Sinoaortic-denervated Rabbits Research Articles

Baroreceptor Denervation Prevents Sympathoinhibition During Angiotensin II–Induced Hypertension

Arterial baroreflexes are well established to provide the basis for short-term control of arterial pressure; however, their role in long-term pressure control is more controversial. We proposed that if the sustained decrease in renal sympathetic nerve activity (RSNA) we observed previously in response to angiotensin II-induced hypertension is baroreflex mediated, then the decrease in RSNA in response to angiotensin II would not occur in sinoaortic-denervated (SAD) animals. Arterial pressure and RSNA were recorded continuously via telemetry in sham and SAD rabbits living in their home cages before, during, and after a 7-day infusion of angiotensin II (50 ng . kg(-1) . min(-1)). The arterial pressure responses in the 2 groups of rabbits were not significantly different (82+/-3 mm Hg sham versus 83+/-3 mm Hg SAD before angiotensin II infusion, and 101+/-6 mm Hg sham versus 100+/-4 mm Hg SAD day 6 of angiotensin II). In sham rabbits, there was a significant sustained decrease in RSNA (53+/-7% of baseline on day 2 and 65+/-7% on day 6 of the angiotensin II). On ceasing the angiotensin II, all variables recovered to baseline. In contrast, RSNA did not change in SAD rabbits with the angiotensin II infusion (RSNA was 98+/-8% of baseline on day 2 and 98+/-8% on day 6 of the angiotensin II infusion). These results support our hypothesis that the reduction in RSNA in response to a pressor dose of angiotensin II is dependent on an intact arterial baroreflex pathway.

Sinoaortic denervation abolishes pressure resetting for daily physical activity in rabbits.

It has been speculated that if baroafferent signals are only related to the negative feedback control of arterial pressure (AP), then physical activity would increase the range of AP fluctuation in baroafferent-denervated animals. Mean AP (MAP), heart rate (HR), and cardiac output (CO) were measured for 24 h in free-moving conscious rabbits. On the basis of hydrostatic pressure and electromyogram, MAP data taken during periods of physical activity and rest were selected from the overall 24-h MAP data and then converted into histograms. During physical activity, the mode of MAP histogram increased in intact rabbits and was unchanged in sinoaortic-denervated (SAD) rabbits. Movement increased the mode of total peripheral resistance (TPR) but did not significantly change CO in intact rabbits. Conversely in SAD rabbits, movement slightly decreased TPR and slightly increased CO. These findings indicate that arterial baroafferent signals are required to shift MAP to a higher pressure level by an increase in TPR but not in CO during a moving phase. These results suggest that baroafferent signals may not only minimize the fluctuating range of MAP through negative feedback control but also be involved in actively resetting MAP toward a higher pressure level during daily physical activity.

Effects of Epidural Ketamine on Blood Pressure and Renal Sympathetic Nerve Activity in Rabbits

The aim of this study was to evaluate the effects of epidural ketamine, an N-methyl-D-aspartic acid (NMDA) antagonist, on haemodynamic responses and renal sympathetic nerve activity (RSNA). Mean arterial blood pressure (MAP), heart rate (HR), RSNA, and integrated RSNA (IRSNA) were recorded in anaesthetized and artificially-ventilated Japanese white rabbits. Epidural ketamine (2 mg kg−1), decreased MAP and IRSNA (P < 0.05), and increased HR (P < 0.05) in intact rabbits (n = 6). Epidural MK-801 (0.024 mg kg−1), another NMDA-receptor antagonist, increased IRSNA, but did not significantly change HR or MAP (n = 6). Pretreatment with epidural naloxone (0.2 mg) did not affect the changes of MAP, HR, and IRSNA induced by ketamine (n = 4), suggesting that opiate receptors were not involved. In sino-aortic denervated rabbits (n = 6), epidural ketamine decreased MAP and IRSNA, but did not significantly change HR, indicating that the ketamine-induced increase in HR occurred via a baroreflex in the neuraxis-intact rabbits. These results indicate that the effects of epidural ketamine on MAP, HR, and RSNA are different from those of MK-801, and suggest that the decrease in sympathetic nerve outflow induced by epidural ketamine is not mediated by NMDA receptors.

Arterial baroreflex modulation of heat-induced vasodilation in the rabbit ear.

The purpose of this study was to determine whether nonthermal baroreflexes arising from cardiopulmonary and/or arterial baroreceptors modulate rabbit ear blood flow (EBF) during hyperthermia. Intact and sinoaortic-denervated (SAD) rabbits were chronically instrumented with a Doppler ultrasonic flow probe for measurement of EBF velocity (kHz). During whole body heating in conscious rabbits, EBF and ear vascular conductance (EVC) increased as core temperature increased until maximal plateau levels of EBF and EVC were reached. The maximal plateau level of EVC attained during heat stress was lower in SAD than in intact rabbits. Subsequent intrapericardial administration of procaine at maximal EBF blocked cardiac afferents but did not alter EVC in either animal group. In a second experiment, ramp decreases in mean arterial pressure were produced by vena caval occlusion at maximal EBF. In intact rabbits, EBF and EVC decreased linearly as mean arterial pressure fell, but EBF and EVC did not decrease during vena caval occlusion in SAD rabbits. Thus neither pharmacological nor mechanical unloading of cardiac baroreceptors results in reflex vasoconstriction in the heat-stressed rabbit ear. However, baroreflexes arising from arterial baroreceptors may modulate EBF in heat-stressed rabbits.

Effects of angiotensin II on myocardial contractility during short-term pressor responses to angiotensin II.

Cardiac angiotensin AT1 receptors have been found in several animal species. In-vitro studies performed on cardiac preparations have shown that angiotensin II (ANG II) exerts a positive inotropic effect; however, in-vivo results have allowed no definitive conclusion to be drawn. The reasons behind these controversial results remain unknown, and could originate both from different experimental conditions and from the techniques used to assess myocardial contractility. To investigate, by means of echocardiographic measurements, whether ANG II, administered to intact and to sinoaortic denervated isoflurane-anesthetized rabbits, was able to directly increase myocardial contractility. The effect of ANG II on cardiac contractility was assessed with the use of simultaneous pressure measurements and Doppler-echocardiographic recordings. Specifically, we used both the relationship between the left ventricular end-systolic wall stress and the velocity of heart-rate-corrected circumferential fiber shortening (VCFC) and the maximum rate of rise of the ventricular pressure as indices of changes in myocardial contractility. Cardiac contractility was evaluated both in intact and in chronically sinoaortic denervated isoflurane-anesthetized rabbits under basal conditions and after ANG II infusion (50 ng/kg per min). After ANG II infusion, increases in mean arterial blood pressure, left ventricular end-diastolic diameter and pressure were observed both in intact and in chronically sinoaortic denervated rabbits. The left ventricular end-systolic wall stress (a function of the mean arterial pressure and chamber size) and the maximum rate of rise of the ventricular pressure rose markedly in rabbits of both groups, whereas the VCFC decreased significantly. However, when compared the VCFC under ANG II infusion with that calculated at the same level of left ventricular afterload under basal conditions, we observed that ANG II infusion induced no significant change in VCFC either in intact of in chronically sinoaortic denervated rabbits. Our results indicate that administration of ANG II to isoflurane-anesthetized rabbits induces a marked rise in ventricular pre- and after-loads and exerts no significant effect on the cardiac contractility. In light of this, it is reasonable to assume that the short-term increase in arterial blood pressure can be ascribed mainly to the increase in peripheral arterial resistance.

Sympathoexcitatory response to cyclosporin A and baroreflex resetting.

We postulate that the sympathoexcitatory response associated with the immunosuppressive agent cyclosporin A is due to an upward resetting of the arterial baroreflex. We performed studies in conscious intact and sinoaortic-denervated rabbits instrumented with catheters and renal nerve electrodes. In intact rabbits, cyclosporin A (20 mg/kg i.v., 30 minutes) produced significant increases in renal sympathetic nerve activity (100% to 269 +/- 74%, P < .05) but did not increase mean arterial pressure. In intact rabbits, we determined arterial baroreflex curves relating renal sympathetic nerve activity and heart rate to mean arterial pressure by producing ramp increases (intravenous phenylephrine) and decreases (intravenous nitroprusside) in mean arterial pressure. Cyclosporin A treatment produced a shift of the midrange of the baroreflex control of heart rate (78.0 +/- 4.1 to 84.6 +/- 4.7 mm Hg, P < .05) and renal sympathetic nerve activity (74.6 +/- 3.9 to 87.0 +/- 4.8 mm Hg, P < .05). Vehicle administration produced no effects on arterial baroreflex curves relating renal sympathetic nerve activity and heart rate to mean arterial pressure. Compared with vehicle treatment, cyclosporin A reduced the maximum gain of heart rate (-5.6 +/- 0.6 versus -3.1 +/- 0.8 beats per minute per millimeter of mercury, P < .05) but had no effect on the maximum gain of renal sympathetic nerve activity. In conscious sinoaortic-denervated rabbits, cyclosporin A had no effect on mean arterial pressure (95.7 +/- 7.3 to 91.8 +/- 10.8 mm Hg), renal sympathetic nerve activity (100% to 110 +/- 6%). and heart rate (287 +/- 10 to 279 +/- 8 beats per minute). However, when the same sinoaortic-denervated rabbits were anesthetized with sodium pentobarbital, cyclosporin A (20 mg/kg i.v.) produced increases in renal sympathetic nerve activity (100% to 189 +/- 27%). These data indicate (1) that the sympathoexcitatory response to cyclosporin A depends on baroreceptor afferent input in the conscious state and (2) that this response involves an upward resetting of the arterial baroreflex.

Effects of NO on baroreflex control of heart rate and renal nerve activity in conscious rabbits.

Recent data suggest that nitric oxide (NO) plays a role in the modulation of sympathetic nerve activity and baroreflex sensitivity. Most of these studies have been carried out in anesthetized preparations, and little if any comparison has been made on the relative role of NO on the baroreflex control of heart rate and sympathetic nerve activity. In the present studies, the effect of the NO synthase inhibitor NG-nitro-L-arginine (L-NNA) on the baroreflex control of heart rate (HR) and renal sympathetic nerve activity (RSNA) were investigated in conscious, instrumented rabbits. Intravenous bolus injections of 13 mg/kg of L-NNA decreased baseline HR (from 205.0 +/- 6.0 to 145.5 +/- 8.2 beats/min; P < 0.05) without significant changes in mean arterial pressure (MAP) and RSNA. L-NNA significantly reduced the lower plateau of the HR-MAP curves and increased the sensitivities of baroreflex control of HR and RSNA. L-Arginine (600 mg/kg i.v.) but not D-arginine reversed the above effects. The effects of L-NNA on baseline HR were not completely blocked by metoprolol (2 mg/kg) or by atropine (0.2 mg/kg). After pretreatment with metoprolol, baroreflex sensitivity was reduced and L-NNA increased baroreflex sensitivity back to the control level. After pretreatment with atropine, L-NNA still reduced the lower plateau but did not significantly affect baroreflex sensitivity. L-NNA increased the HR responses but not the RSNA response to electrical stimulation of the aortic nerve in chloralose-anesthetized, sinoaortic-denervated (SAD) rabbits. L-NNA had no effect on the HR response to right vagal stimulation. In both conscious intact and SAD rabbits, L-NNA did not increase baseline RSNA. These results suggest that endogenous NO decreases baroreflex control of HR and RSNA. Both sympathetic and parasympathetic components play a role in the effects of NO on the baroreflex control of HR. The effects of NO in the central nervous system play a more important role in the baroreflex control of HR than of RSNA.

Naloxone augments sympathetic outflow induced by centrally administered endothelin in conscious rabbits.

We examined the role of endogenous opioids in cardiovascular and neurohormonal responses to intracerebroventricular (icv) endothelin-1 (ET-1) in conscious rabbits. With or without pretreatment with intravenous naloxone (1 mg/kg), the effects of icv ET-1 (25 pmol/kg) on cardiovascular and neurohormonal responses were examined. ET-1 (icv) caused significant increases in mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), plasma catecholamines, and vasopressin levels. Naloxone pretreatment significantly augmented the increases in MAP and RSNA induced by icv ET-1. To clarify the mechanism of these effects, we also examined the effects of naloxone and naloxone methobromide, which does not cross the blood-brain barrier, on baroreceptor reflex. Intravenous naloxone (1 mg/kg) attenuated the baroreflex sensitivity assessed by RSNA (Smax: -9.9 +/- 1.7 vs. -6.9 +/- 1.2, P < 0.01); however, intravenous naloxone methobromide failed to alter the baroreflex sensitivity. Furthermore, in sinoaortic-denervated rabbits, naloxone pretreatment did not affect the increase in MAP induced by (icv) ET-1. These results suggest that intravenous naloxone acts in the central nervous system to attenuate the baroreflex sensitivity, which might augment the increases in MAP and RSNA induced by icv ET-1 in conscious rabbits.

On the role of neural mechanisms in the cardiocirculatory inhibitory action of ??-human atrial natriuretic peptide in the anesthetized rabbit

The effects induced by alpha-human 28-amino acid residue atrial natriuretic peptide (alpha-hANP) on arterial pressure, heart rate and vascular resistance, measured as hindlimb perfusion pressure (HPP), were examined in anesthetized rabbits. In particular, the involvement of the autonomic nervous system in mediating the cardiocirculatory effects of alpha-hANP was investigated. Intravenous alpha-hANP (8 micrograms/kg, bolus injection) in anesthetized rabbits caused a sustained decrease in atrial pressure, a transient decrease in HPP and no significant changes in heart rate. After sinoaortic denervation, alpha-hANP produced a greater decrease in arterial pressure and in hindlimb vascular resistance and also a consistent decrease in heart rate. Bilateral vagotomy did not significantly alter the cardiocirculatory responses to alpha-hANP in either normal or in sinoaortic denervated rabbits. Intravenous infusion of alpha-hANP (2 micrograms/kg bolus + 0.2 micrograms/kg per min) did not substantially change the baroreflex cardiocirculatory responses to loading and unloading carotid and aortic baroreceptors with bilateral carotid occlusion and phenylephrine or nitroglycerin bolus injection. In addition, alpha-hANP infusion did not modify the cardiovascular reflex responses to chemical stimulation of neural receptors (sensory endings of group III and IV somatic afferents) in the hindlimb muscles which are primarily mediated by sympathetic nerves in the anesthetized rabbit. Pharmacological blockade of the autonomic nervous system with atropine and guanethidine did not reduce the hypotensive and bradycardic effects caused by alpha-hANP in sinoaortic denervated animals. The results indicate that in anesthetized rabbits: (1) alpha-hANP can induce inhibitory cardiocirculatory responses (hypotension, bradycardia, musculocutaneous vasodilation) which are consistently offset by the sinoaortic baroreceptor system; (2) alpha-hANP does not alter the reflex control of arterial pressure and heart rate by arterial baroreceptors and muscle chemosensitive receptors; (3) activation of cardiopulmonary vagally-mediated depressor reflexes does not contribute to the inhibitory cardiovascular action of alpha-hANP; and (4) inhibitory effects on sympathetic activity do not constitute a significant component of the cardiocirculatory action of alpha-hANP.

Role of vasopressin in salt-induced hypertension in baroreceptor-denervated uninephrectomized rabbits.

To elucidate the contributions of renal, humoral, and arterial baroreceptor reflex components to salt-induced hypertension, we administered 10% NaCl intravenously for 10 days to sinoaortic-denervated rabbits with unilateral nephrectomy (n = 7), sinoaortic-denervated rabbits with intact kidneys (n = 7), and sham-operated sinoaortic-denervated rabbits with unilateral nephrectomy (n = 7). Serial changes in mean arterial pressure (MAP), heart rate, and blood pressure variability were recorded. In sinoaortic-denervated rabbits with unilateral nephrectomy, MAP increased significantly from 109 +/- 2 to 124 +/- 3 mm Hg (day 4) and remained elevated for the rest of the experiment. This elevation of MAP was accompanied by a reduction in the standard deviation of MAP, with significant elevations in plasma vasopressin, norepinephrine, and atrial natriuretic peptide concentrations and in sodium retention. In the other groups, there were no significant changes in these vasoactive hormones. In the sham-operated sinoaortic-denervated rabbits with unilateral nephrectomy, sodium retention was similar to that of sinoaortic-denervated rabbits with unilateral nephrectomy. Continuous infusion (1 microgram/kg/hr) of a V1 antagonist prevented the elevation of blood pressure and plasma norepinephrine, the accumulation of sodium, and the reduction of blood pressure lability, whereas a bolus injection (10 micrograms/kg) on day 4 reduced blood pressure from 128 +/- 3 to 115 +/- 2 mm Hg (p less than 0.005). These results imply that vasopressin plays a crucial role in the expression of salt-induced hypertension in rabbits with compromised baroreceptor and renal function.

Interaction of vasopressin with area postrema during volume expansion.

Effects of arginine vasopressin (AVP) on inhibition of renal sympathetic nerve activity (RSNA) during activation of cardiopulmonary reflexes by volume expansion were examined in conscious sinoaortic-denervated rabbits. The role of the area postrema in mediating these effects was also evaluated in rabbits subjected to area postrema lesion. Animals were subjected to 12% volume expansion with whole blood alone or during infusion of AVP (0.6 mU . kg-1 . min-1). Volume expansion in area postrema-intact animals caused a progressive reflex inhibition of RSNA (maximum = -36.5 +/- 3.3% delta RSNA). Vasopressin infusion did not significantly alter resting arterial pressure, right atrial pressure, heart rate, or RSNA. However, maximum inhibition of RSNA during volume expansion (-62.6 +/- 3.2% delta RSNA) was significantly augmented during AVP infusion, and the augmentation was reversed by a specific vascular (V1) AVP receptor antagonist. Vagotomy eliminated RSNA responses to volume expansion with or without AVP. In area postrema-lesioned animals, the RSNA response to volume expansion was similar to that of intact animals (-31.8 +/- 2.3% delta RSNA). However, AVP did not augment the RSNA response to volume expansion in lesioned animals (-30.4 +/- 2.5% delta RSNA). Thus exogenous AVP augmented cardiopulmonary reflex-mediated inhibition of RSNA due to volume expansion. This effect appeared to be mediated by the V1 AVP receptor and to require the presence of an intact area postrema.

Absence of sustained hypertension in sinoaortic-denervated rabbits.

In 21 conscious unrestrained rabbits, arterial pressure was continuously recorded up to 11 wk by an indwelling catheter placed in the thoracic aorta. The average and standard deviation of the 24-h mean arterial pressure (MAP) were calculated and compared before and after sinoaortic denervation, sham operation, or cervical sympathectomy. At the time of sinoaortic denervation, the cervical sympathetic nerve was always disrupted bilaterally to remove possible arterial barosensory afferents contained in it. Subsequent to sinoaortic denervation performed on 12 rabbits, the average 24-h MAP was initially elevated but invariably returned to the predenervation level in 5-36 days (average 14 days). Meanwhile, the standard deviation remained elevated in all but one animal. In five sham-operated or four cervical-sympathectomized animals, neither the average 24-h MAP nor the standard deviation was significantly altered from that of controls. Our results are consistent with the view that the arterial baroreceptor reflex by itself does not play a critical role in determining the long-term level of arterial pressure.

Interactions of vasopressin with the area postrema in arterial baroreflex function in conscious rabbits.

This study compares the effect of arginine-vasopressin with phenylephrine on arterial pressure, heart rate, and renal sympathetic nerve activity in conscious rabbits with and without functional arterial baroreflexes and in rabbits with lesions of the area postrema. In intact rabbits, progressive infusions of arginine-vasopressin result in large decreases in renal sympathetic nerve activity and heart rate for a given increase in blood pressure as compared to progressive infusions of phenylephrine. In sinoaortic-denervated rabbits, the responses of arterial pressure on heart rate and renal sympathetic nerve activity to both arginine-vasopressin and phenylephrine are markedly attenuated, indicating the necessity for afferent baroreceptor activity in this response. This observation indicates that arginine-vasopressin is acting centrally to enhance the baroreflex. A central site of action of circulating vasopressin may be the area postrema, since it is the only circumventricular organ in the hindbrain. Lesioning the region of the area postrema resulted in a normalization of the responses evoked with arginine-vasopressin and phenylephrine. There was no difference in the phenylephrine responses of arterial pressure on renal sympathetic nerve activity or heart rate in area postrema-lesioned animals, compared to control rabbits. Therefore, we conclude that the area postrema or its surrounding tissue is either a site of action of circulating arginine-vasopressin or contains fibers of passage from another site where arginine-vasopressin acts to enhance baroreflex activity.

Central and peripheral alpha-adrenoceptor number and responsiveness after sinoaortic denervation in the rabbit.

The effects of bilateral sinoaortic denervation were examined in rabbits in vivo and in vitro. In the in vivo studies the effects of intravenous administration of the alpha-adrenoceptor agonists phenylephrine and guanabenz and the antagonists prazosin and phentolamine on mean arterial pressure, heart rate, and plasma catecholamine levels were investigated in intact sham-operated rabbits and after bilateral baroreceptor deafferentation. In in vitro studies the maximum number of prazosin and clonidine binding sites and their dissociation constants were investigated using radioligand binding techniques. Mean arterial pressure was labile and significantly elevated in the sinoaortic denervated rabbits. Heart rate and basal plasma noradrenaline were also raised, but plasma adrenaline and tissue noradrenaline levels were similar in intact and debuffered animals. alpha-Adrenoceptor antagonists were more effective at lowering mean arterial pressure in the sinoaortic-denervated animals. This was at least in part the result of a lack of compensatory changes in sympathetic activity and heart rate in these animals. Pressor responses to the alpha 1-adrenoceptor agonist and more markedly to the alpha 2-adrenoceptor selective agonist were increased after sinoaortic denervation. These changes could not be related to alteration in alpha 1- or alpha 2-receptor binding in either heart, spleen, or brain tissue as there was no change in number of binding sites or affinity.

Role of baroreceptor reflexes in the hemodynamic and heart rate responses to althesin, ketamine and thiopentone anesthesia

The effects of i.v. infusions of althesin, ketamine and thiopentone were studied in instrumented rabbits, in doses that produced similar levels of light anesthesia. The main hemodynamic differences were in the rises in mean arterial pressure (MAP) and in total peripheral resistance (TPR) which were in the order of ketamine > thiopentone > althesin. These rises in MAP and TPR did not occur in sino-aortic denervated rabbits suggesting that in normal rabbits these depended on the integrity of the arterial baroreceptors and/or chemoreceptors. The heart rate increased with all anesthetics mainly owing to reduction in vagal efferent activity, except with althesin where cardiac sympathetic activity increased also. Baroreceptor—heart rate reflex properties were studied by deriving sogmoid curves relating MAP to heart period (HP, i.e. pulse interval). All drugs depressed the following curve parameters: (i) HP range, i.e. the difference in HP plateaux from maximal tachycardia to maximal bradycardia; and (ii) the reflex gain (sensitivity). The order of depression was ketamine > thiopentone > althesin, i.e. the same as the order in which they evoked pressor effects. The results suggest that the 3 anesthetics produce differing depression of afferent mechanisms related to baroreceptor reflexes and that this accounts for both the differences in pressor effects (through disinhibition of constrictor tone) and in depression of the vagal and sympathetic components of the baroreceptor—heart rate reflex.

Gastrointestinal and mesenteric hemodynamic patterns in neurogenic hypertensive rabbits.

Patterns of blood flow and vascular resistance in the component tissues of the splanchnic region of sinoaortic denervated (SAD) hypertensive rabbits were compared with those of normotensive sham-operated rabbits using previously described methods. SAD rabbits with high total splanchnic resistance showed significant elevations in resistance in all parts of the gastrointestinal tract and mesentery, but the magnitude of the changes compared with values for sham-operated rabbits varied from one area to another, ranging from increases of 34% in the cecum-appendix to 77% in the stomach and jejunum. Blood flow was reduced 22-42% in some areas and less in others. An aboral gradient of mean blood flow was found between and within the small and large intestines in both groups. Intestinal flow pattern differences were found in individual rabbits. We concluded that activation of the sympathetic nervous system by sinoaortic denervation exerted a variable, unpredictable degree of vasoconstrictor tone on different parts of the splanchnic bed which reflected the net result of vascular smooth muscle response to neurogenic and local regulatory factors.

Low arterial pressure in awake rabbits without carotid sinus and aortic nerves.

SummaryArterial pressure records from awake, sinoaortic denervated, normal or experimental renal hypertensive rabbits showed frequent, large pressure falls, average 40 to 50 mm Hg for 10 to 25 seconds. They usually resulted from body movements which, in rabbits were intact moderator nerves, commonly caused a small, brief pressure rise. Additionally, sinoaortic denervated rabbits occasionally had basic mean pressures below predenervated values. These findings supported the concept that, at least in part, the sinoaortic baroreflex system functions to protect against acute episodes of low arterial pressure.