Recording sympathetic nerve activity chronically in rats: surgery techniques, assessment of nerve activity, and quantification.

The sympathetic nervous system plays a pivotal role in homeostasis through its direct innervation and functional impact on a variety of end organs. In rats, a number of methods are available to assess sympathetic nervous system function. Traditionally, direct recording of sympathetic nerve activity (SNA) has been restricted to acute, anesthetized preparations or conscious animals within a few days after electrode implantation. However, these approaches provide short-term data in studies designed to investigate changes in SNA during chronic disease states. Over the last several years, chronic SNA recording has been pioneered in rabbits and more recently in rats. The purpose of this article is to provide insights and a "how to" guide for chronic SNA recordings in rats based on experiences from two independent laboratories. We will present common methodologies used to chronically record SNA, characteristics and methods to distinguish sympathetic bursts versus electrical artifacts (and provide corresponding audio clips when available), and provide suggestions for analysis and presentation of data. In many instances, these same guidelines are applicable to acute SNA recordings. Using the surgical approaches described herein, both laboratories have been able to chronically record SNA in >50% of rats for a duration >3 wk. The ability to record SNA over the time course of several weeks will, undoubtedly, greatly impact the field of autonomic and cardiovascular physiology.

Cafeteria diet increases fat mass and chronically elevates lumbar sympathetic nerve activity in rats.

Obesity causes sympathetic activation that promotes atherosclerosis, end-organ damage, and hypertension. Because high-fat induced weight gain in rats elevates plasma leptin at 1 to 3 days after the onset of calorie-dense diets, we hypothesized that diet-induced overfeeding will increase sympathetic activity within 1 week after the onset of the regimen. To test this, we continuously measured sympathetic activity and blood pressure before and during the onset of diet-induced obesity using a high-calorie, cafeteria-style diet. Female Wistar rats, in which radiotelemeters had been implanted for continuous monitoring of lumbar sympathetic activity, mean arterial pressure, and heart rate, were randomly assigned to groups that received regular chow (control) or a cafeteria diet for a period of 15 days. This short-term, cafeteria-feeding regimen caused modest but nonsignificant increases in body weight (P=0.07) and a doubling of brown and white adipose tissue (P<0.01). The increases in fat mass were accompanied by elevations in plasma leptin (P<0.001) but no change in glucose. Overall heart rates and blood pressure were higher in cafeteria rats compared with controls (P<0.05). Cafeteria diet-induced weight gain caused increases in lumbar sympathetic nerve activity that became significant by the 12th day of the diet (P<0.001). These data show, for the first time, that the high-fat, cafeteria-style diet stimulates sustained increases in lumbar sympathetic neural drive in rats.

Dietary salt loading exacerbates the increase in sympathetic nerve activity caused by intravenous insulin infusion in rats.

Obesity and type 2 diabetes mellitus frequently produce chronic elevations in blood insulin levels. Importantly, hyperinsulinemia stimulates increases in sympathetic nerve activity that may predispose to hypertension, atherosclerosis, and end-organ damage. Because depletion of dietary salt (NaCl) increases angiotensin II levels, which has been shown to enhance sympathetic responses to excitatory stimuli such as thermal stimulation and bicuculline in the hypothalamus, we predicted that insulin-induced elevations in lumbar sympathetic activity would be augmented by low NaCl and suppressed by high dietary NaCl. Adult male Sprague-Dawley rats were randomized into groups receiving low (0.0 mEq/d, n = 10), normal (2.0 mEq/d, n = 10), and high (5.7 mEq/d, n = 10) NaCl for a period of 8 days. After this, the animals were anesthetized for measurement of heart rate, mean arterial pressure, and lumbar sympathetic nerve activity during 110 minutes of intravenous insulin infusion (15 mU/kg per minute) with euglycemic clamp. Insulin administration caused modest blood pressure decreases accompanied by heart rate increases that were similar across the 3 dietary groups. Unexpectedly, sympathetic increases to insulin were lowest in the low-NaCl group (100%-135% +/- 24%), moderate in the normal-NaCl group (100%-170% +/- 23%), and greatest in the high-NaCl group (100%-252% +/- 39%). Dietary NaCl level did not affect baseline blood glucose or insulin sensitivity as assessed by euglycemic clamp. These findings indicate that dietary salt loading exacerbates the lumbar sympathoexcitatory response to intravenous insulin infusion in rats.

Acute homocysteine administration does not elevate sympathetic nerve activity in rats.

Both hyperhomocystenemia and sympathetic overactivity are characterized by increased platelet aggregation, proliferation of vascular smooth muscle, accelerated atherosclerosis, left ventricular hypertrophy, and arterial hypertension. This coexistence of pathophysiological features suggests the possibility that homocysteine may cause increases in sympathetic nerve activity (SNA), which may in turn contribute to vascular and end-organ damage. To test this, we gave continuous intravenous infusion of vehicle (saline) in control experiments, or d,l-homocysteine (2.5 mg/kg, followed by 10 mg/ml at 4 ml/(hkg)) in urethane anesthetized rats while measuring mean arterial pressure, heart rate, and lumbar SNA. We found that a 105 min infusion of homocysteine had no significant effect on lumbar sympathetic outflow. In addition, there was no effect of acute homocysteine on heart rate or blood pressure. These findings indicate that acute administration of homocysteine does not increase the firing rate of the lumbar sympathetic nerves in anesthetized rats.

Captopril does not affect reflex increases in adrenal or lumbar sympathetic nerve activity to hypoglycemia in rats.

Blockade of angiotensin II (ANGII) receptors or converting enzyme inhibition attenuates reflex increases in epinephrine during insulin-induced hypoglycemia. Because ANGII receptors are found in several sites within the central nervous system, the aim of this study was to examine whether acute captopril attenuates the reflex increase in adrenal preganglionic sympathetic nerve activity (SNA) induced by hypoglycemia. We infused vehicle (control) or insulin (30 U/kg IV) in anesthetized rats or in rats pretreated with captopril (Cap-insulin; 2.5 mg/kg, then 1 mg/kg per hour IV) while measuring hemodynamics and SNA from adrenal preganglionic, adrenal postganglionic, and lumbar sympathetic nerves. Hypoglycemia elicited similar adrenal preganglionic SNA increases in insulin-treated (260% +/- 31% from 100% baseline) and Cap-insulin-treated (255% +/- 34%) rats. Likewise, increases in adrenal postganglionic SNA and lumbar SNA were equivalent in the insulin and Cap-insulin groups. Hypoglycemia also elicited a tachycardia in insulin-treated rats that was attenuated in Cap-insulin-treated rats, and corresponding blood pressure decreases in insulin rats were enhanced in Cap-insulin-treated rats. Thus, blockade of ANGII formation by captopril did not affect hypoglycemia-induced activation of adrenal preganglionic SNA, indicating that the renin-angiotensin systems in the brain and spinal cord do not modulate increases in adrenal SNA during hypoglycemia.

Uremia accelerates both atherosclerosis and arterial calcification in apolipoprotein E knockout mice.

Chronic renal failure (CRF) favors the development of atherosclerosis and excessive calcification of atheromatous lesions. CRF was induced in apolipoprotein E knockout (apoE(-/-)) mice to study (1) a possible acceleration of aortic atherosclerosis, (2) the degree and type of vascular calcification, and (3) factors involved in the calcification process. For creating CRF, 8-wk-old apolipoprotein E gene knockout (apoE(-/-)) mice underwent partial kidney ablation. Control animals underwent sham operation. Aortic atherosclerotic plaques and calcification were evaluated using quantitative morphologic image processing. At 6 wk after nephrectomy, CRF mice had significantly higher serum urea, cholesterol, and triglyceride concentrations than non-CRF controls. The serum levels of advanced oxidation protein products were elevated in the uremic group and were correlated with serum urea levels. Atherosclerotic lesions in thoracic aorta were significantly larger in uremic apoE(-/-) mice than in nonuremic controls. The relative proportion of calcified area to total surface area of both atherosclerotic lesions and lesion-free vascular tissue was increased in aortic root of uremic apoE(-/-) mice when compared with controls. The calcium deposits were made of hydroxyapatite and calcite crystals. In addition, plaques from uremic animals showed a significant increase in collagen content, whereas the degree of macrophage infiltration was comparable in both groups. There was no difference in mean arterial BP. These findings demonstrate that CRF aggravates atherosclerosis in apoE(-/-) mice. Moreover, CRF enhances arterial calcification at both atheromatous intimal sites and atheroma-free medial sites. We anticipate that this experimental model will be useful to test treatment strategies aimed at decreasing the accelerated atherosclerosis and arterial calcification in uremia.

Effect of insulin-induced hypokalemia on lumbar sympathetic nerve activity in anesthetized rats.

OBJECTIVE: Acute euglycemic hyperinsulinemia produces sympathoexcitation and a profound fall in plasma potassium levels. Because hypokalemia may activate the renin-angiotensin system to produce the observed increases in sympathetic nerve activity (SNA), the present study was designed to determine whether acute euglycemic-hyperinsulinemia in rats causes decreases in plasma potassium accompanied by increases in plasma renin activity (PRA) as well as elevations in SNA, and whether these alterations would be prevented by maintaining normokalemia with an exogenous potassium infusion. METHODS: We infused vehicle (control; n = 10) or insulin (10 mU/min) in anesthetized untreated rats (insulin; n = 11), or in rats receiving simultaneous KCl infusion (Insulin + K+; n = 10), while measuring mean arterial pressure (MAP), heart rate (HR), SNA, plasma potassium, and PRA during euglycemic clamp. RESULTS: As expected, insulin rats had a large fall in plasma potassium (4.6 +/- 0.1 to 3.9 +/- 0.1 mEq/l), contrasting with no change in the control (4.8 +/- 0.2 to 4.8 +/- 0.2 mEq/l) and insulin + K+ (4.4 +/- 0.1 to 4.6 +/- 0.2 mEq/l) groups. However, PRA levels at study completion were not different in the three experimental groups. In addition, insulin rats had large increases in lumbar SNA (194 +/- 11% from 100% baseline) compared with modest elevations in control rats (122 +/- 10%), and prevention of hypokalemia failed to affect sympathetic increases (213 +/- 20%) in insulin + K+ rats. MAP and HR did not change in any of the experimental groups. CONCLUSIONS: These findings indicate that insulin per se, rather than insulin-induced hypokalemia or hormonal and compensatory adjustments secondary to hypokalemia, is the main mechanism that triggers increases in lumbar SNA.