Effects of sustained flow reduction on postnatal intestinal circulation

Philip T. Nowicki

Department of Pediatrics, The Ohio State University, Columbus 43210; and Wexner Institute for Pediatric Research, Childrens Hospital, Columbus, Ohio 43205

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Studies were conducted to determine the effect of mechanically induced sustained flow reduction on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine. In vitro gut loops were perfused under controlled-pressure conditions from an oxygenated blood reservoir at age-appropriate perfusion pressures; pressure was rapidly reduced to a level that lowered flow rate to ~50% of its baseline value, and pressure was then kept at that level for 2 h. In 3-day-old intestine, vascular resistance (Ri) increased by 20% immediately after pressure and flow reduction but then stabilized for 3-4 min; thereafter, flow began to decrease despite maintenance of perfusion pressure, so that Ri increased an additional 15% by 30 min after flow reduction. Flow continued to diminish over the next 90 min, though at much slower rate. Intestine from 35-day-old swine demonstrated an immediate increase in Ri after pressure and flow reduction, but thereafter Ri increased very little. The protocol was repeated within in vitro gut loops perfused under controlled-flow conditions, and within autoperfused, innervated gut loops developed in vivo and similar observations were made in both preparations. In 3-day-old intestine, pretreatment with the L-arginine analog Nomega -monomethyl-L-arginine (10-4 M) had no effect on the immediate rise in resistance occurring in the first 1 min but substantially attenuated the subsequent slow, progressive rise noted thereafter. Pretreatment with the angiotensin 1A receptor antagonist losartan (2 × 10-6 M) had no effect on hemodynamic changes during the first 60 min after mechanical perfusion pressure reduction but attenuated the very slight increase in resistance noted during the final 60 min of the protocol. The postnatal intestinal circulation demonstrates progressive vasoconstriction when its flow rate is mechanically reduced in a sustained manner, and this effect is age specific, occurring in 3- but not 35-day-old swine. These changes in gut vascular resistance may be consequent to loss of nitric oxide production and/or local production of angiotensin.

nitric oxide; angiotensin; endothelium; intestinal oxygenation

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

PRESSURE-FLOW AUTOREGULATION is absent in the intestinal circulation of 3-day-old swine; accordingly, this vasculature constricts in response to pressure reduction and dilates in response to pressure elevation (22). This circumstance may arise because the intrinsic vascular mechanism responsible for flow-induced dilation (28) is present within the newborn intestinal circulation (21). The net effect of flow-induced dilation is directly opposite that of autoregulation: the former maximizes conductance in the face of increased flow, whereas the latter increases resistance as perfusion pressure rises, a response designed to maintain steady-state hemodynamic conditions (14). The presence of flow-induced dilation within the newborn intestinal circulation may be an adaptation designed to facilitate transition from fetal to neonatal life. During fetal existence, the liver receives abundant blood flow via the umbilical circulation, an arrangement that ensures the degree of hepatic oxygenation requisite to maintain the intense anabolic conditions characteristic of fetal life (1, 11, 12). Abrupt postnatal loss of the umbilical circulation occurs at parturition, but the effect of this loss on hepatic oxygenation is buffered because the rates of intestinal and thus portal venous blood flow are substantially elevated during early neonatal life (23). The existence of flow-induced dilation within the newborn splanchnic bed is one means to achieve this end, since this mechanism is specifically designed to maximize vascular conductance and thus flow rate.

A vascular bed specifically designed to operate at high flow rates might not adapt well to low flow, as might occur during systemic cardiovascular compromise. This predication seems be true for the newborn intestinal circulation, which demonstrates pronounced vasoconstriction in response to pressure or flow reduction (23). To date, the response of this vasculature to reduced pressure or flow has only been determined over very brief periods of time, i.e., several minutes. It is possible, however, that sustained reduction of flow rate might result in progressive vasoconstriction, since the mechanostimulus driving local production of the dilator agents responsible for flow-induced dilation, i.e., flow, is decreased. Loss of these locally produced dilator agents, such as nitric oxide (NO), could have two effects on local vascular tone. First, vascular resistance would increase as a direct consequence of the loss of a dilator stimulus. Second, attenuation of NO production might enhance local production of constrictor agents, such as ANG II, and also increase the contractile efficacy of these agents (3, 26, 30).

The goal of these experiments was to determine the effects of sustained pressure or flow reduction on intestinal hemodynamics and oxygenation in postnatal intestine. To this end, gut loops from 3-day-old swine were exposed to ~50% reduction in flow for 2-4 h, and the effects of this perturbation on gut hemodynamics and oxygenation were determined. Studies were conducted within autoperfused, innervated in vivo gut loops and also denervated in vitro gut loops perfused under controlled-pressure or controlled-flow conditions. The roles of NO and angiotensin in this response were determined by selective blockade of NO production with an L-arginine analog and of angiotensin 1A (AT1A) receptors with losartan. Studies were also carried out within isolated mesenteric rings of mesenteric artery to determine the capacity of this vasculature to complete posttranslational modification of renin substrate to ANG II. Finally, some studies were repeated in 35-day-old weanling swine to determine whether the response to sustained flow reduction was age specific.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animal Acquisition and Handling

Studies were conducted on swine, 2-4 or 33-37 days old, obtained from local swine breeding farms on the day before study. Animals were housed together in a large swine run kept at 30°C and were allowed free access to milk replacer or cereal mash, depending on age, via an automated feeder device. Food, but not water, was withdrawn 8 h before the surgical preparation; this time period is sufficient to ensure preprandial hemodynamic conditions in swine of this postnatal age (23). Anesthesia was induced with tiletamine hydrochloride-zolaxepam hydrochloride (6 mg/kg im) and xylazine (4 mg/kg im) and maintained with pentobarbital sodium (5 mg/kg iv given every 60 min, or sooner if deemed necessary by vivarium staff). All animal care was provided in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHHS Publication No. 85-23) under the auspices of a protocol approved by the Childrens Hospital Research Foundation Institutional Animal Care and Use Committee.

Studies of Isometric Tension Within In Vitro Mesenteric Artery Rings

Ring preparation. The mesenteric artery was removed from its aortic origin to its distal ileal branches and placed in iced Krebs buffer on a dissecting tray. Rings (3 mm) were cut with care taken to avoid intimal contact, except in those rings processed to be endothelium deficient; in these rings, the endothelium was removed by gentle swabbing with a cotton applicator. Rings were mounted between two stainless steel stirrups placed within a water bath-jacketed myograph well. One stirrup was tethered to a force transducer to allow tension measurement (Grass FT-03 and polygraph). The well was filled with Krebs buffer of the following composition (mM): 118.1 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.0 NaHCO3, 11.1 glucose, and 0.026 EDTA. The buffer was maintained at 38°C and continuously aerated with 95% O2-5% CO2. Rings were progressively stretched over 1-2 h to the optimal point of their length-tension curve, as determined by noting maximal contraction to 80 mM KCl. Endothelial integrity was determined in all rings by precontraction with phenylephrine (10-7 M), followed by noting dilation >50% in response to acetylcholine (10-7 M).

Experimental protocol. Four protocols were conducted. In the first protocol, ANG II, ANG I, or renin substrate tetradecapeptide (RST) was administered in increasing concentrations (10-11 to 10-7 M) to endothelium-intact (E+) and endothelium-denuded (E-) rings. Human ANG I and ANG II, which are identical to their porcine counterparts, were obtained from Peninsula Laboratories (Belmont, CA). RST was also obtained from Peninsula as the porcine form, which corresponds to porcine angiotensinogen-(1---14). Each ring was administered only one agonist, and all observations were carried out in paired rings; thus, for a single subject, three pairs of E+ and E- rings were used, one each for ANG II, ANG I, or RST. In the second protocol, the pA2 of the selective AT1A receptor antagonist losartan (29) was determined in newborn swine mesenteric artery by administering varying concentrations of this agent (10-8 to 10-5 M) to mesenteric artery rings, followed by administration of ANG II (10-11 to 10-7 M) to all rings. This concentration was found to be 2 × 10-7 M. The effective blocking concentration was considered to be one order of magnitude above the pA2 (18), i.e., 2 × 10-6 M, and was used in all subsequent blocking studies, in both rings and gut loops. In the third protocol, the effect of losartan on ANG II- and ANG I-induced contractions was determined. Finally, the effects of the angiotensin-converting enzyme inhibitor captopril on contractions induced by ANG I were determined.

Studies Using Innervated, Autoperfused In Vivo Gut Loops

Experimental preparation and measurement techniques. Swine were anesthetized and ventilated to maintain normal blood gas tensions. A femoral artery-vein pair were cannulated; the arterial catheter was connected to a standard pressure transducer to measure systemic arterial pressure, and the venous catheter was used to deliver crystalloid (5% dextrose in 0.9% saline at 15 ml · kg-1 · h-1) and to return blood from the gut loop circuit. A ~25-cm segment of distal jejunum-proximal ileum was vascularly isolated from the remainder of the small bowel so that it was perfused and drained by a single artery-vein pair. The vein was cannulated and the catheter directed to a beaker primed with ~50 ml of heparinized swine blood collected from previous studies. Blood collected in the beaker was returned to the animal at a rate equal to venous outflow, thus maintaining euvolemia with respect to the animal. A flowmeter and pressure transducer were placed within the venous circuit to measure flow and venous pressure, respectively (2.0 mm ID, Gould, Cleveland, OH). The artery and periarterial nerves leading to the gut loop were not disturbed. The intestine and abdominal incisions were covered with plastic film, and the entire subject was kept at 38°C by means of a servo-controlled heating element. The preparation was left undisturbed for ~30 min or until arterial pressure and gut flow reached steady-state (i.e., fluctuation approximately ±5%).

Experimental protocols. A single protocol utilized this preparation. Baseline measurements were obtained once steady-state conditions were present. A silk tie was placed around the mesenteric artery leading to the isolated gut loop and progressively tightened until blood flow was reduced to ~50% of baseline, and the tie was then fixed in this position. Paired arteriovenous blood samples were obtained 30, 60, 90, 120, 180, and 240 min after flow was reduced. At the end of 2-4 h, the animal was killed by pentobarbital overdose. Animals in the control group for this protocol had in vivo gut loops in which the silk tie was placed but not tightened; instead, the hemodynamic and oxygenation parameters of these preparations were simply monitored over 2-4 h.

Studies Using Reservoir-Perfused, Denervated In Vitro Gut Loops

Experimental preparation and measurement techniques. Swine were anesthetized, heparinized, and ventilated as previously described. A segment of distal jejunum-proximal ileum was isolated from the remainder of the gut, the single artery-vein pair serving this loop was cannulated, extracorporeal perfusion of the segment was begun, and the segment was removed from the study subject. The gut lumen was cleansed by repetitive instillation of warm saline and air. A temperature probe was inserted into the lumen and connected to a servo-controlled heating element, which kept tissue temperature at 38°C. Arterial perfusion of the intestinal segment as achieved by means of an extracorporeal perfusion apparatus and blood for the perfusion circuit was obtained from donor swine. These animals were ~90 days old and weighed ~40 kg, and their expected blood volume was 4 liters (8). Donors were anesthetized, heparinized (500 U/kg), and ventilated, and ~1,000 ml of blood were then removed via a carotid artery catheter. During phlebotomy, 0.9% saline was infused into a jugular vein catheter at a rate equal to blood withdrawal; this action was taken to avoid significant activation of the systemic renin-angiotensin axis. Arterial pressure within the donor swine remained stable during phlebotomy (92 ± 5 vs. 85 ± 6 mmHg before and immediately after blood removal). The blood was filtered twice (40 µm), reheparinized (100 U/500 ml), and placed into a flask. Blood within this flask was continuously recirculated (200 ml/min) through a membrane oxygenator (0.6 m2, gassed with 95% O2-5% CO2) to maintain normal arterial blood gas tensions. The hematological characteristics of the arterial perfusate were as follows: hematocrit 27 ± 3%, PO2 90 ± 4 mmHg, PCO2 35 ± 4 mmHg, pH 7.42 ± 0.09, and total O2 content 13.2 ± 0.8 ml O2/dl. At the completion of each experiment the residual arterial perfusate was filtered (20 µm) to determine the presence of microemboli within this blood. A small volume of blood was continuously pumped from this flask to a sealed arterial reservoir, in which it was warmed (38°C, water bath) and stirred (stir plate) before delivery to the gut loop. Delivery of blood to the gut could be achieved in two ways. For controlled-pressure perfusion, the reservoir was pressurized with 95% air-5% CO2 with an air-pressure regulator (Bellofram type 10R, Burlington, MA). This arrangement allowed direct manipulation of arterial pressure as an independent variable (±1 mmHg) and provided pulseless, free-flow perfusion. For controlled-flow perfusion, the arterial limb of the circuit was drawn through a peristaltic pump (Harvard Instruments, Quincy, MA). This arrangement allowed direct manipulation of flow rate as an independent variable (±1 ml/min) and delivered a pulse pressure of 19 ± 2 and 12 ± 2 mmHg at baseline and reduced flow rates, respectively. Venous pressure was kept at 0 mmHg by adjusting the height of the venous outflow cannula. The venous effluent was not recirculated, so that drugs administered via the arterial circuit did not accumulate within the blood perfusate.

Experimental protocols. Three protocols were carried out with this preparation. The first protocol was carried out in 3- and 35-day-old swine. Gut loops were perfused under controlled-pressure conditions at age-appropriate arterial pressures (65 mmHg in 3-day-old and 75 mmHg in 35-day-old swine) while venous pressure was maintained at 0 mmHg throughout the experiment. After steady-state conditions and baseline measurements were obtained, arterial pressure was reduced to the extent necessary to lower gut blood flow by ~50%. Perfusion was continued at this new pressure for 120 min. The control group for this protocol consisted of in vitro gut loops perfused at a consistent arterial pressure of 65 mmHg for 2 h. This protocol was carried out in 3- and 35-day-old swine. In the second protocol, carried out only in 3-day-old swine, gut loops were perfused under controlled-flow conditions and the initial flow rate was set at 90 ml · min-1 · 100 g-1 to duplicate average in vivo conditions. After steady-state conditions and baseline measurements were obtained, the flow rate was rapidly reduced to 50% of baseline by decreasing the pump speed. Perfusion was continued at this new flow rate for 120 min. The control group for this protocol consisted of in vitro gut loops perfused at a constant flow rate of 90 ml · min-1 · 100 g-1 for 2 h. In the third protocol, also carried out only in 3-day-old swine, gut loops were perfused under controlled-pressure conditions, and after steady-state conditions were achieved and baseline measurements were obtained, a continuous infusion of either Nomega -monomethyl-L-arginine (L-NMMA) (10-4 M/min) or losartan (2 × 10-6 M/min) was begun into the arterial limb of the circuit. Both agents were dissolved in 0.9% saline and infused at a rate of 0.1 ml/min. The preparation was allowed to attain new steady-state conditions, and repeat baseline measurements were then obtained. Thereafter, arterial pressure was reduced as described previously, so that blood flow was reduced to ~50% of its baseline value. Drug infusions were continued after flow reduction, but the rate of drug infusion was reduced by 50% in an effort to maintain constant arterial concentration of the drug; further reductions in drug infusion rate were not made during the period of progressive flow reduction, i.e., from t3 to t120. Animals in the control group for this protocol had in vitro gut loops perfused at a continuous perfusion pressure of 65 mmHg and were administered either L-NMMA or losartan for 2 h, without pressure reduction.

Statistical Analysis

Data from the ring studies were expressed as a percentage of the contraction to 80 mM KCl. All myograph observations were made in paired rings, and the mean response was taken; n refers to the number of swine in which the observation was made. Hemodynamic data collected within in vivo and in vitro gut loops were analyzed by ANOVA for repetitive measures. For data sets in which two age groups were studied, the main effects were age (3 vs. 35 days old), condition (control vs. low flow), and time. For data sets in which L-NMMA or losartan were administered, the main effects were treatment (drug vs. no drug), condition (control vs. low flow), and time. In each instance the F statistic for the main effects was first determined. If it was significant, i.e., P < 0.01, then post hoc t-tests were carried out to determine the sites of significance within that data set. For vascular resistance data, three specific post hoc comparisons were made within each data set: resistance at t0 vs. t1; resistance at t30 vs. t1; and final resistance measurement vs. t30.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The first experiment of this study was conducted by using in vitro gut loops perfused under controlled pressure perfusion. In intestine from 3-day-old swine, perfusion pressure was reduced from 65 ± 1 to 38 ± 2 mmHg, causing flow to decrease from its baseline level of 94 ± 8 to 46 ± 4 ml · min-1 · 100 g-1. This action caused a significant (20 ± 3%) increase in vascular resistance. Flow remained relatively constant at this level for ~3-4 min but thereafter began to slowly drift downward despite maintenance of arterial pressure. The early portion of this change can readily be appreciated in Fig. 1. Visual inspection of the resistance data (Fig. 2) suggested that this change was relatively rapid during the first 30 min but then slowed during the final 90 min of low-flow perfusion. This impression was confirmed statistically: the rate of flow reduction averaged 0.13 ± 0.2 ml · min-1 · 100 g-1 between t1 and t30 but only 0.04 ± 0.01 ml · min-1 · 100 g-1 between t30 and t120 (P < 0.01 by t-test). Also, vascular resistance values from t30 to t120 were significantly greater than at t1, whereas the value at t120 was significantly greater than that at t30. The arteriovenous O2 content difference [(a-v)O2] across the gut loop increased 51 ± 6% between t0 and t15 (Table 1). Although significant, this increase was not sufficient to compensate for the effect of flow reduction on the rate of O2 delivery, so that tissue O2 uptake decreased 26 ± 3% at this time. Interestingly, (a-v)O2 did not increase further during the course of the experiment, despite the progressive reduction in blood flow (Table 1). A similar response pattern was noted in intestine from 35-day-old swine, although the magnitude of change was significantly less. Flow was mechanically decreased from 45 ± 3 to 23 ± 2 ml · min-1 · 100 g-1 to initiate low-flow perfusion, achieved by decreasing perfusion pressure from 75 ± 1 to 41 ± 2 mmHg. This action increased vascular resistance by 6 ± 1%. Between t1 and t30 flow declined at a rate of 0.03 ml/min, whereas between t30 and t120, the rate of fall was 0.01 ml/min; resistance rose only 5 ± 1 % between t1 and t120 in older swine (Fig. 3). Sustained flow reduction had no effect on gut O2 uptake in 35-day-old swine (Table 1). Control swine from both age groups demonstrated excellent stability of the measured parameters over the 2-h study period (Table 2).


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Fig. 1.   Reproduction of strip chart from an experiment in which perfusion pressure was reduced in an in vitro gut loop from a 3-day-old swine perfused under controlled-pressure perfusion. Top: arterial pressure (mmHg); bottom: flow rate (ml/min). Note that flow rate falls concomitantly with arterial pressure and reaches its initial lower rate ~30 s after pressure reduction. It then stays stable but begins to fall once again ~3 min after initial pressure reduction despite maintenance of arterial pressure.


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Fig. 2.   Effects of arterial pressure reduction on vascular resistance within in vitro gut loops from 35-day-old (top) and 3-day-old (bottom) swine perfused under controlled-pressure conditions. Time (x-axis) is divided into 2 segments. On left are shown early changes in resistance immediately after pressure reduction (between t0 and t1). On right are shown changes that occurred between t1 and t120. Values are means ± SD; n = 7 for 3-day-old swine, n = 5 for 35-day-old swine. ANOVA (3 way) carried out on these data was significant (F statistic, P < 0.01); specifically, changes noted during low flow were significantly different between 3- and 35-day-old groups. Post hoc t-tests were carried out within each group to determine sites of significant difference as follows: a P < 0.01 vs. t0; b P < 0.01 vs. t1; c P < 0.01 vs. t30; d P < 0.01 vs. low flow.

                              
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Table 1.   Hemodynamic and oxygenation parameters within in vitro gut loops from 3- and 35-day-old swine perfused under controlled-pressure conditions during flow reduction


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Fig. 3.   Effects of sustained reduction of arterial pressure on vascular resistance within autoperfused, innervated in vivo gut loops from 35-day-old (top) and 3-day-old (bottom) swine perfused under controlled-pressure conditions. Time (x-axis) is divided into 2 segments. On left are shown early changes in resistance immediately after pressure reduction (between t0 and t1). On right are shown changes that occurred between t1 and t240. Values are means ± SD; n = 5 for 3-day-old swine for observations between t0 and t120; n = 9 for observations at t180 and t240; n = 5 for 35-day-old swine for low-flow observations; n = 3 for 35-day-old swine for control observations. ANOVA (3 way) carried out on these data was significant (F statistic, P < 0.01); specifically, changes noted during low flow were significantly different between 3- and 35-day-old groups. Post hoc t-tests were carried out within each group to determine sites of significant difference as follows: a P < 0.01 vs. t0; b P < 0.01 vs. t1; c P < 0.01 vs. t30; d P < 0.01 vs. low flow.

                              
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Table 2.   Hemodynamic and oxygenation parameters within in vitro gut loops from 3- and 35-day-old control group swine perfused under controlled-pressure conditions

The second series of experiments were carried out by using pulsatile, controlled-flow perfusion to confirm that the observation of progressive vasoconstriction was not an artifact of pulseless, controlled-pressure perfusion. These experiments were only carried out in 3-day-old swine, since older swine demonstrated significantly less hemodynamic change during flow reduction. The effect of flow reduction on gut vascular resistance was essentially identical to that noted previously (Table 3). Arterial pressure, the dependent variable in a controlled-flow preparation, increased briskly in response to flow reduction over the 1st min and then rose more slowly over the next 30 min, followed thereafter by a much slower rate of rise over the remaining 90 min of perfusion. The effects of flow reduction on (a-v)O2 and gut O2 uptake were similar to those noted under controlled-pressure perfusion, and control gut loops perfused under these conditions demonstrated stable hemodynamic and oxygenation variables over 2 h (Table 3).

                              
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Table 3.   Hemodynamic and oxygenation parameters within in vitro gut loops perfused under controlled-flow conditions

A similar protocol was used in the third series of experiments, this time carried out in autoperfused, innervated in vivo gut loops created in 3- and 35-day-old swine. In some of these experiments the length of flow reduction was increased to 4 h, which was possible because the duration of perfusion was not limited by the volume blood within an arterial reservoir. In 3-day-old swine, the pattern of change in vascular resistance was similar to that noted previously; thus the rate of flow reduction was 0.23 ± 0.03 ml · min-1 · 100 g-1 between t1 and t30, but only 0.02 ml · min-1 · 100 g-1 between t30 and t240. The increase in vascular resistance reached a plateau at t180. As before, older swine demonstrated significantly less increase in vascular resistance during sustained low-flow perfusion; thus vascular resistance increased 36 ± 3% in 3-day-old swine but only 4 ± 1% in 35-day-old swine between t1 and t240, respectively. (a-v)O2 increased in both groups during low flow, although the increase noted in younger swine was not sufficient to offset the magnitude of flow reduction on O2 transport, so that tissue O2 uptake gradually declined in younger intestine (Table 4). Control swine from both age groups demonstrated excellent stability of the measured and calculated variables during the duration of the protocol (Table 5).

                              
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Table 4.   Hemodynamic and oxygenation parameters within in vivo gut loops from 3- and 35-day-old swine during flow reduction

                              
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Table 5.   Hemodynamic and oxygenation parameters within in vivo gut loops from 3- and 35-day-old control swine

A fourth set of experiments investigated the possibility that loss of NO production during sustained flow rate reduction was responsible for the progressive rise in vascular resistance during sustained flow reduction noted in younger swine. These studies were carried out within in vitro gut loops perfused under controlled-pressure conditions and were only performed in 3-day-old swine, since the change in resistance noted in 35-day-old swine was very small. Blockade of NO production with L-NMMA (10-4 M/min) was initiated during perfusion at the baseline arterial pressure of 65 mmHg. This infusion caused vascular resistance to rise from 0.71 ± 0.05 to 0.90 ± 0.9 mmHg · ml-1 · min · 100 g (Table 6). Subsequent reduction of perfusion pressure to a level sufficient to reduce the new baseline flow rate by ~50% led to an initial increase in vascular resistance qualitatively and quantitatively similar to that previously noted in unblocked in vitro gut loops perfused under controlled-pressure conditions (Fig. 4). However, changes in vascular resistance noted during the remainder of the experiment, i.e., from t1 to t120, were significantly different from those noted in unblocked gut loops; specifically, vascular resistance increased only 6 ± 1% during this time compared with the 21 ± 3% increase noted in unblocked gut loops. Furthermore, the relatively rapid increase previously noted between t1 and t30 was not present in L-NMMA-treated gut loops. The final resistances reached in L-NMMA and unblocked gut loops were quite similar, i.e., 1.02 ± 0.02 vs. 1.05 ± 0.02 mmHg · ml-1 · min · 100 g in unblocked vs. L-NMMA-treated gut loops. One interpretation of this observation is that gut loops pretreated with L-NMMA had reached a state of maximal contraction, i.e., that the relative lack of a substantial increase in resistance between t1 and t120 was consequent to an inability of the intestinal circulation to contract further. To clarify this issue, two L-NMMA-treated gut loop preparations were administered phenylephrine (10-7 M/min) at t120. Vascular resistance increased 22 ± 4% further in these gut loops. The effects of sustained flow reduction on intestinal oxygenation in L-NMMA-treated gut loops were similar to that noted in unblocked gut loops (Table 6). Finally, control gut loops administered L-NMMA at a continuous perfusion pressure of 65 mmHg demonstrated no significant change in hemodynamic or oxygenation parameters once steady-state conditions were attained after onset of the drug infusion (Table 6).

                              
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Table 6.   Hemodynamic and oxygenation parameters within in vitro gut loops perfused under controlled-pressure conditions in swine administered L-NMMA


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Fig. 4.   Effects of sustained reduction of arterial pressure on vascular resistance within in vitro gut loops from 3-day-old swine perfused under controlled-pressure perfusion. Some swine were pretreated with Nomega -monomethyl-L-arginine (L--NMMA, 10-4 M/min) before and during pressure and thus flow reduction. Time (x-axis) is divided into 2 segments. On left are shown early changes in resistance immediately after pressure reduction (between t0 and t1). On right are shown changes that occurred between t1 and t120. ANOVA (2 way) carried out on these data was significant (F statistic, P < 0.01). Post hoc t-tests were carried out to determine sites of significance as follows: a P < 0.01 vs. t0; b P < 0.01 vs. t1; c P < 0.01 vs. t30; d P < 0.01 vs. low flow.

A fifth series of experiments was carried out to determined whether locally produced ANG II contributed to the slow, progressive rise in vascular resistance noted during sustained flow reduction. These studies were carried out within in vitro gut loops perfused under controlled-pressure perfusion. Administration of the selective AT1A-receptor antagonist losartan during perfusion at an arterial pressure of 65 mmHg caused an insignificant decrease in vascular resistance from 0.71 ± 0.05 to 0.67 ± 0.07 mmHg · ml-1 · min · 100 g. Subsequent reduction of perfusion pressure to reduce flow rate by ~50% caused an initial increase in vascular resistance virtually identical to that noted in unblocked gut loops (Fig. 5). This similarity continued through t60 but then ended; specifically, no further increase in vascular resistance was noted in losartan-treated gut loops between t60 and t120. The effects of flow reduction on gut oxygenation were similar in losartan and unblocked gut loops (Table 7). Control gut loops administered losartan at a continuous perfusion pressure of 65 mmHg, without pressure reduction, demonstrated stable hemodynamic and oxygenation variables over 2 h (Table 7).


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Fig. 5.   Effects of sustained reduction of arterial pressure on vascular resistance within in vitro gut loops from 3-day-old swine perfused under controlled-pressure perfusion. Some swine were treated with losartan (2 × 10-6 M/min) before and during pressure and thus flow reduction. Time (x-axis) is divided into 2 segments. On left are shown early changes in resistance immediately after pressure reduction (between t0 and t1). On right are shown changes that occurred between t1 and t120. ANOVA (2 way) carried out on these data was significant (F statistic, P < 0.01). Post hoc t-tests were carried out to determine sites of significance as follows: a P < 0.01 vs. t0; b P < 0.01 vs. t1; d P < 0.01 vs. low flow.

                              
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Table 7.   Hemodynamic and oxygenation parameters within in vitro gut loops perfused under controlled-pressure conditions in swine administered losartan

The final set of experiments was designed to determined whether mesenteric artery from 3-day-old swine was capable of converting RST to ANG I, and finally to ANG II. ANG II contracted both E+ and E- rings in a similar fashion, with an ED50 of 5 × 10-9 M (Fig. 6). Losartan caused a significant rightward shift in the dose-response curve for ANG II (Fig. 7). ANG I contracted E+ rings but not E- rings; additionally, the ED50 for ANG I was significantly greater than that for ANG II at 3 × 10-8 M (Fig. 6). The contractile effect of ANG I was significantly attenuated by pretreatment with losartan and virtually eliminated by pretreatment with the angiotensin-converting enzyme inhibitor captopril (Fig. 7). RST caused a very modest contraction of E+ rings, but only at an agonist concentration of 10-6 M (Fig. 6).


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Fig. 6.   Effects of ANG II (left), ANG I (middle), and renin substrate (RS, right) on isometric tension in mesenteric artery rings from 3-day-old swine mounted in aerated Krebs buffer. Each ring was administered only 1 agonist. Contraction is expressed as percentage of maximal contraction noted in response to 80 mM KCl. Agonist concentrations are expressed as -log of molar concentration. bullet , Endothelium-intact rings; open circle , endothelium-denuded rings. Values are means ± SD; n = 5 for all observations. a P < 0.01 vs. endothelium intact.


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Fig. 7.   Effects of losartan (2 × 10-6 M) or captopril (10-5 M) on response of endothelium-intact rings to ANG II (left) or ANG I (with losartan, middle; with captopril, right). Contraction is expressed as percentage of maximal contraction noted in response to 80 mM KCl. Agonist concentrations are expressed as -log of molar concentration. bullet , Endothelium-intact rings; open circle , endothelium-denuded rings. Values are means ± SD; n = 5 for all observations. a P < 0.01 vs. unblocked response.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The principal new observations made in these experiments were that vascular resistance within 3-day-old intestine progressively and significantly increased when flow rate was mechanically reduced to ~50% of baseline and was left at that level for 2-4 h and that this change was age specific, since it did not occur in 35-day-old intestine. The increase in vascular resistance observed in 3-day-old intestine occurred in three distinct phases. First, an initial sharp increase of 16-20% occurred immediately as flow was mechanically reduced by either decreasing perfusion pressure or pump speed. After a brief stabilization at this new resistance for 3-4 min, a second increase of 12-16% was noted, reaching a temporary plateau 30 min after the onset of flow reduction; this increase occurred despite maintenance of perfusion pressure or pump speed at their initial reduced levels. Thereafter, resistance slowly increased 5-16% reaching a final, stable plateau 180 min after the onset of flow reduction. The experimental approach used in this study, i.e., duplication of the observation using multiple perfusion modalities makes it very unlikely that the increase in resistance between t1 and t120-t240 was an artifact of a specific perfusion mode. Furthermore, similar preparations not exposed to pressure or flow rate reduction, i.e., the control gut loops, demonstrated excellent stability of the measured variables over the duration of the protocol, and intestine from older swine demonstrated a minimal, statistically insignificant increase in resistance between t1 and t120-t240. Natural deterioration of the preparations was thus unlikely to have contributed significantly to the observed increase in vascular resistance. One potential confounding variable in the blood perfused in vitro preparations is the formation of microemboli; this concern is especially valid in these experiments, since no effort was made to ensure erythrocyte antigen compatibility between donor and experimental swine. However, several factors make such an occurrence unlikely: first, donor blood was excessively heparinized; second, swine do not express strong erythrocyte antigens (8); and third, the donor blood was perfused through in vitro gut loops without recirculation, diminishing the probability of serum antibody-erythrocyte antigen interaction between the donor and experimental subject. Taken together, these observations strongly suggest that the progressive rise in gut vascular resistance noted in response to sustained flow reduction was not an experimental artifact but, rather, a bona fide physiological response. What might have caused these changes in vascular resistance?

Passive elastic recoil is the most likely basis for the initial rapid rise in resistance noted as pressure was reduced. In many vascular beds, the passive elastic response to intravascular pressure reduction is partly or completely offset by intrinsic myogenic and metabolic vascular responses. The myogenic response is based on the inherent property of vascular smooth muscle to contract in response to a stretch stimulus (19). Change in intravascular pressure alters the stretch of vascular smooth muscle, thus eliciting a change in muscle tone, i.e., decreased pressure offloads the myogenic mechanism causing net vasodilation and vice versa. The myogenic mechanism is present in 3- and 35-day-old intestine but has only been demonstrated as vasoconstriction in response to an increase in intravascular pressure (7). The opposite effect, i.e., vasodilation in response to pressure reduction has not been observed and, on the basis of the present data, does not appear to be present. The metabolic system is based on the parenchymal generation of a vasodilatory feedback signal in response to tissue hypoxia (13). In this paradigm, loss of inflow pressure lowers blood flow and thus O2 delivery, ultimately causing tissue hypoxia. A metabolic feedback signal is generated in response to cellular hypoxia which dilates resistance vessels and opens precapillary sphincters; these actions restore local O2 delivery. The metabolic mechanism is present in 3- and 35-day-old intestine but only appears to participate in local vascular regulation when tissue O2 demand increases, e.g., during postprandial hyperemia (6). In particular, metabolic-based vasodilation in response to reduced O2 delivery does not occur in 3-day-old intestine, although in occurs to a modest degree in 35-day-old intestine (23). The relative absence of the myogenic and metabolic mechanisms leaves passive elastic recoil as the primary initial response to arterial pressure reduction in newborn intestine.

It is tempting to speculate that the progressive increase in gut vascular resistance after t1 occurred, at least in part, because NO production by the endothelial constitutive isoform of NO synthase (ecNOS) decreased as the mechanostimulus of flow rate was reduced. Several lines of evidence support the feasibility of this speculation. First, the importance of flow, or wall shear stress as a mechanostimulus for ecNOS activation has been firmly established (2, 18). Flow activates ecNOS by increasing endothelial intracellular Ca2+ concentration via a G protein-linked, phospholipase C, diacylglycerol, inositol trisphosphate-linked signal transduction pathway (16, 17). Additionally, the mechanostimulus of flow causes phosphorylation of ecNOS (5) and may also activate this enzyme by opening K+ channels (4). Second, the phenomenon of flow-induced dilation occurs within the intestinal circulation of 3-day-old swine and can be blocked with L-NMMA (21). Finally, in the present experiments, pretreatment of in vitro gut loops from 3-day-old swine with L-NMMA significantly attenuated the progressive vasoconstriction between t1 and t120. Definitive proof of this speculation, however, is not yet forthcoming. It will be necessary to demonstrate reduction of NO production by newborn gut endothelium in response to reduction in flow rate. This observation is essential, since the reported observations of flow-induced dilation or flow-induced production of NO have been made after abrupt elevation of flow rate. To the best of my knowledge, the reverse approach, i.e., demonstration of reduced NO production in response to loss or severe reduction of a flow stimulus has not been reported. This explanation is also consistent with the relative lack of resistance increase after t1 noted in older intestine. Blockade of NO production by L-NMMA causes a very minimal increase in vascular resistance in 35-day-old swine intestine, suggesting that NO plays only a minor role in setting vascular resistance at this postnatal age (20).

De novo production of renin substrate, with subsequent posttranslational enzymatic modification to ANG I and then ANG II has been demonstrated in several vascular beds, indicating that these vessels are imbued with the localized version of the renin-angiotensin system (15, 25). Renin substrate has been detected in rat mesenteric artery (9) and canine mesenteric artery is capable of converting ANG I to ANG II (10). The mesenteric artery ring studies conducted herein clearly demonstrate the presence of AT1A receptors within newborn intestine and also confirm that this vasculature is able to convert RST to ANG I and ANG I to ANG II. It remains to be determined, however, if transcription of mRNA for renin substrate occurs in newborn mesenteric artery. Blockade of AT1A receptors with losartan in reservoir-perfused in vitro gut loops had little effect on hemodynamics or oxygenation. This observation suggests that ANG II does not participate in setting basal vascular resistance in this circulation. Losartan-treated gut loops exposed to pressure, and thus flow reduction for 2 h behaved in a fashion similar to untreated gut loops, except during the final hour of the protocol; during that time, vascular resistance in losartan-treated gut loops remained unchanged. One interpretation of this observation is that ANG II participates in creating the progressive increase in gut vascular resistance during sustained low-flow conditions. More definitive proof of this speculation will require measurement of renin, ANG I, and ANG II in the arterial perfusate and venous effluent and also duplication of the observation under in vivo conditions.

Perhaps the most intriguing aspect of the observations presented here is the potential effect of a change in systemic circulatory stability (e.g., occurrence of sustained systemic hypotension) on the balance among locally generated vascular effector mechanisms within the intestinal circulation from 3-day-old swine. It is clear that mechanisms, such as the NO-cGMP axis and renin-angiotensin, interact on a microvascular level, each altering the capacity of the other to affect vascular smooth muscle tone and thus change net vascular resistance. For example, NO attenuates the contractile effect of ANG II, so that reduction of NO increases the contractile efficacy of ANG II (26, 30). Also, it appears that in some instances locally produced vasoactive factors alter the rates of production of other such factors or their receptors (3, 27). The existence of this complex interdependence predicts that a substantial change in the production of one locally produced vasoactive factor, such as NO, might result in a cascade of events whose ultimate impact on vascular resistance is substantially greater than might be predicted on the basis of only the loss of NO. This possibility might assume even greater proportion in a vascular bed whose basal resistance is highly dependent on the constitutive production of NO, e.g., in newborn intestinal circulation (20). Loss of an important stimulus for ecNOS activity, such as flow rate, might initiate a series of events that ultimately have an overwhelming impact on gut perfusion. In this context, it is important to note a possible clinical correlate: preterm newborn infants are particularly susceptible to compromise of intestinal perfusion, a circumstance that may be an important factor in the development of a form of bowel necrosis, necrotizing enterocolitis, unique to infancy (24).

    ACKNOWLEDGEMENTS

Charles E. Miller provided excellent technical support, which was essential to the completion of this project.

    FOOTNOTES

This work was supported by National Institute of Child Health and Human Development Grants HD-25256 and HD-31902.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: P. T. Nowicki, Childrens Hospital, 700 Childrens Dr., Columbus, OH 43205.

Received 22 January 1998; accepted in final form 27 May 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(4):G758-G768
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