Conscious Sheep Research Articles

Neurally-mediated Cardiotoxicity of Local Anesthetics: Direct Effect of Seizures or of Local Anesthetics?

To the Editor:—Ladd et al. 1showed in conscious sheep that the administration of incremental doses of local anesthetics in the central nervous system bloodstream induces minimal systemic recirculation, dose-dependent central nervous system excitatory behavior, and electroencephalographic changes. These authors also observed that bupivacaine is more neurotoxic than levobupivacaine, which is also more neurotoxic than ropivacaine. Finally, they stated that there are no differences among the drugs concerning their arrhythmogenicity.It is well established that large doses of local anesthetics slow cardiac conduction velocity in a dose- and use-dependent manner by inhibiting the fast-inward sodium current. 2The slowing of ventricular conduction velocity is responsible for a proportional QRS widening 3,4and facilitates the occurrence of reentrant ventricular arrhythmias around arcs of conduction block. 5,6In other words, the increase in heart rate by enhancing the slowing of ventricular conduction is one of the main mechanisms facilitating the occurrence of serious reentrant ventricular arrhythmias in the case of high plasma concentration. Moreover, Mazoit et al. 7and our team 8reported that bupivacaine is more use-dependent than levobupivacaine and ropivacaine. This fact explains at least in part the differences in direct cardiotoxicity in these three local anesthetics. As demonstrated by Bernards and Artru 9,10and our team, 11an overdose of local anesthetics induces an increase in the sympathetic outflow by their direct neurotoxicity and therefore an increase in heart rate, facilitating the occurrence of serious reentrant arrhythmias when toxic cardiac concentrations are reached.In the elegant study published by Ladd et al. , 1RR and PR intervals are obviously shortened. In contrast and surprisingly, QRS is shortened by the administration of any local anesthetics at any dose. One might argue that the baseline values are somewhat widened and that the method of measurement, including the paper speed, is not specified in the method section. Nevertheless, because QRS is not widened, one could specially argue that there is no direct cardiotoxicity and that the observed arrhythmias are only due to the pre- and convulsant effects of local anesthetics as any seizures can provoke. In conclusion, the sentence in the summary that “no differences were found in their arrhythmogenic potential”1may be confusing because the analyzed arrhythmogenicity in the present study is only in terms of indirect and neurally mediated cardiotoxicity and not in terms of direct cardiotoxicity.

Near-infrared spectroscopy-guided closed-loop resuscitation of hemorrhage.

Endpoint resuscitation has been suggested as a better means to resuscitate penetrating injury. We performed computer-controlled closed-loop resuscitation using invasive cardiac output (CO) or noninvasive skeletal muscle oxygen saturation (SkMusSO(2)) via near infrared spectroscopy (NIRS). Conscious sheep received a 4.0-mm aortotomy and uncontrolled hemorrhage at t = 0 min (T0) while resuscitation started at T20 using lactated Ringer's solution. The aortotomy rapidly decreased the mean arterial pressure (MAP) to approximately 30 mm Hg and CO to 20% to 30% of baseline. The SkMusSO(2) endpoint group required only half as much fluid through 4 hours of resuscitation as the CO endpoint group (34.9 +/- 8.4 mL/kg vs. 63.1 +/- 9.4 mL/kg). CO and MAP were lower in the SkMusSO(2) group after T60. Mean infusion volumes were 180% and 100% of the bled volume collected at autopsy in the CO and SkMusSO(2) groups. Brain and muscle oxygenation and base excess were as high or higher in the CO endpoint group. Closed-loop resuscitation with either CO or SkMusSO(2) endpoints effectively performs fluid resuscitation of severe uncontrolled hemorrhagic shock. Limited resuscitation may achieve favorable clinical results with volumes less than recommended by Advanced Trauma Life Support guidelines.

Identification of stimulatory and inhibitory inputs to the hypothalamic-pituitary-adrenal axis during hypoglycaemia or transport in ewes.

This study used the novel approach of statistical modelling to investigate the control of hypothalamic-pituitary-adrenal (HPA) axis and quantify temporal relationships between hormones. Two experimental paradigms were chosen, insulin-induced hypoglycaemia and 2 h transport, to assess differences in control between noncognitive and cognitive stimuli. Vasopressin and corticotropin-releasing hormone (CRH) were measured in hypophysial portal plasma, and adrenocorticotropin hormone (ACTH) and cortisol in jugular plasma of conscious sheep, and deconvolution analysis was used to calculate secretory rates, before modelling. During hypoglycaemia, the relationship between plasma glucose and vasopressin or CRH was best described by log10 transforming variables (i.e. a positive power-curve relationship). A negative-feedback relationship with log10 cortisol concentration 2 h previously was detected. Analysis of the "transport" stimulus suggested that the strength of the perceived stimulus decreased over time after accounting for cortisol facilitation and negative-feedback. The time course of vasopressin and CRH responses to each stimulus were different However, at the pituitary level, the data suggested that log10 ACTH secretion rate was related to log10 vasopressin and CRH concentrations with very similar regression coefficients and an identical ratio of actions (2.3 : 1) for both stimuli. Similar magnitude negative-feedback effects of log10 cortisol at -110 min (hypoglycaemia) or -40 min (transport) were detected, and both models contained a stimulatory relationship with cortisol at 0 min (facilitation). At adrenal gland level, cortisol secretory rates were related to simultaneously measured untransformed ACTH concentration but the regression coefficient for the hypoglycaemia model was 2.5-fold greater than for transport. No individual sustained maximum cortisol secretion for longer than 20 min during hypoglycaemia and 40 min during transport. These unique models demonstrate that corticosteroid negative-feedback is a significant control mechanism at both the pituitary and hypothalamus. The amplitude of HPA response may be related to stimulus intensity and corticosteroid negative-feedback, while duration depended on feedback alone.

Circulatory effects of angiotensin II during anaesthesia, evaluated by real-time spectral analysis.

General anaesthesia may stimulate the renin-angiotensin system. Exogenous administration of angiotensin II elevates blood pressure mainly via increased total peripheral resistance caused by direct vasoconstrictor actions. It is also well established that the hypertensive effect of angiotensin II involves a cerebrally mediated component. The hypertensive effect of an intravascular infusion of angiotensin II is substantially reduced by isoflurane anaesthesia. A likely mechanism is that isoflurane anaesthesia reduces the cerebral component of the angiotensin II effect on blood pressure, which involves influences on autonomic nervous activity. In an experimental study in sheep we used real-time spectral analysis of arterial blood pressure signals to obtain information on parasympathetic, respectively, sympathetic autonomic nervous activity in response to angiotensin II administration during isoflurane anaesthesia. The study was performed on conscious and isoflurane-anaesthetized sheep that were subjected to an intracarotid infusion of angiotensin II (85 ng kg(-1) min(-1)) during 20 min followed by a recovery period of 30 min and thereafter an injection of the angiotensin II, AT1-receptor antagonist losartan (10 mg kg(-1)) i.v. Systemic and regional (renal and femoral) circulation was monitored in parallel to real-time spectral analysis of the arterial blood pressure signal. Isoflurane anaesthesia reduced both magnitude and duration of the hypertensive response to angiotensin II infusion. The power spectral density in the frequency band that represents sympathetic activation, correlated to the changes in mean arterial pressure in conscious animals, but not during isoflurane anaesthesia. We conclude that the cerebrally mediated component of the hypertensive effect of circulating angiotensin II is largely eliminated by isoflurane anaesthesia. Spectral power analysis of the blood pressure signal indicates that the cerebral angiotensin II effect involves activation of sympathetic nervous activity.

Cardiac sympathetic nerve activity in conscious sheep: Effect of myocardial infarction

Cardiac sympathetic nerve activity in conscious sheep: Effect of myocardial infarction

The Effect of Hypoxic Hypoxia on the Systemic and Myocardial Pharmacokinetics and Dynamics of Lidocaine in Sheep

It was hypothesized that the increased myocardial blood flow known to occur during some types of hypoxia may alter the kinetics and dynamics of cardio-active drugs such as lidocaine that act directly on the myocardium. In a randomized cross-over design, iv lidocaine (100 mg over 2 min) was administered to conscious instrumented sheep in a control state (C) or when the sheep were rendered hypoxic (H) by the addition of nitrogen to their inspired air (average Pa(O2) = 26 mmHg). Hypoxia caused a significant increase in myocardial blood flow (193% of control, SD = 37%, p < 0.05), a nonsignificant increase in cardiac output, and unchanged heart rate and blood pressure. Peak arterial lidocaine concentrations were unchanged, but peak concentrations in coronary sinus blood (effluent from the myocardium) were increased (maximum 201% of control, SD = 63%, p < or = 0.05), suggesting more rapid uptake of lidocaine into the myocardium in hypoxia. There was a linear relationship between coronary sinus lidocaine concentrations and reductions in myocardial contractility. However, the higher myocardial concentrations associated with H were not associated with overall greater reductions in contractility, as baseline contractility was elevated by H. Thus, hypoxia was not detrimental with respect to this adverse effect of lidocaine on the myocardium.

Endothelin-1 produces no renal vasoconstriction in conscious newborn lambs.

Experiments were carried out to investigate the effects of endothelin-1 (ET-1) on renal vascular tone during development under physiological conditions in conscious lambs. Renal blood flow (RBF), renal vascular resistance (RVR), mean arterial pressure (MAP), and heart rate (HR) were measured in conscious, chronically instrumented lambs aged approximately 1 week and 6 weeks before and after intra-arterial (i.a.) injection of 0, 100, 200, and 400 ng/kg body weight of ET-1. In addition, plasma levels of ET-1 were measured in 39 sheep aged 5-85 days. In 6-week-old lambs, i.a. injection of ET-1 was associated with a rapid dose-dependent decrease in RBF that resulted from a dose-dependent increase in RVR. In 1-week-old lambs, there was no renal vasoconstriction observed after ET-1 administration, even at the highest dose tested. In response to i.a. injection of ET-1 to 1-week-old and 6-week-old lambs, MAP increased and there was a concomitant decrease in HR; these effects were dose dependent but not age dependent. Plasma levels of ET-1 were 10.7+/-4.2 pg/ml at 5-10 days, and remained constant throughout the first 3 months of life in conscious sheep. We conclude that ET-1 is not a renal vasoconstrictor agent in the immediate newborn period, and that the effects of ET-1 on renal vascular tone appear to be developmentally regulated.

31 Effect of different aspirin doses on late preconditioning protection against stunning in conscious sheep

31 Effect of different aspirin doses on late preconditioning protection against stunning in conscious sheep

29 The cyclooxygenase pathway is not involved in late preconditioning protection against arrhythmias in conscious sheep

29 The cyclooxygenase pathway is not involved in late preconditioning protection against arrhythmias in conscious sheep

35 A possible role for the MDR-1 gene-encoded P-glycoprotein (P-170) during post-ischemic myocardial stunning in conscious sheep

35 A possible role for the MDR-1 gene-encoded P-glycoprotein (P-170) during post-ischemic myocardial stunning in conscious sheep

20 Sarcolemmal but not mitochondrial KATP channels account for mechanical recovery from stunning in conscious sheep

20 Sarcolemmal but not mitochondrial KATP channels account for mechanical recovery from stunning in conscious sheep

32 Role of nitroglycerine in late preconditioning against arrhythmias and stunning in conscious sheep

32 Role of nitroglycerine in late preconditioning against arrhythmias and stunning in conscious sheep

30 Late but not early ischemic preconditioning protects against reperfusion-triggered arrhythmias in conscious diabetic sheep

30 Late but not early ischemic preconditioning protects against reperfusion-triggered arrhythmias in conscious diabetic sheep

Differential responses to systemic and local angiotensin II infusions in conscious postnatal sheep.

Angiotensin II (ANG II) increases blood pressure (MAP) via specific ANG II receptors (AT) and is considered important in regulating MAP after birth. In adult animals, AT(1) receptors predominate in vascular smooth muscle (VSM) and mediate vasoconstriction. In newborn sheep, AT(2) receptors, which do not mediate vasoconstriction, predominate in vascular smooth muscle until 2 wk postnatal when they are replaced by AT(1). Thus, the mechanisms whereby ANG II increases MAP after birth are unclear. We examined the effects of ANG II on femoral vascular resistance (FmVR) and blood flow (FmBF) in serial studies of newborn sheep (n = 7) at 7-14 d, 15-21 d, and 22-35 d. Animals had femoral catheters implanted for systemic ANG II infusions and cardiovascular monitoring, and a flow probe was implanted on the contralateral artery proximal to the superficial saphenous artery, which contained a catheter for intra-arterial ANG II infusions. Studies were performed using a range of systemic and intra-arterial ANG II doses. Systemic ANG II increased MAP dose-dependently at all ages (p < 0.001); however, responses were not age dependent. FmBF rose dose dependently at 7-14 d (p < 0.001) and was unchanged at older ages. FmVR was unaffected at 7-14 d, but values increased dose dependently at 15-21 d and 22-3 5d (p < 0.001), although never exceeded relative increases in MAP. Local ANG II did not alter MAP, FmBF, or FmVR at any age. Although systemic ANG II increases MAP and FmVR dose dependently after birth, ANG II-induced vasoconstriction is attenuated. Furthermore, intra-arterial ANG II does not alter FmVR in the absence of systemic responses, suggesting incomplete vascular smooth muscle AT(1) expression, stimulation of local ANG II antagonists, or ANG II-mediated release of another vasoconstrictor.

Differential Responses to Systemic and Local Angiotensin II Infusions in Conscious Postnatal Sheep

Angiotensin II (ANG II) increases blood pressure (MAP) via specific ANG II receptors (AT) and is considered important in regulating MAP after birth. In adult animals, AT1 receptors predominate in vascular smooth muscle (VSM) and mediate vasoconstriction. In newborn sheep, AT2 receptors, which do not mediate vasoconstriction, predominate in vascular smooth muscle until 2 wk postnatal when they are replaced by AT1. Thus, the mechanisms whereby ANG II increases MAP after birth are unclear. We examined the effects of ANG II on femoral vascular resistance (FmVR) and blood flow (FmBF) in serial studies of newborn sheep (n = 7) at 7–14 d, 15–21 d, and 22–35 d. Animals had femoral catheters implanted for systemic ANG II infusions and cardiovascular monitoring, and a flow probe was implanted on the contralateral artery proximal to the superficial saphenous artery, which contained a catheter for intra-arterial ANG II infusions. Studies were performed using a range of systemic and intra-arterial ANG II doses. Systemic ANG II increased MAP dose-dependently at all ages (p < 0.001); however, responses were not age dependent. FmBF rose dose dependently at 7–14 d (p < 0.001) and was unchanged at older ages. FmVR was unaffected at 7–14 d, but values increased dose dependently at 15–21 d and 22–3 5d (p < 0.001), although never exceeded relative increases in MAP. Local ANG II did not alter MAP, FmBF, or FmVR at any age. Although systemic ANG II increases MAP and FmVR dose dependently after birth, ANG II-induced vasoconstriction is attenuated. Furthermore, intra-arterial ANG II does not alter FmVR in the absence of systemic responses, suggesting incomplete vascular smooth muscle AT1 expression, stimulation of local ANG II antagonists, or ANG II-mediated release of another vasoconstrictor.

Hormonal interactions within the hypothalamus and pituitary with respect to stress and reproduction in sheep

Endocrine systems may be used as indicators of stress in two ways. The primary role of a hormone may be as part of the homeostatic response to a stimulus (e.g., adrenaline, corticosteroids). The amplitude of hormone response may correlate with the severity of the stimulus and any change indicate that the body is responding. Alternatively, a hormone may have a key role in normal body function (e.g., reproduction) and stress may deleteriously alter the hormone signal prevent normal function. This demonstrates that the stimulus was sufficiently severe that homeostatic mechanisms were unable to maintain normal function. Stress may effect reproduction by reducing both LH pulse amplitude and frequency. The LH surge may also be delayed. Several mechanisms may account for these effects both at the hypothalamus and pituitary. Corticosteroids have a broad, yet fundamental, role in homeostasis and have been used as primary indicators of stress for many years. Excess corticosteroid can be detrimental so the concentration is controlled via the hypothalamus–pituitary–adrenal (HPA) axis by multi-level feedback mechanisms. Under field and experimental conditions, after an initial large response prolonged stimulation leads to a gradually reducing plasma corticosteroid concentrations. This has been interpreted as a reduction in perceived stimulus severity or habituation to the stimulus and the animal deemed “less stressed” and its welfare “better.” However, this reduction may be due to the intrinsic control mechanisms designed to prevent prolonged increases in corticosteroid concentrations. The stress signal at higher brain levels may still be present and the animal may still be experiencing the stimulus as aversive. Thus, the welfare interpretation of a corticosteroid concentration may differ during the time course of a stress response. A greater understanding of the mechanisms controlling corticosteroid secretion at each level of the HPA is required to determine what is the correct interpretation at any time point. To address these issues, we have used mathematical modelling to produce representations of possible control mechanisms at each level of the HPA. The starting point was to measure AVP and CRH concentrations in hypophysial portal blood and ACTH and cortisol concentrations in jugular blood in conscious sheep during 2 h road transport (a cognitive stimulus). Modelling identified the signal inputs that were most likely to explain the secretion rate of each hormone. Modelling suggested that the reduction in AVP and CRH secretion observed during transport was most likely due to a reduction in stimulus input, with a significant contribution from cortisol negative feedback only on AVP secretion. At the pituitary level, ACTH secretion was stimulated more by AVP than by CRH (ratio 2.3:1) and there was also a stimulatory effect related to cortisol concentration at the time of sampling. However, the responses to both stimuli were curtailed by cortisol negative feedback and an inhibitory effect of prior CRH concentration. These are complex effects, but the modelling does suggest that while “stress” inputs may reduce over time hormone negative feedback is a major factor reducing hormone responses. When interpreting hormone data for animal welfare purposes, it is important not to interpret a reduction in hormone concentration due to intrinsic hormone control mechanisms as a reduction due to a decrease in the stress stimulus.

Aldosterone acts on the kidney, not the brain, to cause mineralocorticoid hypertension in sheep.

To determine the extent to which mineralocortioid hypertension depends on a direct action of aldosterone on the kidney or on the brain. Studies were performed in conscious sheep that were previously uninephrectomized, implanted with silastic cannulae in the renal artery of the remaining kidney, and had guide tubes implanted over the lateral cerebral ventricles. The effect of aldosterone, infused either intrarenally (i.r.; 2 microg/h) or intravenously (i.v.; 2 and 10 microg/h) for 10 days (n = 5), on arterial pressure and fluid and electrolyte balance was determined. The i.r. (2 microg/h) and i.v. (10 microg/h) doses were calculated to give similar intrarenal concentrations of aldosterone. In a further study, the effect of intracerebroventricular (i.c.v.) infusion of aldosterone (2 microg/h for 21 days) on arterial pressure was examined (n = 5). Infusion of aldosterone caused a progressive increase in mean arterial pressure from 83 +/- 3 mmHg to a maximum of 100 +/- 4 mmHg (P < 0.001) with 2 microg/h i.r. and from 84 +/- 3 mmHg to a maximum of 104 +/- 4 mmHg (P < 0.001) with 10 microg/h i.v., both by day 7. With both infusions there were similar increases in plasma [Na+] and decreases in plasma [K+] and total protein concentration (P < 0.05) between days 3 and 5; these were maintained throughout the infusion. There were no significant changes with i.v. aldosterone (2 microg/h). Long-term i.c.v. infusion of aldosterone (2 microg/h for 21 days) caused no change in arterial pressure. In conscious sheep the hypertensive response to aldosterone resulted from an action on the kidney, with no evidence for a direct action on the brain.

Adrenomedullin modulates the neurohumoral response to acute volume loading in normal conscious sheep.

The physiological role of adrenomedullin (ADM) in volume and pressure homeostasis remains unclear. Accordingly, we assessed possible modulatory actions of ADM infusions on the neurohumoral response to acute volume loading with dextran in normal conscious sheep. Dextran (15 ml/kg), given with concurrent ADM (5.5 pmol/kg per min--raising plasma ADM from below detection to approximately 10 pmol/l) or vehicle control infusions, induced matched significant (P<0.001 by ANOVA) falls in hematocrit (27-30%) during both ADM and control and similar increases in right atrial pressure (approximately 10 mmHg). Compared with control, both systemic (P=0.033) and pulmonary (P=0.005) arterial pressure and peripheral resistance (P=0.004) were reduced during ADM but were raised post-infusion. The dextran-induced increase in cardiac output was augmented by ADM (P=0.048). Dextran-induced increases in plasma atrial natriuretic peptide (ANP; P=0.008), brain natriuretic peptide (BNP; P=NS) and cyclic guanosine monophosphate (cGMP; P=0.003) were augmented post-ADM infusions. The dextran-induced fall in plasma renin activity (PRA) was attenuated by ADM (P=0.039) whereas plasma aldosterone levels were unaltered. ADM augmented the increase in urinary volume during the second 2-h clearance period post-dextran. Our data indicate that ADM modifies the hemodynamic and hormonal response to an acute volume challenge, enhances natriuretic peptide secretion and reduces systemic vascular resistance. These results provide further evidence that ADM plays a physiological role in volume and pressure homeostasis.

Volume Kinetic Analysis of the Distribution of 0.9% Saline in Conscious versus Isoflurane-anesthetized Sheep

The distribution and elimination of 0.9% saline given by intravenous infusion has not been compared between the conscious state and during inhalational anesthesia. Six adult sheep received an intravenous infusion of 25 ml/kg of 0.9% saline over 20 min in the conscious state and also during isoflurane anesthesia and mechanical ventilation. The distribution and elimination of infused fluid were studied by volume kinetics based on serial analysis of hemoglobin dilution in arterial blood and by mass balance that incorporated volume calculations derived from volume kinetic analysis and measurements of urinary volumes. The mass balance calculations indicated only minor differences in the time course of plasma volume expansion between the conscious and anesthetized states. However, isoflurane anesthesia markedly reduced urinary volume (median, 9 vs. 863 ml; P < 0.03). In conscious sheep, the central and peripheral volume expansion predicted by volume kinetics agreed well with the calculations based on mass balance. However, during isoflurane anesthesia and mechanical ventilation, calculation using volume kinetic analysis of the variable kr, an elimination factor that, in conscious humans and sheep, is closely related to urinary excretion, represented both urinary excretion and peripheral accumulation of fluid. This suggests that the previous assumption that kr approximates urinary excretion of infused fluid requires modification, i.e., kr simply reflects net fluid movement out of plasma. In both conscious and anesthetized, mechanically ventilated sheep, infusion of 0.9% saline resulted in minimal expansion of plasma volume over a 3-h interval. In conscious sheep, infused 0.9% saline was rapidly eliminated from the plasma volume by urinary excretion; in contrast, the combination of isoflurane anesthesia and mechanical ventilation reduced urinary excretion and promoted peripheral accumulation of fluid.

ATP-sensitive potassium channels do not have a main role in mediating late preconditioning protection against arrhythmias and stunning in conscious sheep.

Although late preconditioning protects against stunning following several short periods of ischemia-reperfusion, it is not clear if it confers protection against stunning and malignant arrhythmias after a sustained reversible ischemia, and whether KATP channels are involved as triggers and/or end effectors of the protective mechanism. The purpose of this work was thus to test these issues in conscious sheep. Five groups were considered: CONT (control): the animals were submitted to 12 min ischemia followed by 2 h reperfusion; SWOP (late preconditioning): on the first day, the animals were preconditioned with 6 periods of 5 min ischemia 5 min reperfusion and 24 h later they were submitted to 12 min ischemia followed by 2 h reperfusion; GLIB: same as CONT with the KATP channel inhibitor glibenclamide (0.4 mg/kg) infused 30 min prior to the 12 min ischemia; SWOPG2: same as SWOP with glibenclamide before the 12 min ischemia; SWOPG1: same as SWOP with glibenclamide prior to the preconditioning stimulus. Percent reperfusion recovery of wall thickening fraction (% WTh) showed late preconditioning protection against stunning throughout reperfusion (SWOP vs CONT, p < 0.01). Arrhythmia severity index (ASI) also demonstrated that late preconditioning protects against malignant arrhythmias at the onset of reperfusion (CONT: 4.87 +/- 1.62 vs SWOP: 1.39 +/- 0.93, p < 0.01). Glibenclamide was unable to prevent preconditioning, both against stunning and arrhythmia incidence, when administered either before the preconditioning stimulus (SWOPG1 vs CONT, p < 0.01) or before the sustained ischemia (SWOPG2 vs GLI, p < 0.01). Results indicate that late preconditioning protects against stunning and arrhythmias following a reversible, sustained ischemia in conscious sheep and that KATP channel participation is negligible as triggers and end effectors of both types of protection.