Unanaesthetized Sheep Research Articles

Effects of birth on baroreceptor-mediated changes in heart rate variability in lambs and fetal sheep.

1. In adult unanaesthetized sheep, there is a V-shaped relationship between mean arterial pressure (MAP) and heart rate variability (HRV), measured in both time and frequency domains. In contrast, in fetal sheep, there is only a positive direct relationship between MAP and HRV, which is determined by the cardiac vagus. We postulated that by the time lambs were 8-10 days old, the 'adult like' V-shaped relationship between MAP and HRV would be present and it may appear at or after birth. To test these hypotheses, experiments were performed in six chronically catheterized fetal sheep (aged 132-138 days gestation), 10 newborn sheep (within 10 h of birth) and 10 lambs (aged 8-10 days). The relationships between MAP and HRV (in both time and frequency domains) were studied before and during beta-adrenoreceptor blockade with propranolol and before and during cardiac vagal blockade with atropine. 2. In 8-10-day-old lambs, V-shaped relationships between MAP and HRV (measured in both time and frequency domain) were obtained. The negative limb of this V-shaped relationship between MAP and HRV was present after cardiac vagal blockade. The positive slope of the V was present after beta-adrenoreceptor blockade. 3. In 4-h-old newborn sheep, there was no relationship between MAP and HRV (measured in the time domain), but between 7 and 10 h of age a negative relationship was found during treatment with atropine and a positive relationship was found during beta-adrenoreceptor blockade, when HRV was measured in both time and frequency domains. 4. As described previously, there was only a positive relationship between MAP and HRV in fetal sheep, which was abolished by atropine but not affected by beta-adrenoreceptor blockade. 5. Thus, until relatively late in fetal life, baroreceptor- modulated changes in efferent cardiac sympathetic tone, determined by measuring the effects of autonomic blockade on HRV, could not be elicited, although reflex vagal pathways were active. By 7-10 h after birth, baroreceptor-modulated changes in efferent cardiac sympathetic tone were present. It was possible using measurements of HRV made in the time domain to show that these baroreceptor-modulated cardiac sympathetic effects became stronger over the first 10 days of life (P < 0.01). These studies are the first to show that the influence of baroreceptor-mediated changes in cardiac sympathetic tone on HRV increases in early life. This is probably because maturation of sympathetic innervation of the fetal sheep heart is occurring at this age.

Lymph Flow Pattern in the Intact Thoracic Duct in Sheep

1. To study the lymph flow dynamics in the intact thoracic duct, we applied an ultrasound transit-time flow probe in seven anaesthetized and four unanaesthetized adult sheep (approximately 60 kg). In unanaesthetized non-fasting animals we found that lymph flow in the thoracic duct was always regular pulsatile (pulsation frequency, 5.2 +/- 0.8 min-1) with no relation to heart or respiratory activity. At baseline the peak level of the thoracic duct pulse flow was 11.6-20.7 ml min-1 with a nadir of 0-3.6 ml min-1. Mean lymph flow was 5.4 +/- 3.1 ml min-1. The flow pattern of lymph in the thoracic duct was essentially the same in the anaesthetized animals. 2. In both the anaesthetized and unanaesthetized animals, the lymph flow response to a stepwise increase in the outflow venous pressure showed interindividual variation. Some were sensitive to any increase in outflow venous pressure, but others were resistant in that lymph flow did not decrease until outflow venous pressure was increased to higher levels. This resistance was also observed in the high lymph flow condition produced by fluid infusion in the anaesthetized animal and mechanical constriction of the caudal vena cava in the unaesthetized animals. Pulsation frequency of the thoracic duct flow initially increased and then decreased with a stepwise increase in the outflow venous pressure. This initial increase might be a compensatory response to maintain lymph flow against elevated outflow venous pressure. 3. To test the effect of long-term outflow venous pressure elevation in unanaesthetized sheep, outflow venous pressure was increased by inflation of a cuff around the cranial vena cava for 1, 5 or 25 h. The cuff was inflated to a level where lymph flow was reduced. Lymph flow remained low or decreased further during the entire cuff-inflation period. We calculated the lymph debt caused by the outflow venous pressure elevation and the amount 'repaid' when venous pressure returned to normal. Lymph debt for 25 h was 6400 ml but only 200 ml was repaid. Since we observed no visible oedema formation in the lower body of the sheep, the non-colloidal components of the lymph must have been reabsorbed into the bloodstream, most likely in the lymph nodes.

GABAergic and glutamatergic effects on behaviour in fetal sheep.

1. Studies were carried out in unanaesthetized fetal sheep at 125-135 days gestation to investigate neurotransmitters involved in behavioural state. 2. Catheters and electrodes were chronically placed to record tracheal and arterial pressure, electrocortical activity (ECoG), nuchal muscle activity and to instill drugs into the cerebrospinal fluid (CSF) of the fourth ventricle. 3. Administration of the N-methyl-D-aspartate receptor antagonists DL-2-amino-5-phosphopentanoic acid (AP5) or (+)-5-methyl-10,11-dihydro-5H-dibenzol[a,d]cyclohepten-5,10- iminemoleate (MK-801) increased the incidence of fetal behaviour characterized by low voltage ECoG, nuchal muscle activity and an increase in mean arterial blood pressure from 4.1 +/- 6 to 60.6 +/- 6.2% (mean +/- S.E.M.) (AP5; P = 0.003) and from 7.6 +/- 3.6 to 50.8 +/- 7.0% (MK-801; P = 0.004; values are expressed as the percentage of each 60 min period in which the state was present). 4. The incidence of fetal breathing during periods of low voltage (LV)-ECoG and nuchal muscle activity was 83.1 +/- 5.6%. The incidence of fetal breathing during LV-ECoG associated with nuchal muscle atonia was 63.1 +/- 5.0% before AP5 or MK-801 and 64.4 +/- 9.8% after instillation of these drugs. The amplitude of fetal breaths increased from 4.0 +/- 0.3 mmHg in low voltage ECoG periods to 6.7 +/- 0.8 mmHg (P = 0.006) during periods of low voltage with nuchal muscle activity. There was no significant change in breath timing during these periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of central adenosine on brainstem blood flow in fetal sheep.

The effect of the adenosine analogue R-N6-(phenylisopropyl)adenosine (R-PIA) on blood flow to the medulla and pons was examined in unanaesthetized fetal sheep. Microspheres labelled with isotopes were used to determine blood flow before and after instillation of 0.2 or 0.5 microgram R-PIA into the cerebrospinal fluid of the fourth ventricle. Blood flow to the medulla, which had a mean value (+/- S.E.M.) of 285 +/- 41 ml min-1 (100 g)-1 during the control period, was not changed by the central instillation of R-PIA. Blood flow to the pons was also not affected. These data indicate that central adenosine, which depresses respiratory drive in fetal sheep, acts by mechanisms independent of removal of carbon dioxide from the brainstem.

Time Course of Systemic and Renal Plasma Prostanoid Concentrations and Renal Function in Ovine Hyperdynamic Sepsis

1. We continuously recorded systemic and renal haemodynamic changes, and arterial, renal venous and urinary concentrations of thromboxane B2, 6-keto-prostaglandin F1 alpha and prostaglandin E2, and determined their relationship to renal function in an ovine model of progressive hyperdynamic sepsis. 2. Nine chronically instrumented unanaesthetized sheep were given a continuous intravenous infusion of Escherichia coli endotoxin (20 ng min-1 kg-1) for 3 days. 3. Within the first 12 h of infusion, endotoxin induced a major hypotensive septic syndrome, including a persistent 30% reduction in mean arterial pressure, a 50% decrease in systemic vascular resistance and a 50% increase in mean pulmonary artery pressure, associated with severe lactacidaemia. 4. Renal blood flow decreased by 40%, and creatinine clearance, urine flow, and fractional sodium excretion decreased by more than 75%, of baseline values. After 12 h of endotoxin infusion, cardiac output increased two-fold and renal blood flow recovered to baseline values, whereas creatinine clearance remained depressed. Four sheep died between 13 and 22 h of endotoxaemia; these animals (allocated to group 1) presented a significantly and persistently more reduced renal blood flow (-23%) and creatinine clearance (-77%) after 4 h than the remaining five sheep (allocated to group 2), which survived more than 36 h (-16% and -21%, respectively), whereas systemic and pulmonary haemodynamic and gas exchange data remained similar in both groups. 5. The more pronounced decreases in renal blood flow, creatinine clearance and urine flow in group 1 were associated with higher plasma renin activity and plasma 6-keto-prostaglandin F1 alpha concentrations and a lower fractional urinary excretion of 6-keto-prostaglandin F1 alpha than in group 2, whereas plasma thromboxane B2 concentrations were similarly increased in both groups. Plasma prostaglandin E2 concentrations and urinary excretion were not notably affected by endotoxin infusion in either group. 6. Our results are not in favour of a significant renal production of any of these three prostanoids during endotoxaemia. In both groups, values of creatinine clearance were linearly correlated with simultaneous mean arterial pressure values after starting endotoxin infusion (group 1: creatinine clearance = 1.99 x mean arterial pressure--105, r = 0.95; group 2: creatinine clearance = 2.06 x mean arterial pressure--104, r = 0.80).(ABSTRACT TRUNCATED AT 400 WORDS)

Control of Lymphatic Pumping: Interest of Daflon 500 mg

Objective: To determine the effects of Daflon 500 mg on lymph flow, known to be modulated by lymph formation and contractility of the collecting ducts. Experimental design: The effect of Daflon 500 mg was investigated in vitro on isolated rings of sheep mesenteric lymphatics, at concentrations of 10-7 M and higher. In vivo, lymph flow was measured in unanaesthetized sheep before and after 5 days of administration of Daflon 500 mg at the oral dose of 10 mg/kg/day. Results: Daflon 500 mg significantly increased the frequency of spontaneous contractions in vitro, and in vivo increased the lymph flow when compared with pre-drug levels. Conclusion: It is suggested that Daflon 500 mg increases lymph flow under these conditions by stimulating the lymph pump directly, a result that is consistent with previous studies.

Autonomic modulation of insulin levels in foetal sheep

The autonomic nervous system plays an important part in metabolic and circulatory adaptation of the foetus to changes in its intrauterine environment and after delivery. Foetal and neonatal glucose metabolism and insulin secretion are influenced by changes in humoral catecholamine levels as they may occur during asphyxia. To assess the role of neuronal and humoral sympathetic activity in foetal endocrine pancreatic regulation, chronically catheterized foetal sheep near term were chemically sympathectomized with 6-hydroxydopamine. Experiments were carried out in unanaesthetized foetal sheep in utero in the absence of uterine contractions. Insulin and glucose levels, blood gases, acid-base status and catecholamines were measured before, during and after a 2 min occlusion of uterine blood flow caused by mechanical constriction of the maternal aorta. Pancreatic blood flow was determined using radioactive labelled microspheres. During normoxaemia, insulin levels, pancreatic blood flow and glucose transport to the organ in sympathectomized foetuses were elevated compared with intact animals, whereas glucose concentrations did not show any significant differences. After the onset of asphyxia humoral catecholamine levels rose significantly in both groups. Insulin concentrations in the plasma of both intact and sympathectomized foetuses were no longer different indicating both indirect (blood flow and humoral catecholamine related) and direct (neurally mediated) sympathetic effects on pancreatic beta cells.

Fetal breathing, sleep state and cardiovascular adaptations to anaemia in sheep.

1. In unanaesthetized fetal sheep (greater than 0.8 term) prolonged anaemia initially reduced the incidences of low-voltage electrocortical activity, rapid eye movements and breathing activity; but the incidence of each returned to normal within 4-7 h. 2. Anaemia induced a persistent rise in fetal heart rate and plasma concentrations of adrenaline, noradrenaline and cortisol. 3. After 16 h the fetal haematocrit was returned to normal. Isocapnic hypoxia induced less than 1 h later also inhibited eye and breathing activity. 4. After 1 h fetal arterial PO2 (Pa,O2) was returned to normal. This rise in O2 tension was associated with an elevation in the incidence of low-voltage electrocortical activity, eye movements and breathing. Breathing movements also occurred during high-voltage electrocortical activity. 5. It is concluded that the brain PO2 set-point for hypoxic inhibition adapts rapidly to alterations in O2-carrying capacity and is probably due to changes in the concentration and/or receptor affinity of a central neuromodulator. Secondly, a rise in brain PO2 at birth may contribute to the onset of continuous breathing.

Haemodynamic and humoral responses to repeated hypotensive haemorrhage in conscious sheep

Haemodynamic and humoral responses to two subsequent hypotensive haemorrhages, separated by 3 hours and each followed by retransfusion, were studied in unanaesthetized sheep. Haemorrhage was induced by removal of blood from a jugular vein at a rate of 0.7 ml kg-1 min-1 until the mean systemic arterial pressure suddenly decreased by 35 mmHg or more. In addition to the mean systemic arterial pressure, the cardiac output, the mean pulmonary arterial pressure, the central venous pressure and the pulmonary capillary wedge pressure decreased in response to each haemorrhage. The recovery of the systemic and pulmonary arterial pressure was slower and/or less efficient after the second haemorrhage, due to a less pronounced increase of the vascular resistance. Relative bradycardia, in association with the abrupt fall of the mean systemic arterial pressure, was more apparent during the first haemorrhage. The plasma levels of vasopressin, renin activity and angiotensin II were increased by each blood removal, but the vasopressin response to the second haemorrhage was significantly reduced. The plasma noradrenaline concentration was slightly and transiently elevated only in response to the second haemorrhage. The concentration of neuropeptide Y-like immunoreactivity in plasma was unaffected by both haemorrhages. It is suggested that the reduced and delayed increase in the systemic vascular resistance, accompanied by impaired recovery of the arterial pressure, and the relative absence of 'bleeding bradycardia', during the second haemorrhage, were due to the diminished vasopressin response.

Influence of the plasma protein concentration on renal function

1. The effect of the plasma protein concentration on renal function remains controversial. Most, but not all, experimental studies suggest that a reduced plasma protein concentration perfusing the kidney may reduce tubular sodium reabsorption. Hypoproteinaemic disease states are usually associated with sodium retention, which is not always volume-dependent. 2. We induced a 21% and 24% reduction in plasma total protein and plasma albumin, respectively, in unanaesthetized sheep by acute extracorporeal plasmapheresis. Arterial pressure did not change, and changes in circulatory volume were minimised by infusion of crystalloid to maintain pulmonary artery occlusion pressure, measured using a Swann-Ganz pulmonary artery catheter. 3. After plasmapheresis, there was no significant change in creatinine clearance, sodium excretion, plasma renin activity or urinary kallikrein excretion. 4. After plasmapheresis, there was no significant reduction in plasma osmolality, increase in urine osmolality and fall in free water clearance. 5. The results suggest that in the absence of detectable changes in circulating volume or arterial pressure, acute hypoproteinaemia is associated with significant changes in renal water handling, but has no direct effect on sodium excretion or on renal release of renin and kallikrein.

Acute effect of a thromboxane synthetase inhibitor on renal function in unanaesthetized sheep.

1. Eleven healthy, unanaesthetized sheep were given either a single intravenous bolus infusion of U63,577A (Upjohn), a selective thromboxane synthetase inhibitor, at a dose of 30 mg/kg (group 1, n = 6), or vehicle alone (group 2, n = 5). Animals were maintained in metabolic cages during the study, and received 150 ml of water/h and 7.5 mmol of sodium/h as Ringers lactate by intravenous infusion for 24 h before and during the study. During two 1 h control urine collections via bladder catheter, urine volume and sodium excretion closely paralleled these infusion rates. 2. In the first hour after injection of U63,577A, there were significant two- to three-fold increases in urine volume, urinary sodium excretion and fractional sodium excretion, compared with the control collections. During the subsequent 4 h, urine volume, urinary sodium excretion, and fractional sodium excretion fell to values significantly lower than in the control period. Creatinine clearance was reduced 1, 2 and 4 h post injection and returned to control values at 5 h. Urinary excretion of thromboxane B2 was significantly reduced compared with control values during the 5 h after injection of U63,577A. Excretion of 6-keto-prostaglandin F1 alpha did not change. Plasma renin activity was significantly increased 1, 3 and 5 h after injection of U63,577A. Vehicle controls showed no change in any of the above parameters. 3. The results indicate that in healthy conscious sheep, sodium and water replete, U63,577A has a transient but significant diuretic and natriuretic effect, followed by sodium and water retention and increased plasma renin activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects and interactions of angiotensin and vasopressin on the heart of unanaesthetized sheep.

The effects of infusions of angiotensin and vasopressin, in stepwise concentrations, on the cardiac baroreflex and on cardiac output were studied in seven adult unanaesthetized sheep and compared with those obtained with infusions of phenylephrine. Six animals were treated with the beta-adrenoceptor blocking drug, propranolol (in order to inhibit the effects of the sympathetic nervous system on the heart). One animal was not treated with propranolol. In those animals in which arterial pressure increased during infusion of vasopressin, the slope of the systolic pressure-pulse interval relation was greater than that seen when phenylephrine was used to increase arterial pressure. Compared with the cardiac response to pressor doses of phenylephrine, infusions of angiotensin were associated with a lesser degree of cardiac slowing and a lesser reduction in cardiac output. The effects of combined infusions of angiotensin and vasopressin on the cardiac baroreflex were studied. In five sheep which were infused with a pressor dose of angiotensin (1.1 microgram/min), the stimulatory effect of vasopressin (1.0 u./min) on pulse interval and its depressant effect on cardiac output were attenuated. In seven sheep infused with 0.5 u./min of vasopressin, I.V. infusion of angiotensin (0.2-5.0 micrograms), produced a progressive decrease in pulse interval and increase in cardiac output as the dose was increased. Therefore, angiotensin can offset the cardioinhibitory effects of vasopressin. Since cardiac sympathetic activity was blocked and neither drug has any direct chronotropic effect on the heart, it would appear that these interactions between the two drugs affect the cardiac vagus either at a peripheral or central level.

Potentiation of cardiac vagal action by cold.

The effect of cold on vagal action at the heart was studied in sheep, dogs and an isolated guinea pig atrial preparation. During cardiac output measurements in unanaesthetized sheep, by the thermodilution method, bradycardia was evoked on injection of cold indicator in eight of 12 sheep studied. This bradycardia was consistently evoked when blood pressure was increased, but not at normal blood pressure levels. In the guinea pig atrial preparation, which has one vagus nerve attached, bradycardia was evoked by electrical stimulation of the vagus nerve. When the preparation was cooled this bradycardia was potentiated. In anaesthetized dogs, the cut peripheral end of one vagus was stimulated electrically at different frequencies. The linear relationship between pulse interval and vagal frequency was then compared at deep body temperatures of 35, 37, 39 and 41 degrees C. This comparison showed that the vagus prolonged pulse interval more effectively when the animal was cool (35 degrees C) than when it was warm (41 degrees C).

Excitation of the cardiac vagus by vasopressin in mammals.

In unanaesthetized sheep, the sensitivity of the baroreceptor-cardio-inhibitory reflex was greater when intravenous vasopressin was used to raise blood pressure, than when intravenous phenylephrine was used to raise blood pressure. This difference was still evident in animals in which beta-adrenergic blockade had been carried out using propranolol. In the presence of combined beta-adrenergic and muscarinic blockade, a direct negative chronotropic effect of intravenous vasopressin could not be demonstrated. It was concluded, therefore, that intravenous vasopressin enhanced cardiac vagal tone. This effect of vasopressin on efferent cardiac vagal tone was confirmed directly in anaesthetized dogs by recording from single cardiac vagal efferent fibres. Furthermore, recordings from single carotid sinus baroreceptor fibres did not demonstrate a direct action of vasopressin on the sensitivity of the baroreceptors. However, the pressor effect of vasopressin is associated with a greater increase in efferent cardiac vagal discharge than that seen when equipressor doses of phenylephrine are given, or when blood pressure is raised by a similar amount by inflation of an intra-aortic balloon. Studies in isolated guinea-pig atrial preparations and in anaesthetized rabbits and dogs, revealed no consistent peripheral action of vasopressin on the action of the vagus at the heart.

Inhibition of the vagal component of the baroreceptor-cardioinhibitory reflex by angiotensin III in dogs and sheep.

Angiotensin II inhibits baroreceptor-evoked activity in cardiac vagal motoneurones. Because angiotensin III [( des-1-Asp ]angiotensin II) is a less potent pressor agent than angiotensin II, and has been reported not to share some of the actions of angiotensin II within the central nervous system, its influence on central vagal pathways was examined here. In unanaesthetized sheep equipressor doses of angiotensins II and III similarly inhibited the baroreceptor-cardioinhibitory reflex, a reflex which is almost wholly dependent on cardiac vagal activity. In anaesthetized dogs, equipressor doses of angiotensin II and III were equally effective in inhibiting the cardiac vagal activity usually evoked by rises in arterial pressure. This was established by direct recordings of activity in single cardiac vagal efferent nerve fibres. Direct recordings from single baroreceptor nerves in anaesthetized dogs showed that the angiotensins do not depress the receptor responses to elevations in blood pressure. It is concluded that equipressor doses of angiotensin III and angiotensin II inhibit central vagal pathways to the same extent.

A method for the collection of lymph from the prescapular lymph node of unanaethetized sheep.

A method for cannulating the efferent duct of the prescapular lymph node of sheep was developed and carried out on over 100 occasions so that lymph could be collected from unanaesthetized sheep under physiological conditions. On average the lymph flowed at 9 ml/h and contained 15 X 10(6) lymphocytes/ml. The surgical procedure and relevant anatomical details are described.

The origin of immunoglobulins in intestinal secretion of sheep.

SummaryStudies were made on the origin and mechanism of transfer of IgG1, IgG2, IgA and IgM into the secretion of isolated loops of jejunum of unanaesthetized sheep. IgA was the major immunoglobulin in intestinal secretion, although considerable quantities of IgG1 and IgG2 were also present. Secretion: serum radioactivity ratios for IgG1 and IgG2 were similar, indicating that they were transferred into intestinal secretion with equal facility. Specific radioactivities of the various immunoglobulins in serum and secretion were compared and the results suggested that all the IgG2 and most of the IgG1 were derived from blood plasma, whereas only a trivial amount of the IgA was plasma‐derived. The slow equilibration of IgM, together with its relatively short biological ‘half‐life', prevented accurate assessment of plasma contribution of this immunoglobulin.

A method for recording unit neural activity in unanaesthetized sheep and goats.

Abstract A chronic preparation has been made in horned sheep and goats for recording unit activity in unanaesthetized animals. The micro-electrode manipulator is supported by means of a metal bar permanently attached to the animal's horns. A perforated lucite block, implanted into the roof of the cranium, rendered a large part of the brain accessible for recording. A brief outline is given of the advantages of sheep and goats for experimental work on brain function and behavior.

Effect of Benzetimide (R 4929) On Ruminal and Intestinal Motility in Sheep

The effects of benzetimide on ruminal and intestinal motility in unanaesthetized sheep were evaluated. Benzetimide is a particularly potent and long-acting anticholinergic agent in sheep after oral (0·1 mg/kg) and subcutaneous (0·0063—0·0250 mg/kg) administration. On a évalué les effets de la benzetimide sur la motilité du rumen et de l’intestin chez des moutons non anesthésiés. La benzetimide est un agent particulièrement actif et anticholinergique de longue durée chez les moutons après administration par voie buccale (0,1 mg/kg) et par voie sous-cutanée (0,0063-0, 0250 mg/kg). Eine Ermittlung der Wirkungen von Benzetimid auf die Aktivität des Rumens und des Darms nichtnarkotisierter Schafe. Benzetimid ist ein besonders kräftiges und lang wirkendes Anticholinergikum, wenn es Schafen entweder per os (0,1 mg pro kg Körpergewicht) oder subkutan (0,0063–0,0250 mg pro kg) gegeben wird. Se valoran los efectos de la benzetimida sobre la ruminación y la motilidad intestinal en los corderos no anestesiados. La benzetimida es un agente de larga acción anticolinérgica y especial potencia en los corderos después de su administración oral (0.1 mg/kg) y por vía subcutánea (0,0063–0,0250 mg/kg).

Oxygen transport by blood in relation to body size.

AN understanding of the mechanisms of oxygen transport in mammals requires a knowledge of the rate of oxygen consumption (which represents the quantity of oxygen transported), the arteriovenous oxygen concentration difference (which expresses the amount of oxygen delivered per unit of circulatory load), and the oxygen tension in mixed venous blood (which expresses the “activity” of oxygen in capillary blood). These factors have previously been compared in resting, unanaesthetized sheep and humans and a striking similarity of all three measurements was noted despite great species differences in blood haemoglobin concentration and oxygen affinity1.