Factors contributing to neurogenic hypertension in genetically hypertensive mice

Hypertension affects approximately 1 in 4 people worldwide and whilst it is typically asymptomatic, it is an insidious disease contributing to life threatening conditions including heart failure, stroke and renal failure (Wolf et al., 1991; Levy et al., 1996; Kearney et al., 2005; Ravera et al., 2006). Recent clinical studies show renal sympathetic nerve ablation is an effective treatment in uncontrolled hypertensive patients (Esler et al., 2010) which has strengthened research interest into the contribution of the sympathetic nervous system (SNS) to hypertension. However, investigation of the neural pathophysiology in human hypertension has obvious boundaries, particularly those related to examining the role of the central nervous system (CNS). Therefore it is necessary to assess neurogenic hypertension in animal models such as BPH/2J hypertensive mice. BPH/2J mice are a genetic model of hypertension which were selectively bred to exhibit elevated blood pressure (BP) compared with concurrently bred hypotensive (BPL/1J) and normotensive (BPN/3J) controls. Whilst originally bred to evaluate the genetics of hypertension, they have since been studied in a wide range of areas. Importantly recent findings indicate that BPH/2J mice have a neurogenic form of hypertension, primarily based on findings demonstrating that the hypertension is abolished by ganglion blockade, indicating a considerable contribution from the SNS (Davern et al., 2009b). Early studies also demonstrate abnormal brain catecholamine levels in BPH/2J mice compared with normotensive BPN/3J counterparts and more recently BPH/2J mice were reported to have greater neuronal activity in limbic and hypothalamic brain regions associated with autonomic regulation of the cardiovascular system (Schlager & Freeman, 1983; Strazielle et al., 2004; Davern et al., 2009b). As such there is mounting evidence to suggest that the CNS may be an important mediator of the hypertension. Consequently hypertensive BPH/2J mice appear to be a suitable model to study the potential causes contributing to overactivity of the SNS in hypertension. In the present thesis a range of potential mechanistic paradigms were explored to identify the major factors contributing to hypertension in BPH/2J mice. In the first instance an extensive characterization of the metabolic profile in BPH/2J mice was assessed to determine the potential influence of metabolic factors on BP. Additionally the known interactions between the SNS and peripheral and central renin-angiotensin system (RAS) on cardiovascular regulation were investigated using pharmacological inhibition, immunohistochemical indicators of sympathetic innervation and measurement of renal gene expression. Finally the role of the CNS in the sympathetically mediated hypertension was explored by means of pharmacological inhibition and excitotoxic lesions. Together the aim was to provide additional insight into the factors contributing to hypertension in BPH/2J mice. A range of characteristics indicate that BPH/2J mice may have metabolic abnormalities including low body weight (BW), hyperactivity and abnormal thermoregulation (Rosenberg et al., 1982; Malo et al., 1989; Davern et al., 2009b). Importantly metabolic rate positively correlates with BP, yet there has been no systematic assessment of the metabolic profile of BPH/2J mice so it is unknown whether metabolic differences may be contributing to the hypertension (Luke et al., 2004). Consequently the first study of this thesis (chapter 3) was conducted to determine whether there are differences in energy metabolism in BPH/2J mice and whether any identified differences may be associated with hypertension in BPH/2J mice. This was examined by characterizing the metabolic profile of hypertensive BPH/2J mice in comparison with normotensive BPN/3J and C57Bl6 mice. Whole body metabolic and cardiovascular parameters were measured over 24 hours by indirect calorimetry and radiotelemetry respectively in conscious young (10-13 week) and older (22-23 week) BPH/2J, normotensive BPN/3J and C57Bl6 mice. The main finding was that whilst metabolic rate was greater in BPH/2J compared with BPN/3J mice, it was actually similar to the normotensive C57Bl6 mice. This indicates that high metabolic rate is not axiomatically related to the hypertension. Importantly, at a time when metabolic rate was comparable between all three strains, hypertension was still apparent in BPH/2J mice. Furthermore whilst there was a strong positive correlation between BP and metabolic rate in BPH/2J mice, this was shown to be driven by differences in physical activity rather than metabolic rate. Echo magnetic resonance imaging (EchoMRI) also revealed that strain differences in BW between BPH/2J and BPN/3J mice were not accompanied by differences in body composition. Taken together, the findings suggest that the hypertension is largely unrelated to differences in energy metabolism in BPH/2J mice. The RAS is another factor crucial for BP regulation and involved in many forms of hypertension, yet there are conflicting reports on the contribution of the RAS to the hypertension in BPH/2J mice. Furthermore it is possible that the RAS interacts with the augmented SNS activity in BPH/2J mice via the actions of either the central or peripheral RAS. Yet the potential involvement of these systems has not been fully delineated in BPH/2J mice. Consequently the second study of this thesis (chapter 4) details an investigation of the hypothesis that the hypertension may be mediated by an interaction between the peripheral RAS and the overactive SNS in BPH/2J mice (Davern et al., 2009b). BPH/2J and BPN/3J mice were pre-implanted with radio-telemetry devices to measure BP. Depressor responses to the ganglion blocker pentolinium in mice pre-treated with the angiotensin converting enzyme inhibitor enalaprilat revealed a 2-fold greater sympathetic contribution to BP in BPH/2J mice during both the light and dark periods of the 24-hour light cycle, when mice are predominantly inactive and active respectively. However, the depressor response to enalaprilat was 4-fold greater in BPH/2J compared with BPN/3J mice, but only during the dark (active) period when hypertension is at its greatest in BPH/2J mice. Importantly, the greater RAS contribution during the active period was associated with 1.6-fold higher renal Ren1 mRNA expression and also a lower abundance of renal miR-181a, a negative regulator of human renin mRNA. Ren1 mRNA levels were positively associated with depressor responses to pentolinium and furthermore TH staining indicated a 1.4-fold greater abundance of renal sympathetic innervation in BPH/2J compared with BPN/3J mice. Taken together these findings suggest that tonic overactivity of the SNS is a major contributor to hypertension in BPH/2J mice, yet the RAS also contributes, doing so to a greater extent during the active period and less during the inactive period. Therefore it is possible that renal hyper-innervation and enhanced sympathetically-induced renin synthesis mediated by lower levels of the microRNA miR-181a is contributing to the hypertension in BPH/2J mice. To complement the first study which assessed the contribution of the peripheral RAS, the focus of the second study was on the role of the central RAS, as overactivity of this system is implicated in other models of neurogenic hypertension (Ye et al., 2002a). Thus the aim of the third study (chapter 5) was to determine the influence of AngII activation of central angiotensin receptor type 1 (AT1R) on the hypertension in BPH/2J mice, as well as the possible contribution of reactive oxygen species (ROS). To determine the contribution to hypertension in BPH/2J mice, pre-implanted telemetry devices were used to measure the cardiovascular response to acute and chronic intracerebroventricular (ICV) administration of AT1R antagonists, candesartan and losartan in BPH/2J and BPN/3J mice. Additionally the acute cardiovascular response to ICV administration of superoxide dismutase mimetic, tempol and ROS scavenger resveratrol were measured. ICV injection of tempol or resveratrol had minimal effect on BP in either strain during periods of either inactivity or activity. During the dark (active) period acute ICV injection of an AT1R antagonist induced depressor responses which were 40% smaller in BPH/2J compared with BPN/3J mice, whereas during the inactive period the effect was minimal in both strains. Furthermore during this active period, ICV injection of AngII produced a smaller pressor response in BPH/2J compared with BPN/3J mice. Chronic ICV administration of losartan for 7 days via an osmotic minipump induced a greater decrease in BP in BPH/2J mice and had minimal effect in BPN/3J mice. However, the effect was comparable to that observed following systemic administration, suggesting that this chronic hypotensive effect in BPH/2J mice was likely to be peripherally mediated rather than central. Collectively, based on acute and steady state inhibition of central AT1Rs, these findings suggest that greater activity of the central RAS does not appear to contribute to hypertension in BPH/2J mice. Regardless of the lack of contribution of the central RAS to hypertension in BPH/2J mice, the hypertension is mediated by a persistent overactivity of the SNS and as such it is likely that these mice are sensitive to centrally acting sympatholytic agents (Davern et al., 2009b)(Study 2). Therefore the aim of the fourth study (chapter 6) was to assess whether BPH/2J mice were indeed sensitive to the antihypertensive effect of rilmenidine, an antihypertensive agent which has centrally mediated sympatholytic effects predominantly via actions at premotor neurons located within the RVLM (Head et al., 1998). Chronic oral administration of rilmenidine at a wide range of doses had minimal effect on 24-hour average BP in both strains ( ...