Acta Physiol Scand 1991, 143, 55-64

ADONIS

000167729100163M

Haemodynamic and humoral responses t o repeated hypotensive haemorrhage in conscious sheep H. HJELMQVIST, J. U L L M A N " , U. G U N N A R S S O N , J. M . LUNDBERGT and M. R U N D G R E N Departments of Physiology and .I. Pharmacology, Karolinska Institute, and Department of Anaesthesiology, Karolinska Hospital, Stockholm, Sweden.

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H., ULLMAN,J., GUNNARSSON, U., LUNDBERG, J. M. & RUNDGREN, M. 1991. Haemodynamic and humoral responses to repeated hypotensive haemorrhage in conscious sheep. Acta Physiol Scand 1991, 143, 55-64. Received 11 March 1991, accepted 17 May 1991. ISSN 0001-6772. Departments of Physiology and Pharmacology, Karolinska Institute and Department of Anaesthesiology, Karolinska Hospital, Stockholm, Sweden. HJELMQVIST,

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-' min-' 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 I1 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 haernorrhage, were due to the diminished vasopressin response. Key words: haemorrhage, hypotension, vasopressin, angiotensin, renin, noradrenaline, neuropeptide Y, sheep. Haemorrhagic hypotension activates a number of cardiovascular compensatory mechanisms, all of which are aimed at providing sufficient blood flow to 'immediately' vital organs (the central nervous system and the heart) by restoring the arterial pressure towards normal and redistributing the reduced blood volume. T h e acute Correspondence : Mats Rundgren, Dept. of Physiology, Karolinska institutet, S-104 01 Stockholm, Sweden

compensatory adjustments are not only mediated via activation of autonomic reflexes, but also via increased plasma levels of hormones and other substances such as vasopressin (AVP) (Share 1988), the components of the renin-angiotensin-aldosterone system (Cameron et al. 1985), ACTH-cortisol (DeMaria et al. 1987), catecholamines (Lilly et al. 1986), and neuropeptide Y (NPY) (Rudehill et al. 1987). Many of these substances are known to mediate several of the characteristic haemodynamic and metabolic

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H. Hjelmqzist et al.

responses to blood loss. I n that respect, the paramount importance of the sympathoadrenal system, the renin-angiotensin system and AYP is well established. However, the relative contribution of each of these systems in different haemorrhagic situations, and possible interactions between them, remain unclear (Share 1988). Furthermore, the haemodynamic and ' endocrine' effects of blood loss have mostly been studied in response to a single haemorrhage, although under w-idely different conditions regarding species, anaesthesia, surgical trauma, degree and rate of blood removal, nutritional state, etc. Apart from some studies of hormonal responses to repeated blood loss (see DeMaria et a!. 1987), cardiovascular and humoral responses to closely repeated haemorrhages (performed after full or partial substitution of the shed blood) have apparently received relatively little attention. T h i s experimental approach may correspond to common clinical situations where intermittent bleeding occurs concomitant with blood and/or fluid replacement therapy. We have previously observed that the AVP response to a hypotensive haemorrhage, repeated 3 h after a corresponding blood withdrawal (followed by retransfusion), is markedly reduced and accompanied by impaired recovery of the mean systemic arterial blood pressure (MS.L\P) Uonasson et a!. 1989). This led us to further investigate the haemodynamic effects and the release of vasoactive substances in response to repeated hypotensive haemorrhage in conscious sheep. Parts of this study have been presented at the XXXI International Congress of Physiological Sciences in Helsinki, July 1989 (Hjelmqvist et a / . 1989, Rundgren et a / . 1989).

X1I.ATERIALS A N D METHODS .4rzimals und surgical preparatcon. Twelve adult Texel cross hred ewes (bodj- wt 5.63 k2.9 kg; range: 43-72 kg) were used. The animals were housed in metabolism cages which permitted free access to water and they were fed hay and commercial grain mix (100 g with 4 g NaCl added) daily at 16.00 h. At least 4 weeks prior to experimentation the animals had their carotid arteries exteriorized into bilateral cervical skin loops while under general anaesthesia (sodium thiopental intravenously, 10 mg kg-', and maintenance with a gaseous mixture of O,, N,O and 2-300 enflurane (Efrane, Abbott, Campoverde, Italy) via an endotracheal tube). Bensylpenicillinprocain (20000 IE kg ') and dihydrostreptomycin (0.025 g kg-') (Strep-

tocillin yet, Novo, Denmark) were given intramuscularl!- on the day of surgery and for the following 4 days. Experiniental procedure. The experiments commenced at about 10.00 h and they were performed with the animals standing in their habitual environment with no extra restraint during the haemorrhages. At least 90 min prior to the experiments intravascular cannulations and catheterizations were made. A jugular cannula (0.d. 1.6 mm) was used for blood withdrawal and retransfusion. After local anaesthesia, a flow-directed thermodilution catheter (Swan-Ganz, Edwards Lab., Santa Ana, U.S.A.) was introduced via the contralateral jugular vein into a pulmonary arterial branch by guidance of continuous pressure monitoring. The catheter was connected to a Gould (P23 ID) pressure transducer and the blood pressures were displayed on a Grass polygraph (model 7D). The catheter was used for monitoring the mean pulmonarl- arterial pressure (MPAP), the pulmonary capillary wedge pressure (PCWP) and the central venous pressure (CVP). It was also used for measurements of the cardiac output (CO) by thermodilution technique (injections of 10 ml of iced saline). An Edwards Laboratory cardiac output computer (model 9510-4) was used for processing of the signals from the catheter. For each determination of the CO the mean value of three subsequent measurements was used. Finally, one of the carotid arteries was supplied M-ith a cannula (0.d. 1.0 mm) connected to a similar pressure transducer as mentioned above, and the mean systemic arterial pressure (MSAP), alternatively the systolic and diastolic pressures, were displayed on the Grass polygraph. The transducers used for the different intravascular pressure recordings were positioned at heart level. The heart rate (HR) was measured by counting the pulsations from the systemic arterial blood pressure recording. Hypotensive haemorrhage was induced by withdrawing blood from a jugular vein at about 0.7 ml kg-' min-' until the MSAP suddenly dropped by 35 mmHg, or more, below pre-haemorrhage level. The blood was collected in sterile plastic bags (Travenol, Sweden) and stored at 38 "C until retransfused 1 h later. The same haemorrhage procedure, followed by retransfusion, was performed 2.5 h after the end of the first haemorrhage. Measurements of cardiovascular parameters were made in all animals at 30 and 5 min before commencement of haemorrhage, at cessation of blood removal, 30 and 60 min later, and immediately after retransfusion. In 9 of the animals the MSAP and CVP were continuously recorded during the experiment and in 8 of these animals the heart rate was measured at shorter intervals during the haemorrhages and for 30 min after the end of blood removal. Blood samples (15-30 ml) were taken from the jugular vein before haemorrhage, at a blood loss of

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Repeated haemorrhage 12.5 ml kg-l, at the end of blood removal, and 30 respectively 60 min thereafter. The blood was collected in pre-chilled tubes with heparin or EDTA added as anticoagulant. The protease inhibitor phenantroline (25 mM) and neomycine (0.2%) were also added to tubes in which blood was collected for analysis of angiotensin I1 (ANG 11). After centrifugation (3000 rpm for 10 min at 4 "C), aliquotes of plasma were collected for determination of sodium, osmolality and protein concentration, or stored at - 20 or - 70 "C for later 'hormone' analyses. Analyses. The total plasma protein concentration was determined by refractometry, osmolality by freezing point depression (Auto & Stat Om 6010 osmometer, Kagaku Co., Japan) and "a] by using an IL 343 (Instrumentation Labs, Paderno, Italy) flame photometer. The plasma concentrations of AVP and ANG I1 were measured by radioimmunoassay (RIA) after extraction of EDTA-plasma with acetone and petroleum benzine. Apart from this extraction procedure, the assays were performed as previously described (AVP, Lishajko 1983; ANG 11, Gray & Simon 1985). The sensitivity of the assays, as used in the present study, was 0.6 pmoll-' for AVP and 1.2 pmol I-' for ANG 11. A commercial RIA kit (Rianen Angiotensin I(125) RIA kit; NEN) was used for measurements of plasma renin activity (PRA). PRA values are given as picokatal per litre (pkat I-') - for transformation to traditional units (ng ml-' h-') divide by 0.212. Ethanol-extracted samples of heparin-plasma were used for RIA determination of neuropeptide Y-like immunoreactivity (NPY-LI) as previously described by Theodorsson-Norheim et al. (1985). The plasma concentration of noradrenaline (NA) was measured by cation exchange high-performance liquid chromatography with electrochemical detection (Hjemdahl et al. 1979). Calculations and statistical analysis. The systemic vascular resistance (SVR) was calculated by dividing the difference between the MSAP and the CVP (mmHg) by the CO (ml min-' kg-'). Correspondingly, the pulmonary vascular resistance (PVR) was calculated by dividing the difference between MPAP and the PCWP by the CO. Results are presented as means standard error of the means (SE). The data were analysed with one- or two-way analysis of variance (ANOVA), repeated measures design. Post hoc tests with F test or Newman-Keuls multiple-range test (NK) were used for comparisons of means within and between haemorrhage experiments. Significant differences (P< 0.05) are indicated in figures and/or text.

RESULTS Vohme balance. T h e rate of blood withdrawal

did not differ between the two haemorrhages (0.69 k 0.03 vs 0.73 & 0.03 ml kg-' min-l). T o obtain the predefined degree of hypotension, significantly (P< 0.001) more blood had to be shed during the second (22.0_+1.4 ml kg-') than during the first haemorrhage (16.9k 0.9 ml kg-'). T h e total volume of the blood samples taken in association with each haemorrhage was 150ml while about 2 0 0 m l of isotonic saline was administered during CO measurements. T h e plasma protein concentration decreased significantly in response to each haemorrhage as judged from measurements in five of the animals (Fig. 1). T h e haemodilution was only marginally affected by retransfusion. Consequently, the plasma protein concentration at commencement of the second haemorrhage was significantly lower than before the first blood removal. Both plasma osmolality and "a] remained unchanged during the course of the experiments. Cardiovascular effects. T h e MSAP did not differ significantly before the first and second haemorrhages and it decreased gradually after the loss of 5-7 ml kg-' of blood in response to each haemorrhage, as judged from continuous pressure recordings in nine of the animals (Fig. 2). However, a significant lowering of the MSAP, compared to pre-haemorrhage levels, was obtained after a smaller blood loss during the

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Fig. 1. Changes in plasma protein concentration in response to repeated hypotensive haemorrhage, spaced 3 h apart, in five euhydrated sheep. Both the first (0) and second (@) haemorrhage was followed by retransfusion one h after the end of blood removal. Measurements were made in plasma from blood samples taken before each haemorrhage (BH), a t cessation of blood removal (0), and 30 respectively 60 min later. *** P < 0.001, significance of difference from pre-haemorrhage level (ANOVA, NK). The protein concentration was significantly (P< 0.05) lower before the second compared to the first haemorrhage (not indicated in the Fig.).

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Fig. 2. Effects on mean systemic arterial pressure (MS.%P)h?- the same haemorrhage procedure as in and second haemorrhages Fig. 1 . The first (0) were separated by 3 h. Blood was removed at a constant rate until the XISAP dropped by 35 mmHg, or more, below pre-haemorrhage level (BH). The MSAP after 2.5, 7.5 and 12.5 ml kg-' of blood removal, and at the stage immediately before the acute pressure fall (BPF) are shown. The nadir of the XISAP is also illustrated. * = P < 0.05 ; **" P < 0.001 - significance of differences from pre-haemorrhage level (A44NOV,4,NK).

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second (7.5 ml kg-') than during the first (1 2.5 ml kg..') haemorrhage. Immediately before the abrupt fall of the blood pressure, the MSAP had decreased by about 10 m m H g during both haemorrhages and a nadir of about 40 m m H g was reached at the termination of each bleeding (Fig. 2 ) . As evident from Figure 3, where sl-stemic cardiovascular parameters are plotted against time, some recover!- of the MSAP was seen during the following hour, but it remained significantly lower than pre-haemorrhage levels until the blood was retransfused. T h i s recovery was somew-hat slower and less efficient after the second haemorrhage. Statistical evaluation of the MSAP, plotted at 5 min intervals, during the 60 min post-haemorrhage observation period in the animals with continuous blood pressure recordings (n = 9) revealed a significantly (P < 0.0.5, ANOVA, NK) lower MSAP after the second than after the first haemorrhage. T h e CO decreased by about 5Oolb concomitant w-ith the abrupt fall of the MSAP in both haemorrhages (Fig. 3). Some recovery of the CO was seen, but it remained significantly lowered compared to the pre-haemorrhage level until the start of retransfusion. Similar relative changes in the CO were obtained when this parameter was calculated on a body-weight basis to compensate for differences in size of the animals. Consequent to the changes in MSAP and CO, there was a

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Fig. 3. Changes in heart rate (HR), cardiac output (CO), mean systemic arterial pressure (MSAP) and systemic vascular resistance (SVR) in response to repeated haemorrhagic hypotension in conscious sheep (n = 12). Haemorrhage procedure is described in --- 0) ; second Figure 1. First haemorrhage : (0 Measurements were made haemorrhage .).-.( before haemorrhage (BH), at cessation of blood removal (O), and 30 and 60 min thereafter, and after retransfusion (PT). Asterisks at symbols represent significance of difference from pre-haemorrhage values, while asterisks between symbols show differences between the two haemorrhages. " P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA, NK). faster and more pronounced increase in the SVR in response to the first haemorrhage (Fig. 3). T h e first bleeding caused a modest tachycardia, but the HR rose significantly more during and after the second haemorrhage (Fig. 3 ) . I n

Repeated haemorrhage

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served in three animals in response to the second blood loss. When present, the lowering of the heart rate lasted for 3-5 min. A comparison of the differences between the maximal and minimal heart rates observed during the haemorrhages in these animals revealed that they differed significantly during the first (104 & 7 us 85 6 beats min-'; P < 0.02) but not during the second (114+8 us 1 0 3 f l l ; n.s.) haemorrhage experiment. The effects on the CVP and the pulmonary vascular parameters are illustrated in Figure 4. The CVP decreased significantly, and to the same degree, in response to both haemorrhages. Similarly, the MPAP and the PCWP invariably fell in response to blood withdrawal. However, both these parameters were fully restored within 30 min after the first haemorrhage, whereas a slow and incomplete recovery was observed after the second haemorrhage. All cardiovascular parameters were restored to, or slightly above, pre-haemorrhage levels by retransfusion after each haemorrhage (Figs 3 &

4).

Humoral effects

AVP. Effects on the plasma AVP concentration induced by the first and second haemorrhage were studied in all animals ( a = 12) and are shown in Figure 5. There was no obvious I I I I I difference in the AVP level before each haemBH 0 30 60 PT orrhage (2.7f0.7 U S 4.9k0.9 pmol I-'; (n.s.). Time (min) The AVP concentration was significantly inFig. 4. Changes in central venous pressure (CVP), creased at the end of each hypotensive haempulmonary capillary wedge pressure (PCWP), mean orrhage, but the response was quite variable with pulmonary arterial pressure (MPAP) and calculated increases ranging from 10 to over 1000 times the pulmonary vascular resistance (PVR) in response to basal plasma levels, in spite of similar degrees of repeated hypotensive haemorrhage (n = 12). Symbols blood pressure fall. Peak levels were usually and measure points as in Figure 3. Asterisks between obtained in the sample taken at the end of symbols represent significance of differences between haemorrhage, but in 3 of the animals somewhat, haemorrhages, while asterisks above or below symbols shoR- significance from pre-haemorrhage levels. or clearly higher, plasma AVP levels were found * P < 0.05 ; ** P < 0.01 ; *** P < 0.001 (ANOVA, 30 min after the first as well as the second haemorrhage. In accordance with our previous NK). findings (J6nasson et al. 1989) the plasma AVP concentration was significantly lower (P< 0.05; eight of the animals the HR was more frequently ANOVA, NK) immediately after the second measured, concomitant with the sudden drop in haemorrhage than after the first. MSAP and during the following 30 min. In 6 of The plasma AVP concentration was also these animals, the abrupt fall of the MSAP was measured at a blood loss of 12.5 ml kg-' (not associated with a relative bradycardia during the shown in Fig. 5). Then, in all but one animal, first haemorrhage, whereas this was only ob- only small increases in AVP levels were seen

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The effect of repeated hypotensive haemorrhage on plasma vasopressin concentration (AVP ; n = 12), plasma renin activity (PRA; n = 9) and plasma angiotensin I1 concentration (ASG 11; N = 5) in conscious sheep. Haemorrhage procedure and measure points as in Figure 1, symbols as in Figure 3. Asterisks at symbols represent degree of significance from prehaernorrhage values, while asterisks between symbols show difference between haemorrhages. * P < 0.05 ; **P < 0.01 (AYOV.4; F test and NK).

during both haemorrhages. T h e exception was one sheep in which the MSAP had started to drop already at this stage during the first haemorrhage, which was accompanied by a rise in plasma AVP to 203 pmol 1-'. Renin-angiotensin. PRA measurements were performed in 9 of the animals whereas the plasma ANG I1 concentration was measured in 5 animals. The changes in these parameters during the two consecutive haemorrhages are illustrated in Figure 5. .Also here, the levels had normalized before the second haemorrhage, and thus, did not differ from those seen before the first haemorrhage (PRA : 0.11 f0.02 i's 0.10 & 0.03 pkat 1-I; ANG I1 : 14.0 f3.0 'cs 11.7 F 2.0 pmol 1-I). The PRti increased significantly in response to both haemorrhages.

BH

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The effect of repeated hypotensive haemFig. orrhage on plasma concentrations of noradrenaline (N.1; n = 5) and neuropeptide Y-like immunoreactivity (NPY-LI; n = 5 ) . Haemorrhage procedure and measure points as in Figure 1, symbols as in Figure 3. ** P < 0.01 (difference from pre-haemorrhage level; ANOiTA,NK).

The peak PRA level occurred somewhat later after the second, than after the first blood removal, but there were no differences in the PRA response to the two haemorrhages at any time. Similarly, the plasma ANG I1 concentration was significantly increased by both haemorrhages and with no difference in the response between the two consecutive procedures. Like AVP, PRA and ANG I1 were also measured in the blood samples taken at 12.5 ml kg-' of blood loss (not shown in Fig. 5). The PRA had increased somewhat at this stage of both haemorrhages (0.19 f 0 . 0 3 and 0.18 f0.05 pkat 1-', respectively) but the increase was not statistically different from basal levels. No corresponding increase in plasma ANG I1 concentration was observed. Noradrenaline and NP Y-LI. The plasma levels of NA and NPY-LI were measured in five of the

Repeated haemorrhage animals and the effects of repeated haemorrhage on these parameters are shown in Figure 6. The NA concentration did not change in response to the first haemorrhage, whereas a significant, but transient, increase was seen at termination of the second haemorrhage. In contrast, the concentration of NPY-LI remained unchanged during both haemorrhages.

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is indirectly supported by the higher CVP (Fig.

4) and the tendency towards as increased CO

(Fig. 3) before the second haemorrhage. Thus, in spite of larger amounts of blood removed during the second haemorrhage, about the same percentage of the total blood volume may have been withdrawn during each haemorrhage. Data on the blood volume of sheep ranges from 55-65 ml kg-' body wt (Torrington et al. 1989), which means that removal of about 25-30°/, of the estimated blood volume was needed to DISCUSSION induce an abrupt fall in MSAP during haemThe haemorrhage procedure used in this study, orrhage. This degree of sensitivity to slow with blood removal to the point of an abrupt fall venous haemorrhage is in close agreement with in MSAP, was designed to assure an effective other studies in sheep (Starc & Stalcup 1987) ' volumetric ' stimulus for the AVP release in and goats (Olsson et al. 1987). both haemorrhages. This was considered of The changes in MSAP, C O and SVR in particular importance with regard to the species response to the first haemorrhage were almost used, since in small ruminants haemorrhage- identical to those observed earlier at similar rate induced AVP release does not become significant and degree of blood loss in the same species until pronounced arterial hypotension occurs (Starc & Stalcup 1987). The haemorrhage(Larsson et al. 1978). However, such a practice induced effects on the low-pressure side of the may complicate the interpretation of effects on cardiovascular system are accordant with another parameters during the two haemorrhages. ticipated consequences of reduced cardioSignificantly more blood (on average about pulmonary filling. The percentage decrease of 300 ml) had to be withdrawn to obtain the the MPAP was less than that of the MSAP, predefined degree of hypotension during the which agrees with numerous observations in second haemorrhage. During hypotension, a different models of haemorrhagic shock (see rapid transfer of interstitial fluid to the plasma Chien 1967). This more efficient maintenance of compartment is expected to occur (Mellander the MPAP has been found to be associated with 1960), and the significantly decreased plasma a drastic elevation of the PVR during severe protein concentration after each haemorrhage haemorrhagic shock in anaesthetized dogs (Fig. I) indicates that this autotransfusion (Klllay et al. 1961, Rothe & Selkurt 1964). This mechanism was operative. Apparently, the fluid was not particularly evident during moderate transfer was not facilitated by concomitant hypotensive bleeding of conscious sheep in the hyperglycaemia, as has been suggested from present study. Previous studies of repeated haemorrhage studies in anaesthetized dogs (Knott et al. 1969) and cats (Jarhult 1973), since the plasma with interexperimental intervals of 1 week (Knott osmolality remained unchanged during the et al. 1969) or 24 h (Jonasson et al. 1989) did not experiments. This is in accordance with the reveal any differences on the cardiovascular observations in the same species by Grimes et al. effects. Simulated 'haemorrhage ' in rabbits, by (1987), who also found that during the first h constriction of the thoracic inferior vena cava, following a rapid haemorrhage of 19% of the was also recently shown to be accompanied by blood volume, about one third of the removed largely unchanged haemodynamic responses volume was spontaneously recovered, partly via when repeated three times with 90 min intervals a recovery of the total plasma protein mass. With (Ludbrook et al. 1988). Furthermore, only minor regard to this, and the fact that sheep can release differences in the blood pressure response to a substantial amounts of erythrocytes from the small (10%) haemorrhage were observed in spleen (Torrington et al. 1989), it is impossible conscious dogs when bled twice with an interval to calculate fluid shifts from changes in plasma of 5 h (DeMaria et al. 1987). In the present protein concentration and haematocrit. Still, it is study, where a moderately large, hypotensive, conceivable that the animals were slightly haemorrhage was repeated with a shorter time hypervolaemic after the first retransfusion, which interval, the recovery of the MSAP was slower

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and less efficient after the second blood loss, apparently due to a delayed and impaired increase of the SVR. Largely similar differences were obtained in the pulmonary circulation, although they were not statistically significant. Also the heart rate response was affected b!the iterated haemorrhage. Blood loss causes increasing tachycardia, followed by absolute or relative bradycardia during the decompensatory phase of haemorrhage, as originally described in cats by Oberg & White (197O), and subsequently also observed in humans in association mith haemorrhagic h!-potension (Sander-Jensen 1986). It has been suggested that, here. a paradoxical increase in afferent input from ventricular vagal afferents mediates a reflex bradycardia (see Thoren 1979). T h e same eflect of haemorrhage on the heart rate was el-ident in six out of eight sheep after the first blood loss. However, only three of them showed the response during the second haemorrhage. Interestingl!-, from circumstantial evidence Sjijstrand (1973) has suggested that bleeding bradycardia might he elicited via effects of AVP on cardiovascular retlexes. In support of that idea is the presently observed reduction of both the A\.-P and bradycardia responses during the second haemorrhage. .4 fatigue of the AVP secretory response after prolonged or closely repeated hypotensive stimuli was originally observed by Sachs et al. (1967) in dogs, and confirmed by Oyama et al. (1978) in the same species 10 years later. On the basis of studies on intact dogs and isolated pituitarv glands, Sachs et u1. (1967) suggested that the reduced AVP response to repeated stimuli reflects the inability of the neurohypophj-sis to release more than a fraction (lO-2Oo0) of its stored amount of hormone within a certain time span. Hon-ever,circumstantial evidence indicates that hypersecretion of cortisol during the second haemorrhage ma)- also have contributed to the reduced ‘4VP response observed in this study. .L\dministration of the synthetic glucocorticoid methylprednisolone to anaesthetized dogs (Oyama et al. 1978), or cortisol to conscious dogs ( R a g et nl. 1990), has been found to reelice hypotension-induced AVP release. Furthermore, iterated haemorrhage potentiates the cortisol response in that species (DeMaria et al. 1987). Since cortisol was not measured in our experiments, a negative correlation between AL-P and cortisol l e d s upon repeated hypotensive haemorrhage remains unknown. In fact, the obseri-a-

tions in conscious sheep that the cortisol response is not potentiated by iterated haemorrhages, spaced 24 h apart (Smith et a/. 1988), and glucocorticoid infusion does not reduce a moderatel!- large AVP response to nitroprussideinduced hvpotension (Wood & Silbiger 1988), call such a relationship between AVP and cortisol into question in this species. However, since our haemorrhages wcre larger and more closely repeated than those of Smith et al. (1988), and that glucocorticoids apparently mainly affect the ‘maximal’ A\-P secretory response (Raff et al. 1990), further studies on the possible role of cortisol secretion for the reduced AVP response to repeated hypotensive haemorrhage in sheep are needed. The PRA and ANG I1 levels seen here (Fig. 5 ) arc similar to those observed by others in response to a slow venous haemorrhage of the same degree in sheep (Cameron e t a l . 1985, Starc & Stalcup 1987). Apparently, haemorrhage is a relatively weak stimulus for the renin-angiotensin svstem in this species, since much higher PRA and ANG I1 levels are induced by moderate sodium deficiency, when they also are kept high for several days (Blair-West et a / . 1971). An unexpended renin secretory capacity may thus have enabled a normal responsiveness to a closely renewed hypotensive blood loss. In view of the reported mutual compensatory relationship between AVP and renin-angiotensin (McNeill 1983), the reduced AVP response to our second haemorrhage could instead have been expected to be accompanied by accentuated PRA and XNG I1 responses. However, this was apparently not the case. The immediate and intense activation of the sympathoadrenal system is undoubtedly one of the major compensatory mechanisms during haemorrhagic hypotension (Chien 1967). Still, large variations in effects on plasma catecholamine levels have been obtained in response to haemorrhage, which are not readily explained by differences in rate and degree of blood loss. T h e lack of effect of the first haemorrhage on the plasma N.4 concentration (Fig. 6) is consistent with observations in other species, like goats (Olsson e t a l . 1987), rats (Darlington et a / . 1986), and cats (Bereiter & Gann 1986). However, small but significant increases in plasma NA levels have previously been reported as an effect of haemorrhage to about the same degree in conscious sheep (Cameron ef a / . 1985, Starc & Stalcup 1987). Apparently, both rate (Starc &

Repeated haemorrhage

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Stalcup 1987) and amount of blood removal, but infusion in normovolaemic animals (Rascher not necessary degree of hypotension (Darlington et al. 1983). et af. 1986), do influence the effects on plasma catecholamine levels. Therefore, rapid induction This study was supported by grants from the Swedish of severe haemorrhagic hypovolaemia usually Medical Research Council (project nos. 6553 and causes huge elevations of both plasma NA and 6554) and the Karolinska institute. We thank Fjodor adrenaline concentration (Rudehill et al. 1987). Lishajko, Alice Skogholm and Carina Soderblom for T h e second haemorrhage in the present study expert technical assistance. The study was approved caused a significant increase in the plasma NA by the Ethical Committee for Experiments in Animals. concentration, which agrees with the increased N A response to paired haemorrhage observed by Lilly et al. (1986) in dogs. T h e bleeding rate and R E F E R E N C E S degree of hypotension was similar to that during BEREITER, D.A. & GANN,D.S. 1986. Potentiation of the preceding haemorrhage, but a larger amount haemorrhage-evoked catecholamine release by prior blood loss in cats. Am 3Physiol 205, E18-E23. of blood was withdrawn, which might have been J.R., CAIN,M. D., CATT,K.J., COGHthe ultimate cause of the NA response. However, BLAIR-WEST, LAN, J. P., DENTON, D.A., FUNDER, J.W., SCOGGINS, for reasons discussed above, a more pronounced B.A. & WRIGHT,R.D. 1971. The dissociation of hypovolaemia was not necessarily induced during aldosterone secretion and systemic renin and the second haemorrhage. T h e increased plasma angiotensin I1 levels during the correction of sodium N A levels during the second haemorrhage deficiency. Acta endocrinol 66, 229-247. probably reflects a more intense activation of the CAMERON,V., ESPINER,E.A., NICHOLLS,M.G., sympathoadrenal system, which apparently was DONALD, R.A. & MACFARLANE, M.R. 1985. Stress insufficient to cause increased plasma levels of hormones in blood and cerebrospinal fluid in NPY-LI, which has been shown to be coconscious sheep : Effects of haemorrhage. Endoreleased with NA from peripheral sympathetic crinology 116, 1460-1465. neurons upon strong stimulation (Lundberg CHIEN,S. 1967. Role of the sympathetic nervous et al. 1986). This reason for the lack of effects on system in haemorrhage. Physiol Rev 47, 214-288. J. & DALLMAN, M. F. D.N., SHINSAKO, plasma NPY-LI levels is supported by recent DARLINGTON, 1986. Responses of ACTH, epinephrine, norstudies on graded exercise in man, which has the epinephrine, and cardiovascular system to haemsame basal plasma N A and NPY-LI levels at rest orrhage. Am 3 Physiol251, H612-H618. as sheep, showing that increased NPY-LI concentration does not become apparent until DEMARIA,E., LILLY,M.P. & GANN,D. S. 1987. Potentiated hormonal responses in a model of the N A levels have increased to 5-10 nM (Pernow traumatic injury. 3. Surg Res 43, 45-51. et al. 1988). GRAY,D.A. & SIMON,E. 1985. Control of plasma In summary, the reduced AVP response and angiotensin I1 in a bird with salt glands (Anas the increase in plasma NA levels after the second platyrhyncos). Gen Comp Endocrinol 60, 1-1 3. haemorrhage were the only significant differences GRIMES, J.M., Buss, L.A. & BRACE, R.A. 1987. Blood in effects of repeated hypotensive blood loss on volume restitution after haemorrhage in adult sheep. the plasma concentration of selected vasoactive Am 3 Physiol253, R541-R544. substances. T h e main haemodynamic difference HJELMQVIST, H., ULLMAN,J., LUNDBERG, J.M. & was a slower and less efficient recovery of the RUNDGREN, M. 1989. Release of vasoactive substances in response to repeated hypotensive haemmean arterial blood pressure after the second orrhage in conscious sheep. (abstract) Proc Int Uni haemorrhage, apparently due to a blunted Physiol Sci XXXI, 489. increase of the systemic vascular resistance. M. & KAHAN,T. 1979. P., DALESKOG, Further, ' bleeding bradycardia ' was more evi- HJEMDAHL, Determination of plasma catecholamines by high dent during the first haemorrhage, and the heart performance liquid chromatography with electrorate was significantly higher after the second chemical detection : comparison with a radiothan after the first bleeding. These changes are enzymatic method. Lfe Sci 25, 131-138. in accordance with those expected to occur when JONASSON, H., HJELMQVIST, H. & RUNDGREN, M. there is a, relatively seen, diminished AVP 1989. Repeated hypotension induced by nitroinfluence upon the cardiovascular system, as prusside and haemorrhage in the sheep : effects on vasopressin release and recovery of arterial blood indicated from studies with pharmacological pressure. Acta Physiol Scand 137, 427436. blockade of vascular AVP receptors during J. 1973. Osmotic fluid transfer from tissue to haemorrhage (Zerbe et al. 1982) and AVP JARHULT,

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