Ghrelin Preserves Ischemia-Induced Vasodilation of Male Rat Coronary Vessels Following ß-Adrenergic Receptor Blockade.

Acute myocardial infarction (MI) triggers an adverse increase in cardiac sympathetic nerve activity (SNA). Whereas ß-adrenergic receptor (ß-AR) blockers are routinely used for the management of MI, they may also counter ß-AR-mediated vasodilation of coronary vessels. We have reported that ghrelin prevents sympathetic activation following MI. Whether ghrelin modulates coronary vascular tone following MI, either through the modulation of SNA or directly as a vasoactive mediator, has never been addressed. We used synchrotron microangiography to image coronary perfusion and vessel internal diameter (ID) in anesthetized Sprague-Dawley rats, before and then again 30 minutes after induction of an MI (left coronary artery ligation). Rats were injected with either saline or ghrelin (150 µg/kg, subcutaneously), immediately following the MI or sham surgery. Coronary angiograms were also recorded following ß-AR blockade (propranolol, 2 mg/kg, intravenously). Finally, wire myography was used to assess the effect of ghrelin on vascular tone in isolated human internal mammary arteries (IMAs). Acute MI enhanced coronary perfusion to nonischemicregions through dilation of small arterioles (ID 50 to 250 µm) and microvessel recruitment, irrespective of ghrelin treatment. In ghrelin-treated rats, ß-AR blockade did not alter the ischemia-induced vasodilation, yet in saline-treated rats, ß-AR blockade abolished the vasodilation of small arterioles. Finally, ghrelin caused a dose-dependent vasodilation of IMA rings (preconstricted with phenylephrine). In summary, this study highlights ghrelin as a promising adjunct therapy that can be used in combination with routine ß-AR blockade treatment for preserving coronary blood flow and cardiac performance in patients who suffer an acute MI.

Pulmonary vascular tone is dependent on the central modulation of sympathetic nerve activity following chronic intermittent hypoxia.

Chronic intermittent hypoxia (IH) provokes a centrally mediated increase in sympathetic nerve activity (SNA). Although this sympathetic hyperexcitation has been linked to systemic hypertension, its effect on the pulmonary vasculature is unclear. This study aimed to assess IH-mediated sympathetic excitation in modulating pulmonary vasculature tone, particularly acute hypoxia vasoconstrictor response (HPV), and the central ß-adrenergic signaling pathway for facilitating the increase in SNA. Sprague-Dawley rats were exposed to IH (cycle of 4% O2 for 90 s/air for 90 s) for 8 h/day for 6 weeks. Subsequently, rats were anesthetized and either pulmonary SNA was recorded (electrophysiology), or the pulmonary vasculature was visualized using microangiography. Pulmonary sympathetic and vascular responses to acute hypoxia were assessed before and after central ß1-adrenergic receptor blockade (Metoprolol, 200 nmol i.c.v.). Chronic IH increased baseline SNA (110% increase), and exacerbated the sympathetic response to acute hypoxia. Moreover, the magnitude of HPV in IH rats was blunted compared to control rats (e.g., 10 and 20% vasoconstriction, respectively). In only the IH rats, ß1-receptor blockade with metoprolol attenuated the hypoxia-induced increase in pSNA and exacerbated the magnitude of acute HPV, so that both sympathetic and HPV responses were similar to that of control rats. Interestingly, the expression of ß1-receptors within the brainstem was similar between both control and IH rats. These results suggest that the centrally mediated increase in SNA following IH acts to blunt the local vasoconstrictor effect of acute hypoxia, which reflects an inherent difference between vasodilator and vasoconstrictor actions of SNA in pulmonary and systemic circulations.

Role of cardiac sympathetic nerves in blood pressure regulation.

Stellate ganglionectomy (SGx) was used to assess the contribution of cardiac sympathetic nerves to neurogenic hypertension in deoxycorticosterone (DOCA)-salt treated rats. Experiments were conducted in two substrains of Sprague-Dawley (SD) rats since previous studies reported bradycardia in Charles River-SD (CR-SD) rats and tachycardia in SASCO-SD (SA-SD) rats with DOCA treatment suggesting different underlying neural mechanisms. Uninephrectomized male rats underwent SGx or SHAM surgery and were instrumented for telemetric monitoring of mean arterial pressure (MAP) and heart rate (HR). After recovery, 0.9% saline solution and DOCA (50mg) were administered. Baseline MAP (Days 0-5 average) after SGx in CR-SD rats (96±2mmHg; n=7) was not significantly different (p=0.08) than CR-SD SHAM rats (103±3mmHg; n=9); however, there was a significantly lower HR during the baseline period (377±7 vs. 432±7bpm, p<0.05) in SGx rats. In SA-SD rats baseline MAP was not different between SGx and SHAM rats and HR was lower in SGx rats (428±8 vs. 371±5bpm, p<0.05). After DOCA treatment in both substrains, MAP and HR were elevated similarly in SHAM and SGx groups showing minimal impact in both groups of SGx on hypertension development. However, overall MAP in SA-SD SHAM rats reached a significantly higher level (155±10mmHg vs 135±5mmHg, p<0.05) than that observed in CR-SD SHAM rats demonstrating that the magnitude of hypertensive response to DOCA-salt treatment varies between substrains. In conclusion, removal of cardiac sympathetic nerves did not alter the development or maintenance of DOCA-salt hypertension in SD rats.

Acute Rho-kinase inhibition improves coronary dysfunction in vivo, in the early diabetic microcirculation.

OBJECTIVES: Activation of RhoA/Rho-kinase (ROCK) is increasingly implicated in acute vasospasm and chronic vasoconstriction in major organ systems. Therefore we aimed to ascertain whether an increase in ROCK activity plays a role in the deterioration of coronary vascular function in early stage diabetes. METHODS: Synchrotron radiation microangiography was used to determine in vivo coronary responses in diabetic (3 weeks post streptozotocin 65 mg/kg ip) and vehicle treated male Sprague-Dawley rats (n = 8 and 6). Changes in vessel number and calibre during vasodilator stimulation before and after blockade of nitric oxide synthase and cyclooxygenase were compared between rats. Acute responses to ROCK inhibitor, fasudil (10 mg/kg iv) was evaluated. Further, perivascular and myocardial fibrosis, arterial intimal thickening were assessed by histology, and capillary density, nitrotyrosine and ROCK1/2 expressions were evaluated by immunohistochemical staining. RESULTS: Diabetic rats had significantly elevated plasma glucose (P < 0.001 vs control), but did not differ in fibrotic scores, media to lumen ratio, capillary density or baseline visible vessel number or calibre. Responses to acetylcholine and sodium nitroprusside stimulation were similar between groups. However, in comparison to control rats the diabetic rats showed more segmental constrictions during blockade, which were not completely alleviated by acetylcholine, but were alleviated by fasudil. Further, second order vessel branches in diabetic rats were significantly more dilated relative to baseline (37% vs 12% increase, P < 0.05) after fasudil treatment compared to control rats, while visible vessel number increased in both groups. ROCK2 expression was borderline greater in diabetic rat hearts (P < 0.053). CONCLUSIONS: We found that ahead of the reported decline in coronary endothelial vasodilator function in diabetic rats there was moderate elevation in ROCK expression, more widespread segmental constriction when nitric oxide and prostacyclin production were inhibited and notably, increased calibre in second and third order small arteries-arterioles following ROCK inhibition. Based on nitrotyrosine staining oxidative stress was not significantly elevated in early diabetic rats. We conclude that tonic ROCK mediated vasoconstriction contributes to coronary vasomotor tone in early diabetes.

Assessment of the serotonin pathway as a therapeutic target for pulmonary hypertension.

Blockade of the serotonin reuptake transporter (5-HTT), using fluoxetine, has been identified as a potential therapeutic target for preventing and, importantly, reversing pulmonary hypertension (PH). This study utilized synchrotron radiation microangiography to determine whether fluoxetine could prevent or reverse endothelial dysfunction and vessel rarefaction, which underpin PH. PH was induced by a single injection of monocrotaline (MCT; 60 mg kg(-1)). Following MCT administration, rats received daily injections of either saline or fluoxetine (MCT+Fluox; 10 mg kg(-1)) for three weeks. A third group of rats also received the fluoxetine regime, but only three weeks after MCT (MCT+FluoxDelay). Control rats received daily injections of saline. Pulmonary microangiography was performed to assess vessel branching density and visualize dynamic changes in vessel diameter following (i) acute fluoxetine or (ii) acetylcholine, sodium nitroprusside, BQ-123 (ET-1A receptor blocker) and L-NAME (NOS inhibitor). Monocrotaline induced PH that was inevitably terminal. `Delayed' treatment of fluoxetine (MCT+FluoxDelay) was unable to reverse the progression of PH. Early fluoxetine treatment pre-PH (i.e. MCT+Fluox) attenuated but did not completely prevent vascular remodeling, vessel rarefaction and an increase in pulmonary pressure, and it did not prevent pulmonary endothelial dysfunction. Interestingly, fluoxetine treatment did counter-intuitively prevent the onset of right ventricular hypertrophy. Using synchrotron radiation microangiography, selective blockade of the serotonin reuptake transporter alone is highlighted as not being sufficient to prevent pulmonary endothelial dysfunction, which is the primary instigator for the inevitable onset of vascular remodeling and vessel rarefaction. Accordingly, potential therapeutic strategies should aim to target multiple pathways to ensure an optimal outcome.

Imaging of the closed-chest mouse pulmonary circulation using synchrotron radiation microangiography.

Structural and functional changes of pulmonary circulation related to pathophysiology of pulmonary arterial hypertension (PAH) remain to be fully elucidated. Angiographic visualization in in vivo animals provided a powerful tool for assessing the major indexes associated with the pathogenesis of PAH. In this study, we have exploited the full potential of synchrotron radiation (SR) microangiography to show the ability to visualize pulmonary hemodynamics in a closed-chest mouse. Male adult mice were anesthetized and cannulated with a customized 24-gauge catheter into the right ventricle via the jugular vein for administering iodine contrast agent. The microangiography was performed on the left lung. We measured dynamic changes in vessel diameter in response to acetylcholine (ACh) and acute exposure to hypoxic gas (10% O(2)). Moreover, the pulmonary transit time was estimated by the time of contrast agent circulating. We were able to visualize the pulmonary arteries from the left pulmonary artery (LPA) to the third generation of branching (inner diameter <100 µm). ACh and acute hypoxia induced vascular responses chiefly in the second and third branching vessels rather than the LPA and the first branching vessels. The transit time was only 0.83 s. These results demonstrate the effectiveness of SR for visualizing the pulmonary circulation in a closed-chest mouse. Future studies using SR microangiography on specific gene-targeted knockout and transgenic mice will provide new insights into the pathophysiology of pulmonary dysfunction and functional adaptation to survive in hypoxic condition.

Effect of stellate ganglionectomy on basal cardiovascular function and responses to beta1-adrenoceptor blockade in the rat.

Cardiac sympathetic nerve activity is an important short-term controller of cardiac function and arterial pressure. Studies also suggest that long-term increases in cardiac sympathetic nerve activity may contribute to hypertension, coronary artery disease, and cardiac remodeling in heart failure. However, our understanding of the role of cardiac sympathetic nerves in chronic models of cardiovascular disease has been limited by inadequate experimental approaches. The present study was conducted to develop a surgical method to surgically denervate the sympathetic nerves of the rat heart for long-term cardiovascular studies. We characterized the effect of cardiac sympathetic denervation on basal levels of mean arterial pressure (MAP) and heart rate (HR) and the responses to a chronic administration of atenolol, a beta1-adrenoceptor antagonist. Rats were instrumented with telemetry transmitters for continuous recording of MAP and HR. After a 4-day baseline period, the rats were subjected to bilateral stellate ganglionectomy (SGX; n=9) or sham surgery (Sham; n=8). Seven days following SGX or Sham, the rats were administered atenolol for 5 days, followed by a 7-day recovery period. Following a transient decrease, SGX had no effect on basal MAP but decreased HR compared with baseline and Sham rats. Five days of atenolol treatment decreased MAP similarly in SGX and Sham rats. Atenolol resulted in a marked bradycardia in Sham rats but had a neglible effects on HR in SGX rats. The measurement of the content of cardiac catecholamines in all cardiac chambers at the end of the study verified a successful sympathetic denervation. This study confirms that bilateral SGX is a useful method to study the contribution of cardiac sympathetic nerves on the regulation of cardiac function. Moreover, these results suggest that cardiac sympathetic nerves are relatively unimportant in maintaining the basal level of MAP or the depressor response to atenolol in conscious, unrestrained rats.