Renal denervation prevents stroke and brain injury via attenuation of oxidative stress in… – europe pmc article – europe pmc anoxic anoxia

BACKGROUND: although renal denervation (RD) is shown to reduce blood pressure significantly in patients with resistant hypertension, the benefit of RD in prevention of stroke is unknown. We hypothesized that RD can prevent the incidence of stroke and brain injury in hypertensive rats beyond blood pressure lowering. METHODS AND RESULTS: high-salt-loaded, stroke-prone, spontaneously hypertensive rats (SHRSP) were divided into 4 groups: (1) control; (2) sham operation; (3) bilateral RD; and (4) hydralazine administration to examine the effect of RD on stroke and brain injury of SHRSP. RD significantly reduced the onset of neurological deficit and death in SHRSP, and this protection against stroke by RD was associated with the increase in cerebral blood flow (CBF), the suppression of blood-brain barrier disruption, the limitation of white matter (WM) lesions, and the attenuation of macrophage infiltration and activated microglia.Anoxic anoxia furthermore, RD significantly attenuated brain oxidative stress, and NADPH oxidase subunits, P67 and rac1 in SHRSP. On the other hand, hydralazine, with similar blood pressure lowering to RD, did not significantly suppress the onset of stroke and brain injury in SHRSP. Furthermore, RD prevented cardiac remodeling and vascular endothelial impairment in SHRSP. CONCLUSIONS: our present work provided the first experimental evidence that RD can prevent hypertensive stroke and brain injury, beyond blood pressure lowering, thereby highlighting RD as a promising therapeutic strategy for stroke as well as hypertension.

Introduction

Multiple lines of evidence indicate that renal nerves, composed of efferent sympathetic nerves and afferent sensory nerves, are involved in the pathophysiology of hypertension and chronic kidney disease. 1– 5 renal efferent sympathetic nerve activation enhances volume retention and sodium reabsorption in the kidney, reduces renal blood flow, and activates renin‐angiotensin‐aldosterone system. 4– 5 on the other hand, renal afferent sensory nerves transmit important sensory information to the paraventricular nucleus of hypothalamus (PVN), and in turn PVN transmits the information to the rostral ventrolateral medulla (RVLM), the vasomotor center that determines basal sympathetic nerve activity. 2– 3, 6

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A proof‐of‐concept study 7 and a subsequent randomized‐controlled trial 8 have demonstrated that catheter‐based bilateral renal denervation (RD) can cause a significant and sustained reduction of blood pressure (BP) in patients with treatment‐resistant hypertension. Furthermore, it has been demonstrated that muscle sympathetic nerve activity and systemic norepinephrine spillover are significantly reduced after RD in a single patient with resistant hypertension. 9 RD reduces left ventricular hypertrophy and improves cardiac function 10 in patients with resistant hypertension. Furthermore, RD provides rate control and reduces susceptibility to arterial fibrillation (AF) in patients with permanent AF. 11 these previous reports support the notion that reduction of renal afferent nerve traffic caused by RD might elicit inhibition of central sympathetic activity, thereby suggesting the possibility that RD may have the benefit in prevention of cerebrovascular and cardiac events independently of BP.Anoxic anoxia

The present study, using a rat model of hypertensive stroke, was undertaken to demonstrate our hypothesis that RD can prevent stroke and brain injury independently of BP‐lowering effect. We obtained the first experimental evidence that RD can prevent hypertensive stroke and brain injury beyond BP‐lowering effect.

Measurement of cerebral blood flow

After 3 weeks of RD, the cerebral blood flow (CBF) of SHRSP was recorded by a laser speckle blood flow imager (omega zone; omegawave), as described previously. 15 briefly, the rats were anesthetized with 2% isoflurane and the rectal temperature was kept at 37.0±0.5°C. After the rats were placed in the prone position, the skull was exposed by a midline scalp incision.Anoxic anoxia then, the surface of the region of bilateral cerebral hemispheres was diffusely illuminated by 780 nm semiconductor laser light. Color‐coded blood flow images obtained in high‐resolution mode (638×480 pixels; 1 image/second) were captured by a CCD camera positioned above head and transferred to a computer for analysis. The settings of CCD camera and color image program were kept the same among all the measurements. Images were analyzed by the color image program incorporated in the flowmetry system to obtain the average value of blood flow. The mean CBF of 10 measurements in each group was determined. The value of CBF was expressed as a percentage of low‐salt group. All measurements were performed in a blinded fashion.Anoxic anoxia

Immunohistochemical staining of macrophage and activated microglia and astrocyte

The brain and heart samples were fixed with 4% (w/v) paraformaldehyde overnight, embedded in paraffin, cut into 5‐μm thick sections. The samples were incubated with blocking solution for 30 minutes, and then incubated overnight at 4°C with the primary antibodies. For assessment of macrophage in the white matter and myocardial muscle, the samples were immunostained with anti‐ED‐1 antibody (working dilution 1:500; BMA biomedicals AG), as described. 15 positive staining was detected using horseradish peroxidase—conjugated secondary antibodies (nichirei) by incubating the sections with diaminobenzidine (DAKO). The number of ED‐1‐positive cells was counted in 4 fields of the bilateral white matter and in 10 fields of the myocardial muscle.Anoxic anoxia

For assessment of activated microglia and activated astrocyte in the white matter, the samples were immunostained with anti‐ionized calcium binding adaptor molecule‐1 (iba‐1, 1:200; abcam) and anti‐glial fibrillary acidic protein (GFAP, 1:200), in the same manner described above. All measurements were performed in a blinded fashion and expressed as the mean number of the positive cells/mm 2.

Statistical analysis

All assays and measurements in this study were performed in a blinded fashion. The method of statistical analysis used in each experiment is described in all figure legends. Results were expressed as mean±SEM. The onset of stroke symptom and survival rate were analyzed by the standard kaplan–meier analysis with a log rank test and χ 2 analysis, using graphpad prism version 5 for windows (graphpad software).Anoxic anoxia the data on SBP and DBP over 14 consecutive days after RD measured by telemetry, and vascular relaxation were analyzed by 1‐way analysis of variance (ANOVA) with repeated measures followed by bonferroni’s post‐hoc test for multiple comparisons. The data on SBP, DBP, HR, and locomotor activity during 24 hours (12‐hour dark period and 12‐hour light period) measured by telemetry were analyzed by 2‐way ANOVA with repeated measures, followed by bonferroni’s post‐hoc test for multiple comparisons. The other normal distribution data were analyzed by 1‐way ANOVA with bartlett’s test for equal variances, followed by bonferroni’s multiple comparison test. Otherwise non‐normal distribution data were analyzed by nonparametric test with kruskal‐wallis test, followed by dunn’s multiple comparison test.Anoxic anoxia in all tests, differences were considered statistically significant at a value of P0.05.

Discussion

Although RD significantly reduces BP in patients with resistant hypertension, 5, 7– 9, 22 it is unknown whether RD can prevent cerebrovascular events beyond BP‐lowering effect. The major findings of our present work were that RD significantly prevented the onset of stroke and the progression of brain injury in hypertensive rats, and these brain‐protective effects of RD were at least in part, mediated by BP‐independent effects, including attenuation of oxidative stress and inflammation, and suppression of BBB disruption. Therefore, our present work provided the first evidence supporting that RD seems to be a promising therapeutic strategy for stroke in hypertension.Anoxic anoxia

In the present study, to determine the effectiveness of RD on stroke in hypertension, we used high‐salt‐loaded SHRSP, since SHRSP is regarded as an established and popular model of hypertensive stroke. 12, 15, 20, 23 in agreement with our previous reports, 12, 15, 20, 23 high‐salt intake significantly accelerated stroke incidence and brain injury in SHRSP, which is consistent with clinical evidence 24– 28 that excessive salt intake is an important risk factor for stroke. Of note, RD significantly reduced the incidence of neurological deficit and death in high‐salt‐loaded SHRSP, despite the very small BP‐lowering effect of RD in SHRSP. On the other hand, hydralazine treatment with similar small BP‐lowering effect to RD did not significantly prevent stroke in SHRSP.Anoxic anoxia these results demonstrate that the mechanism underlying prevention of stroke by RD in SHRSP is mediated by BP‐independent effect. Furthermore, RD significantly attenuated the decrease in cerebral blood flow and significantly prevented the progression of white matter lesion in high‐salt‐loaded SHRSP. On the contrary, hydralazine failed to attenuate these changes induced by high‐salt in SHRSP. These results indicate that RD prevented the disturbance of cerebral blood flow and progression of white matter lesion independently of BP. Furthermore, RD significantly limited disruption of BBB in high‐salt‐loaded SHRSP, being accompanied by the suppression of downregulation of occludin, 29– 30 a key tight junction protein involved in intact BBB function, while hydralazine treatment failed to limit these changes in high‐salt‐loaded SHRSP.Anoxic anoxia therefore, the protective effect of RD against stroke in high‐salt‐loaded SHRSP seems to be at least partially mediated by the improvement of cerebral blood flow and inhibition of BBB disruption.

In the present work, it is worthy to note that RD significantly ameliorated the increase in inflammatory cells such as macrophage, and activated microglia and astrocyte in cortex, white matter, and PVN regions in high‐salt‐loaded SHRSP, while hydralazine did not attenuate them. These findings imply that RD exerts anti‐inflammatory effects in the brain of SHRSP. Accumulating evidence 31– 34 supports the notion that oxidative stress and inflammation play a key role in the pathogenesis of stroke and brain injury, through formation of a vicious cycle.Anoxic anoxia previously, we have reported that antioxidant significantly slows the incidence of stroke in high salt‐loaded SHRSP, and this is associated with attenuation of cerebral inflammation, thereby indicating the critical role of brain oxidative stress in the mechanism of stoke in high‐salt‐loaded SHRSP. 20 therefore, in the present study, we examined the effect of RD on brain oxidative stress in SHRSP. RD, but not hydralazine, significantly reduced oxidative stress in cortex, white matter, and PVN of SHRSP. Furthermore, RD, but not hydralazine, significantly prevented the increase in NADPH oxidase subunits, P67 and rac1 in SHRSP. These results show that RD attenuated brain oxidative stress beyond BP‐lowering effect.Anoxic anoxia collectively, our present work supports the notion that the attenuation of brain oxidative stress by RD is involved in the prevention of stroke in high‐salt‐loaded SHRSP.

A growing body of clinical evidence 35– 36 and experimental work including SHRSP 15, 20, 23 show that renin‐angiotensin system participates in the pathophysiology of stroke and brain injury. In the present study, plasma renin activity was significantly reduced by RD in high‐salt‐loaded SHRSP, indicating the suppression of circulating renin‐angiotensin system by RD. Previously we have found that angiotensin II is directly involved in the progression of stroke in high‐salt‐loaded SHRSP. 15, 20, 23 therefore, not only attenuation of brain oxidative stress but also suppression of circulating renin‐angiotensin system appears to account for the protective effects of RD against stroke and brain injury.Anoxic anoxia

The method of RD used in the present work causes denervation of afferent renal sensory nerves as well as efferent renal sympathetic nerves. Substantial evidence indicates that afferent renal sensory nerves project directly to various areas in the central nervous system involved in the regulation of cardiovascular system. 4 previous experimental studies 37– 39 show that renal injury such as ischemia, through afferent renal nerve, significantly activates the central sympathetic nervous system, thereby leading to the enhancement of systemic sympathetic nerve activity. Furthermore, patients with chronic renal failure are characterized by the enhancement of muscle sympathetic nerve activity through renal afferent nerves. 40– 41 thus, it is proposed that renal afferent nerves are a key regulator of peripheral sympathetic nerve activity as well as the central sympathetic nervous system.Anoxic anoxia recent clinical studies show that RD significantly attenuates muscle sympathetic nerve activity 42 and improves cardiac hypertrophy and function, 10 atrial fibrillation, 11 glucose intolerance 43 in patients with resistant hypertension, thereby supporting the concept that RD can have the benefit in prevention of cardiovascular injury through afferent renal nerve ablation. However, it remains to be determined whether these potential organ protective effects of RD in hypertensive patients are secondary to BP‐lowering or not. In the present work, notably, RD, but not hydralazine, significantly attenuated cardiac hypertrophy, cardiac macrophage infiltration, and cardiac fibrosis and also prevented the impairment of vascular endothelial function in high‐salt‐loaded SHRSP.Anoxic anoxia these results suggest that BP‐independent protective effects of RD against stroke and cardiovascular injury observed in the present work might be attributed, at least partially, to the ablation of renal afferent nerves.