and 12 weeks illustrate the capillary profile (rhodamine-70kDa, red), the FITC-10 kDa leakage into the brain parenchyma (green) and the colocalization of both (white). The superimposed rectangle over both the ventromedial nucleus of the PVN (PVNvm) and NTS and the square over the RVLM are ROIs in which measurements were made. 3v, third ventricle, cc, central canal, A, ambiguous nucleus. Scale bar = 50 μm. Graphs on the right depict FITC-10 kDa leakage values into the brain parenchyma of SHRs and Wistar groups within the PVNvm, NTS, and RVLM. Values are the means of 6–8 slices/rat, three rats/age/group. Comparisons made by two-way factorial ANOVA. PVNvm: group F (1,20) = 615 P < 0.001, age F (4,20) = 252 P < 0.001, interaction F (4,20) = 195 P < 0.001; NTS: group F (1,20) = 6305 P < 0.001, age F (4,20) = 1995 P < 0.001, interaction F (4,20) = 1750 P < 0.001; RVLM: group F (1,20) = 1293 P < 0.001, age F (4,20) = 323 P < 0.001, and interaction F (4,20) = 298 P < 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 3

Microglia density changes within autonomic nuclei during the development of hypertension. Representative images of SHRs (PVN, NTS, and RVLM) and Wistar rats (PVN) aged 4 weeks, 6 weeks, and 12 weeks illustrate the morphological changes on microglial cells. The superimposed rectangle over both the ventromedial nucleus of the PVN (PVNvm) and the NTS and the square over the RVLM are ROIs in which measurements were made. Insets in the upper right or left corners show the morphology of a respective microglial cell in higher magnification. 3v, third ventricle, cc, central canal, A, ambiguous nucleus. Scale bar = 50 μm. Graphs on the right depict the values of microglia density of SHR and Wistar groups within the PVNvm, NTS, and RVLM. Values are the means of 6–8 slices/rat, 4–5 rats/age/group. Comparisons made by two-way factorial ANOVA. PVNvm: group F (1,40) = 101 P < 0.001, age F (4,40) = 23 P < 0.001, interaction F (4,40) = 21 P < 0.001; NTS: group F (1,40) = 1158 P < 0.001, age F (4,40) = 489 P < 0.001, interaction F (4,40) = 299 P < 0.001; RVLM: group F (1,34) = 469 P < 0.001, age F (4,34) = 141 P < 0.001, interaction F (4,34) = 138 P < 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 4

Morphological changes of the microglia within the PVNvm during the development of hypertension. (A). Images of single microglial cells of the SHRs and Wistar rats aged 4 weeks, 6 weeks, and 12 weeks showing the soma size (blue), length and number of processes (red), and endpoints (green) that were obtained and quantified by the NeurphologyJ plugin to ImageJ. Graphs compare the sequential changes in cell number (B), soma size index (C), number of processes (D), length of processes (E), and endpoints (F) measured within the ROI superimposed over the PVNvm. n = 5 rats/age/group. Comparisons made by two-way factorial ANOVA. Cell number: group F (1,40) = 11,1 P = 0.002, age F (4,40) = 1.4 P = 0.251, interaction F (4,40) = 1.4 P = 0.256; Soma size index: group F (1,40) = 92.0 P < 0.001, age F (4,40) = 46.6 P < 0.001, interaction F (4,40) = 42.9 P < 0.001; number of processes: group F (1,40) = 475.0 P < 0.001, age F (4,40) = 41.7 P < 0.001, interaction F (4,40) = 102.6 P < 0.001; length of processes: group F (1,40) = 92.2 P < 0.001, age F (4,40) = 5.32 P = 0.002, interaction F (4,40) = 17.1 P < 0.001; endpoints: group F (1,40) = 271.6 P < 0.001, age F (4,40) = 20.8 P < 0.001, interaction F (4,40) = 26.4 P < 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 5

Morphological changes of microglia within the NTS during the development of hypertension. (A). Images of single microglial cells of SHRs and Wistar rats aged 4 weeks, 6 weeks, and 12 weeks showing the soma size (blue), number and length of processes (red), and endpoints (green) were obtained and quantified by the NeurphologyJ plugin to ImageJ. Graphs compare the sequential changes in cell number (B), soma size index (C), number of processes (D), length of processes (E), and endpoints (F) within the NTS of the SHR and Wistar groups during the development of hypertension. n = 5 rats/age/group. Comparisons made by two-way factorial ANOVA. Cell number: group F (1,40) = 17.09 P < 0.001, age F (4,40) = 3.62 P = 0.013, interaction F (4,40) = 7.87 P < 0.001; Soma size index: group F (1,40) = 679.2 p < 0.001, age F (4,40) = 237.0 p < 0.001, interaction F (4,40) = 303.7 P < 0.001; number of processes: group F (1,40) = 81.67 P < 0.001, age F (4,40) = 8.76 P < 0.001, interaction F (4,40) = 18.20 P < 0.001; length of processes: group F (1,40) = 70.89 P < 0.001, age F (4,40) = 7.57 P < 0.001, interaction F (4,40) = 5.51 P = 0.001; Endpoints: group F (1,40) = 161.9 P < 0.001, age F (4,40) = 14.93 P < 0.001, interaction F (4,40) = 32.47 P < 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 6

Morphological changes of microglia within the RVLM during the development of hypertension. (A). Images of single microglial cells of SHRs and Wistar rats aged 4 weeks, 6 weeks, and 12 weeks showing the soma size (blue), number and length of processes (red), and endpoints (green) were obtained and quantified by the NeurphologyJ plugin to ImageJ. Graphs compare the sequential changes in cell number (B), soma size index (C), number of processes (D), length of processes (E), and endpoints (F) within the RVLM of SHRs and Wistar groups during the development of hypertension. n = 4-5 rats/age/group. Comparisons made by two-way factorial ANOVA. Cell number: group F (1,34) = 0.34 P = 0.562, age F (4,34) = 65.67 P < 0.001, interaction F (4,34) = 27.64 P < 0.001; Soma size index: group F (1,34) = 300.6 P < 0.001, age F (4,34) = 119.9 P < 0.001, interaction F (4,34) = 158.7 P < 0.001; number of processes: group F (1,34) = 0.17 P = 0.683, age F (4,34) = 0.30 P = 0.878, interaction F (4,34) = 7.37 P = 0.005; length of processes: group F (1,34) = 35.96 P < 0.001, age F (4,34) = 13.73 P < 0.001, interaction F (4,34) = 6.78 P < 0.001; Endpoints: group F (1,34) = 35.32 P < 0.001, age F (4,34) = 7.21 P < 0.001, interaction F (4,34) = 29.85 P < 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 7

Changes of Ang II density within autonomic nuclei during the development of hypertension. Representative images of SHRs (PVN, NTS, and RVLM) and Wistar rats (PVN) aged 4 weeks, 5 weeks, and 12 weeks depict Ang II availability in these nuclei. The superimposed rectangle over both the ventromedial nucleus of the PVN (PVNvm) and NTS and the square over the RVLM are the ROIs in which measurements were made. 3v, third ventricle, cc, central canal, A, ambiguous nucleus. Scale bar = 50 μm. Graphs on the right depict the values of Ang II density within the PVNvm, NTS, and RVLM. Values are the means of 6–8 slices/rat, 4–5 rats/age/group. Comparisons made by two-way factorial ANOVA. PVNvm: group F (1,40) = 184.6 P < 0.001, age F (4,40) = 27.8 P < 0.001, interaction F (4,40) = 18.4 P < 0.001; NTS: group F (1,40) = 4403 P < 0.001, age F (4,40) = 248.4 P < 0.001, interaction F (4,40) = 307.2 P < 0.001; RVLM: group F (1,34) = 1040.0 P < 0.001, age F (4,34) = 133.0 P < 0.001, interaction F (4,34) = 93.6 P < 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 8

Colocalization of Ang II with microglia within the autonomic nuclei during the development of hypertension. Images show Ang II immunofluorescence (red), microglia (green), and their colocalization (yellow) in both soma and processes (white arrowheads) within the PVNvm, NTS, and RVLM of SHRs aged 4 weeks, 5 weeks, and 12 weeks. Magnified binary images in insets in the upper left corners depict Ang II-IBA-1 colocalization within the soma and processes of a microglial cell (small, dashed square) and also show the intensification of Ang II-IBA-1 colocalization during the experimental protocol. Scale bar = 50 μm. Graphs at the right depict the quantitative analysis of Ang II-microglia colocalization into specified ROIs of PVNvm, NTS, and RVLM during the experimental protocol in the SHR and Wistar groups. n = 4–5 rats/age/group. Comparisons made by two-way factorial ANOVA. PVNvm: group F (1,40) = 342.6 P < 0.001, age F (4,40) = 28.1 P < 0.001, interaction F (4,40) = 23.1 P < 0.001; NTS: group F (1,40) = 25.4 P < 0.001, age F (4,40) = 7.9 P < 0.001, interaction F (4,40) = 3.7 P = 0.013; RVLM: group F (1,34) = 575.1 P < 0.001, age F (4,34) = 24.5 P < 0.001, interaction F (4,34) = 22.5 p < 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 9

Time-course changes of Ang II immunofluorescence, BBB permeability, microglia density, and their morphological states within the somatosensory cortex during the development of hypertension. Images of SHRs aged 4 weeks, 5 weeks, 6 weeks, and 12 weeks show Ang II availability (A), FITC leakage (B), and microglia immunofluorescence (C-insets in the right upper corner show the morphology of a respective microglial cell in higher magnification); Scale bar = 50 μm. The superimposed rectangle indicates the ROI in which measurements were made. Graphs compare the temporal changes on Ang II immunofluorescence (D), BBB permeability (E), microglia density (F), soma size (G), number of processes (H), length of processes (I), and endpoints (J) of microglial cells of the SHR and Wistar groups during the experimental protocol. Values are the means of 6–8 slices/rat, 3–4 rats/age/group. Comparisons made by two-way factorial ANOVA. Ang II density: group F (1,30) = 140.1 P < 0.001, age F (4,30) = 21.81 P < 0.001, interaction F (4,30) = 18.74 P < 0.001; FITC leakage: group F (1,16) = 195.5 P < 0.001, age F (3,16) = 142.8 P < 0.001, interaction F (3,16) = 120.3 P < 0.001; microglia density: group F (1,30) = 118.6 P < 0.001, age F (4,30) = 23.01 P < 0.001, interaction F (4,30) = 11.98 P < 0.001; soma size index: group F (1,30) = 35.97 P < 0.001, age F (4,30) = 17.54 P < 0.001, interaction F (4,30) = 14.32 P < 0.001; number of processes: group F (1,30) = 36.31 P < 0.001, age F (4,30) = 1.55 P = 0.213, interaction F (4,30) = 9.90 P < 0.001; length of processes: group F (1,30) = 85.78 P < 0.001, age F (4,30) = 4.04 P = 0.010, interaction F (4,30) = 6.61 P = 0.001; Endpoints: group F (1,30) = 138.6 P < 0.001, age F (4,30) = 14.59 P < 0.001, interaction F (4,30) = 45.90 P < 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 10

Time-course changes of Ang II immunofluorescence, BBB permeability, microglia density, and their morphological states within the hypoglossal nucleus during the development of hypertension. Images of SHRs aged 4 weeks, 6 weeks, and 12 weeks show Ang II availability (A), FITC leakage (B), and microglia immunofluorescence (C)-insets in the lower right corner show the morphology of a respective microglial cell in higher magnification). Scale bar = 50 μm. The superimposed squares indicate the ROIs in which measurements were made. Graphs compare the temporal changes on Ang II immunofluorescence (D), BBB permeability (E), microglia density (F), soma size (G), number of processes (H), length of processes (I), and endpoints (J) of microglial cells of the SHR and Wistar groups during the experimental protocol. Values are the means of 6–8 slices/rat, 3–4 rats/age/group. Comparisons made by two-way factorial ANOVA. Ang II density: group F (1,30) = 61.82 P < 0.001, age F (4,30) = 12.80 P < 0.001, interaction F (4,30) = 10.42 P < 0.001; FITC leakage: group F (1,16) = 142.1 P < 0.001, age F (3,16) = 83.36 P < 0.001, interaction F (3,16) = 69.49 P < 0.001; microglia density: group F (1,30) = 65.74 P < 0.001, age F (4,30) = 10.10 P < 0.001, interaction F (4,30) = 7.00 P = 0.001; soma size index: group F (1,30) = 34.57 P < 0.001, age F (4,30) = 12.39 P < 0.001, interaction F (4,30) = 5.98 P = 0.001; number of processes: group F (1,30) = 20.55 P < 0.001, age F (4,30) = 0.11 P = 0.979, interaction F (4,30) = 3.46 P = 0.021; length of processes: group F (1,30) = 24.90 P < 0.001, age F (4,30) = 2.91 P = 0.004, interaction F (4,30) = 2.13 P = 0.104; endpoints: group F (1,30) = 10.74 P = 0.003, age F (4,30) = 1.28 P = 0.303, interaction F (4,30) = 7.65 P = 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.

FIGURE 11

Colocalization of angiotensin II and microglia within the somatosensory cortex (CSS) and hypoglossal nucleus (12N) during the development of hypertension. Images show angiotensin II immunofluorescence (red), microglia (green), and their colocalization (yellow) in both soma and processes (white arrowheads) in SHRs aged 4 weeks, 6 weeks or 8 weeks, and 12 weeks. Magnified binary images in the upper left corner insets depict Ang II-IBA-1 colocalization within soma and processes of a microglial cell (small, dashed square) showing, in addition, the intensification of Ang II-IBA-1 colocalization during the experimental protocol. Scale bar = 50 μm. Values are the means of 6–8 slices/rat, 3–4 rats/age/group. Comparisons made by two-way factorial ANOVA. C_SS: group F (1,30) = 712.4 P < 0.001, age F (4,30) = 313.4 P < 0.001, interaction F (4,30) = 173.3 P < 0.001; 12N: group F (1,30) = 80.7 P < 0.001, age F (4,30) = 20.3 P < 0.001, interaction F (4,30) = 17.0 P < 0.001. Significances (P < 0.05): * vs. age-matched Wistar; † vs. respective week 4.