Department of Pediatrics, The Ohio State University, Columbus
43210; and Wexner Institute for Pediatric Research, Childrens
Hospital, Columbus, Ohio 43205
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
N
-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.
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INTRODUCTION |
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 |
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
N
-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 |
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
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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
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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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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
N -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.
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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
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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. , Endothelium-intact rings; , 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. , Endothelium-intact rings; , endothelium-denuded
rings. Values are means ± SD; n = 5 for all observations.
a P < 0.01 vs. unblocked response.
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DISCUSSION |
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).
Charles E. Miller provided excellent technical support, which was
essential to the completion of this project.
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.