Trained hypertensive rats exhibit decreased transcellular vesicle trafficking, increased tight junctions' density, restored blood-brain barrier permeability and normalized autonomic control of the circulation

Abstract

Introduction: Chronic hypertension is accompanied by either blood-brain barrier (BBB) leakage and autonomic dysfunction. There is no consensus on the mechanism determining increased BBB permeability within autonomic areas. While some reports suggested tight junction's breakdown, others indicated the involvement of transcytosis rather than paracellular transport changes. Interestingly, exercise training was able to restore both BBB permeability and autonomic control of the circulation. We sought now to clarify the mechanism(s) governing hypertension- and exercise-induced BBB permeability. Methods: Spontaneously hypertensive rats (SHR) and normotensive controls submitted to 4-week aerobic training (T) or sedentary protocol (S) were chronically cannulated for baseline hemodynamic and autonomic recordings and evaluation of BBB permeability. Brains were harvested for measurement of BBB function (FITC-10 kDa leakage), ultrastructural analysis of BBB constituents (transmission electron microscopy) and caveolin-1 expression (immunofluorescence). Results: In SHR-S the increased pressure, augmented sympathetic vasomotor activity, higher sympathetic and lower parasympathetic modulation of the heart and the reduced baroreflex sensitivity were accompanied by robust FITC-10kDa leakage, large increase in transcytotic vesicles number/capillary, but no change in tight junctions' density within the paraventricular nucleus of the hypothalamus, the nucleus of the solitary tract and the rostral ventrolateral medulla. SHR-T exhibited restored BBB permeability and normalized vesicles counting/capillary simultaneously with a normal autonomic modulation of heart and vessels, resting bradycardia and partial pressure reduction. Caveolin-1 expression ratified the counting of transcellular, not other cytoplasmatic vesicles. Additionally, T caused in both groups significant increases in tight junctions' extension/capillary border. Discussion: Data indicate that transcytosis, not the paracellular transport, is the primary mechanism underlying both hypertension- and exercise-induced BBB permeability changes within autonomic areas. The reduced BBB permeability contributes to normalize the autonomic control of the circulation, which suppresses pressure variability and reduces the occurrence of end-organ damage in the trained SHR. Data also disclose that hypertension does not change but exercise training strengthens the resistance of the paracellular pathway in both strains.

Keywords: aerobic exercise training; autonomic control; blood-brain barrier; paracellular transport; spontaneously hypertensive rats (SHR); transcytosis.

FIGURE 1

Comparison of BBB permeability within the PVN, NTS, and RVLM of sedentary (S) and trained (T) SHR and Wistar rats. Left photomicrographs show the capillary network (Rhodamine-70kDa, red), the FITC-10kDa leakage (green) into the brain parenchyma and the colocalization of both inside capillaries (white) within the three autonomic areas. Scale bars = 50 μm. Right bar graphs depict the effects of hypertension and exercise training on the BBB leakage into the PVN, NTS and RVLM. Values are means of 5-7 slices/area/rat, 3-4 rats/group. Comparisons made by 2-way factorial ANOVA. PVN: group F (1,12) = 23.57, p < 0.001, condition F (1,12) = 23.98, p < 0.001, group x condition F (1,12) = 13.06, p = 0.004; NTS: group F (1,12) = 5.96, p = 0.031, condition F (1,12) = 28.53, p < 0.001, group x condition F (1,12) = 6.27, p = 0.028; RVLM: group F (1,10) = 3.40, p = 0.090, condition F (1,10) = 19.79, p < 0.001, group x condition F (1,10) = 3.85, p = 0.073. Significances (p < 0.05) are * vs. respective Wistar group; † vs. respective S control.

FIGURE 2

Effects of hypertension and exercise training on transcellular vesicles number/capillary. (A). Electron micrographs depicting the transcellular vesicles (yellow arrows) being formed in the luminal and abluminal borders of the endothelial cell within PVN capillaries of sedentary (S) and trained (T) SHR and Wistar rats. Scale bars = 200 nm Right panels show the quantification of vesicle number in the 4 experimental groups at the end of protocols within the PVN (B), NTS (C), and RVLM (D) capillaries. n = 9–11 capillaries/rat, 3 rats/group. Comparisons made by 2-way factorial ANOVA. Significances (p < 0.05) are * vs. respective Wistar group; † vs. respective S control.

FIGURE 3

Effects of hypertension and exercise training on caveolin-1 content within the PVN. (A). Photomicrographs show caveolin-1 expression (green) within PVN capillaries (RECA-1, red) and the colocalization of both (yellow) in sedentary (S) and trained (T) SHR and Wistar rats. Scale bar = 50 μm, 3V, third ventricle. (B). Comparison of PVN caveolin-1 immunofluorescence in the 4 experimental groups at the end of protocols. n = 5–7 slices/rat, 6 rats/group. Comparisons made by 2-way factorial ANOVA: group F (1,20) = 13.36, p = 0.002, condition (F1,20) = 19.03, p < 0.001, group x condition (F1,20) = 13.74, p = 0.001. Significances (p < 0.05) are * vs. respective Wistar group; † vs. respective S control.

FIGURE 4

Effects of hypertension and exercise training on tight junction (TJ) occupancy of capillary border. (A). Electron micrographs depicting TJ extension (yellow bars) in the border of neighboring endothelial cells within PVN capillaries of sedentary (S) and trained (T) SHR and Wistar rats. Scale bars = 200 nm. Right panels show the quantification of TJ extension in the 4 experimental groups at the end of protocols within the PVN (B), NTS (C), and RVLM (D) capillaries. n = 5–7 capillaries/rat, 3 rats/group. Comparisons made by 2-way factorial ANOVA. Significance (p < 0.05) is † vs. respective S control.

FIGURE 5

Effects of hypertension and exercise training on pericytes coverage of endothelial cells within the PVN (A), NTS (B),and RVLM (C) capillaries. n = 5–11 capillaries/area/rat, 3 rats/group. Comparisons made by two-way factorial ANOVA. Significances (p < 0.05) are * vs. respective Wistar group; † vs. respective S control.

FIGURE 6

Correlations between the number of vesicles/capillary and BBB permeability (A,C,E) and between tight junctions/capillary and BBB permeability (B,D,F) within the PVN (A,B), NTS (C,D) and RVLM (E,F) of sedentary and trained SHR and Wistar rats. BBB permeability and respective vesicles/capillary values, were obtained in 3 rats/group. Linear regression equations, correlation coefficients and p values are: PVN vesicles x BBB permeability Y = 1.10x–0.16, r = 0.905, p < 0.001; NTS vesicles x BBB permeability Y = 1.40x–1.66, r = 0.808, p = 0.002; RVLM vesicles x BBB permeability Y = 1.12x + 0.44, r = 0.743, p = 0.006; PVN TJs/capillary border x BBB permeability Y = −0.10x +10.9, r = − 0.461, p = 0.131; NTS TJs/capillary border x BBB permeability Y = −0.09x +9.7, r = −0.469, p = 0.124; RVLM TJs/capillary border x BBB permeability Y = −0.07x +10.7, r = −0.479, p = 0.229. * denotes a significant correlation.