Pressure and flow characteristics of terminal mesenteric arteries in postnatal intestine

Kristina M. Reber and Philip T. Nowicki

Department of Pediatrics and The Wexner Institute for Pediatric Research, Children's Hospital, Columbus, Ohio 43205

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The responses of buffer-perfused terminal mesenteric arteries from 3- and 35-day-old swine to manipulation of intravascular pressure and flow rate were determined. Under in vivo conditions, these vessels demonstrated age-dependent differences in resting diameter (182 vs. 301 µm in 3- vs. 35-day-old swine). A proximal-to-distal pressure gradient was present in vessels from both age groups (Delta 13 vs. Delta 16 mmHg in 3- vs. 35-day-old swine), suggesting their functional role as resistance vessels. Vessels were mounted within an in vitro perfusion apparatus that allowed independent regulation of inflow and outflow pressure. Vessels from both age groups demonstrated the development of active tone in response to an incremental rise in pressure, applied in the absence of flow. However, myogenic vasoconstriction was only observed in younger arterioles. Similarly, both groups demonstrated dilation in response to a flow stimulus generated in the absence of a net change in intravascular pressure, although the magnitude of this response was significantly greater in younger vessels (+27 vs. +7% in 3- vs. 35-day-old swine). The dilatory response to flow was eliminated by NG-monomethyl-L-arginine (10-4 M) but restored by coadministration of L-arginine (10-3 M). Myogenic vasoconstriction was overridden by flow-mediated dilation in terminal mesenteric arteries from 3- but not 35-day-old swine after concomitant application of pressure and flow stimuli. We conclude that the hemodynamic characteristics of terminal mesenteric arteries are age dependent in postnatal swine.

intestinal circulation; newborn; microvasculature; oxygen delivery; blood flow

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IN CONTRAST to its relatively dormant fetal existence, the early postnatal gastrointestinal tract is a site of intense metabolic and anabolic activity as it assumes complete responsibility for assimilation of water and nutrients and also experiences a stage of prolific growth (18). It is thus not surprising that the rate of oxygen utilization per gram of tissue is nearly twofold greater within in vivo intestine from 3-day-old swine compared with 35-day-old swine (20, 21), a developmental pattern also noted in lambs (7). This oxygen demand is met by maintaining a very high rate of gut blood flow and thus oxygen delivery, a circumstance facilitated by low vascular resistance. Indeed, newborn gut vascular resistance, when expressed as a function of tissue weight, is approximately one-third that noted just a few months later (20, 21). These conditions may contribute to age-dependent variations in such vascular responses as autoregulation, flow-induced dilation, and the myogenic response to venous pressure elevation.

These postnatal circulatory conditions have been measured within whole organ preparations, either in vivo or in vitro, and thus represent the combined contributions of arterial, capillary, and venous portions of the gut circulation to total intestinal vascular resistance. Although an essential means to delineate the physiological relevance of vascular responses, this approach does not allow isolation of the sites or mechanisms of resistance regulation. This limitation might be especially germane to the gut circulation, wherein the bulk of resistance, and thus flow regulation, generally occurs in relatively discrete areas, e.g., the terminal mesenteric vessels, the intramural microvasculature, or both (9). Direct observation of those segments of the gut circulation responsible for resistance regulation might afford substantial insight into the mechanisms responsible for the postnatal changes characteristic of this vascular bed.

To this end, the goal of the present experiments was to determine the pressure and flow characteristics of terminal mesenteric arteries from 3- and 35-day-old swine. These vessels were chosen for study on the basis of preliminary in vivo observations that confirmed their functional role as resistance vessels in this species. These vessels were studied in vitro, within a perfusion chamber that allowed independent regulation of inflow and outflow pressures. We hypothesized that newborn terminal mesenteric arteries would demonstrate a greater degree of conductance in response to perturbations of pressure or flow. To test this hypothesis, four protocols were carried out: 1) incremental elevation of intravascular pressure, applied in the absence of flow; 2) elevation of flow rate, applied in absence of a significant change of intravascular pressure; 3) concomitant elevation of pressure and flow; and 4) generation of pressure-flow curves.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Care and Handling of Experimental Animals

Studies were conducted on swine of two postnatal ages, 3 days old (range 2-6 days) and 35 days old (range 32-40 days). These ages were selected on the basis of our previous work, which demonstrated significant differences in intestinal hemodynamics and oxygenation at these two postnatal ages (20, 21). Animals were obtained from local swine farms on the day before use. Anesthesia was induced with xylazine (5 mg/kg im) and telezol (7.5 mg/kg im) and maintained with pentobarbital sodium (5 mg/kg iv at intervals <60 min, based on need as assessed by vivarium staff). Animal care was provided in accordance with the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985], and all experimental protocols were approved by the Institutional Animal Care and Use Committee of the Children's Hospital Research Foundation.

In Vivo Studies

Anatomy of the swine mesenteric artery arcade. The swine mesenteric vasculature is substantially different from that of other species commonly used in studies of the intestinal circulation and so merits a brief description. Intestinal perfusion is derived from a single mesenteric artery, which gives rise to a series of short first-order branches whose initial diameters become progressively smaller in a proximal-to-distal gradient. All first-order branches enter an arterial plexus, which in 1- to 35-day-old swine is approximately 1-2 cm lateral to the mesenteric arterial trunk and is a site of extensive arterial collateralization (Fig. 1). This plexus is generally not appreciated on initial visual inspection of the swine mesentery because it lies buried beneath a compact network of lymph nodes. Arising from this plexus are a dense series of terminal arteries that run, unbranched, directly to their insertion sites within the gut wall. In contrast to the anatomic pattern characteristic of the dog, cat, and rat intestine, collateralization among arteries at their insertion site into the gut wall does not occur in swine. Instead, arterial collateralization is limited to the arterial plexus, from which arise the terminal arteries.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Anatomic configuration of the mesenteric arterial tree in swine. Note that first-order branches arising from mesenteric artery trunk are short and form a dense, highly collateralized arterial plexus. Terminal mesenteric arteries arise from the arterial plexus and course, unbranched, to their insertion site at the mesenteric border of the gut wall.

Measurement of vascular pressures in vivo. Artificial ventilation was initiated via tracheostomy and adjusted to maintain normal blood gas tensions, and arterial and venous catheters were placed to permit blood pressure monitoring and infusion of fluids (15 ml · kg-1 · h-1 of lactated Ringer solution). The pressure drop along the mesenteric artery system was determined by measuring pressure at six sites: 1) systemic arterial pressure, measured within the thoracic aorta just distal to the aortic valve; 2) the mesenteric artery; 3) a first-order branch of the mesenteric artery; 4) the arterial plexus; 5) the proximal end of the terminal mesenteric artery, within 0.5 cm of its origin in the arterial plexus; and 6) the distal end of the terminal mesenteric artery, just proximal to its site of gut wall penetration (Fig. 1). Different means were used to impale the vessel lumina, depending on their diameter. Systemic arterial pressure was measured by means of a plastic catheter inserted into the thoracic aorta via a carotid artery (V/12, 2 mm ID; Bolab, Lake Havasu, AZ). Pressures within the mesenteric artery trunk, first-order branch, and arterial plexus were measured with a narrow steel needle (27-gauge "butterfly" needle, 0.3 mm ID; Becton-Dickinson, East Rutherford, NJ) inserted into the vessel lumen and held in place by a hydraulic micromanipulator (MMO-203; Narishige, Tokyo, Japan). Pressures within the terminal mesenteric artery were measured with a glass micropipette, tip diameter 0.1 mm, inserted with the aid of a dissecting microscope and micromanipulator. In all instances the insertion devices were directly connected to a low-compliance pressure transducer (Gould, Cleveland, OH) by means of heparin and saline-filled, thick-walled Tygon tubing (1/16th in. ID, 3/16th in. OD). It should be noted that the glass micropipette tip diameter was substantially smaller than the arteriole lumen, even in the 3-day-old swine, by at least 0.75 mm, i.e., the pipette tip did not occlude the arteriolar lumen. In all instances, pressures were measured using the phasic mode on the amplifier until steady state was noted, and then the amplifier was switched to record mean pressure. All six sites were measured within each subject used in this protocol, and duplicate measurements were carried out in the smaller vessels. Measurements were made from smallest to largest vessels, and different arteries were impaled for proximal and distal measurements. These in vivo pressure measurements were made within the mesenteric vasculature of the distal small bowel, at the same anatomic site we have previously used to create in vivo or in vitro gut loops for whole organ perfusion studies (20, 21).

Measurement of mesenteric artery diameters in vivo. Measurement of the in vivo diameter of the mesenteric arteries was made before insertion of the glass pipette by means of a micrometer scale within the eyepiece of the dissecting microscope. Assessment of the diameters of larger mesenteric vessels was not carried out.

In Vitro Studies: Experimental Preparation

Vessel removal. Terminal mesenteric arteries were removed from the vasculature of the distal small bowel in anesthetized, ventilated animals and placed into oxygenated phosphate-buffered saline (23°C). Arteries were mounted in the proper proximal-to-distal anatomic orientation between two glass micropipettes seated within a plastic vessel chamber (CH/2/AS; Living Systems, Burlington, VT). The inflow pipette was fixed within the chamber, whereas the outflow pipette was mounted on a micrometer, which allowed adjustment along the long axis of the vessel. The vessel was secured to the pipette tips by 11-0 ophthalmic suture.

Terminal mesenteric artery perfusion. Arterial pressure and flow were adjusted by means of a pressure-servo system (PS/200/Q; Living Systems), using the configuration described by Koller et al. (15). This pressure-servo device consisted of a peristaltic pump, pressure transducer, and servo mechanism, which regulates pump speed to maintain a preset pressure at the transducer site. Two pressure-servo devices were incorporated in this preparation, one placed on either side of the arteriole so that inflow pressure (P1) and outflow pressure (P2) across the terminal mesenteric artery could be regulated independently. This arrangement allowed discrete manipulation of pressure and flow within the terminal mesenteric artery. The inflow and outflow circuits were set in mirror symmetry between the inflow and outflow pressure transducers, with the axis of symmetry taken as the center of the arteriole; thus, resistance generated by the apparatus between the cannulating pipette and pressure transducer was equal on both sides of the arteriole. Standard Krebs buffer of the following composition was used for arteriolar perfusion (in mM): 118.1 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2HPO4, 25.0 NaHCO3, 11.1 glucose, and 0.026 EDTA. The buffer was aerated with 95% O2-5% CO2, giving it a pH of 7.4 at 38°C.

Chamber suffusion. The vessel chamber, and thus the exterior surface of the terminal mesenteric artery, was continuously suffused with warm (38°C), aerated Krebs buffer. The temperature of the suffusion buffer was controlled by means of a heat exchanger. Suffusion buffer temperature was initially set at 23°C, and this temperature was slowly increased to 38°C during vessel stabilization. The suffusion buffer was recirculated at a rate of 50 ml/min into the vessel chamber, and the total volume of the suffusion system (i.e., reservoir, tubing, and chamber) was 200 ml. All vasoactive agents used in these experiments were added to the suffusion buffer reservoir in a final volume of 1 ml.

Measurement of terminal mesenteric artery diameter, pressure, and flow rate. The microvascular perfusion/suffusion chamber was mounted on the stage of an inverted microscope set in line with a video camera. Vascular dimensions were determined by a precalibrated video dimension analyzer (V94; Living Systems), which displayed wall thickness and lumen diameter (10). Flow rate across the terminal mesenteric artery was measured by a ball flowmeter (Omega, Putnam, CT) set in line with the outflow circuit. P1 and P2 were measured with standard transducers. The outputs from the video dimension analyzer and pressure transducers were recorded (Grass 7D polygraph, Quincy, MA).

In Vitro Studies: Protocols

Stabilization and determination of vessel viability. All terminal mesenteric arteries underwent the same stabilization, preconditioning, and viability assessment, regardless of the protocol in which they were eventually used. Arteries were initially pressurized to 20 mmHg in the absence of flow (P1 = P2 = 20 mmHg) and allowed to stabilize for 45 min, during which time the suffusion buffer temperature was slowly raised from 23°C to 38°C. P1 and P2 were then simultaneously increased to 80 mmHg and then decreased to 0 mmHg, both in steps of 20 mmHg. This initial series of incremental pressure steps was carried out quickly (<5 min) and was done to ensure the absence of leaks, kinks, or twists in the vessel at higher pressure, as well as to "precondition" the vessel (10). P1 and P2 were then set at 45 mmHg (3-day-old swine) or 53 mmHg (35-day-old swine). These basal pressures were chosen to duplicate in vivo conditions. Thereafter, three tests of vessel viability were carried out in each vessel: 1) constriction >25% in response to 10-7 M phenylephrine, 2) subsequent dilation of the phenylephrine-precontracted artery in response to 10-8 M substance P (>15%), and 3) >35% contraction in response to suffusion buffer containing 40 mM KCl. Terminal mesenteric arteries that did not meet all of these criteria were discarded. Each viable terminal mesenteric artery was used in only one of the following four protocols.

Protocol I. Incremental rise in pressure in the absence of flow. This protocol was designed to elicit a myogenic response to a change in intravascular pressure. P1 and P2 were first reduced to 0 mmHg. After vessel diameter reached steady state, P1 and P2 were increased in 20-mmHg increments to a maximal pressure of 100 mmHg. Each new pressure was maintained until arterial diameter reached a new steady-state level (generally 5-7 min), and in all instances flow across the terminal mesenteric artery remained at 0 µl/min; inflow and outflow pressures remained equal throughout the protocol. At the completion of the first series of incremental pressure steps P1 and P2 were returned to 0 mmHg, and the suffusion buffer was changed from standard Krebs to a Ca2+-free physiological salt solution containing ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (1.0 mM). Once the vessel had attained a new steady-state diameter, the pressure ramp was repeated to determine the passive characteristics of these vessels.

Protocol II. Increase in flow rate. This protocol was designed to elicit flow-mediated vasodilation. We were particularly interested in rapidly inducing flow from an initial rate of 0 µl/min, without causing a net change in the intraluminal pressure within the artery. To this end, the system was first pressurized in the absence of flow (i.e., P1 = P2), and then P1 was increased and P2 decreased by 5 mmHg. This action established a 10-mmHg pressure gradient across the vessel while, it was assumed, the net pressure within the vessel remained unchanged because of the symmetry of the perfusion system. This assumption was tested by mounting a plastic tube (0.2 mm ID) with a T-branch at its midpoint within the vessel chamber, in place of an artery. Thereafter, pressures across the tubing were changed as described: P1 from 45 to 50 mmHg and P2 from 45 to 40 mmHg. This action established flow (60 µl/min), while the pressure within the tube remained between 38 and 41 mmHg, validating our assumption. Arteries were initially pressurized in the absence of flow (i.e., P1 = P1) at age-appropriate pressures (i.e., 45 vs. 53 mmHg for 3- vs. 35-day-old swine) until diameter reached steady state. A 10-mmHg pressure gradient was then established as previously described, which caused flow rate to increase. The final flow rate established in each terminal mesenteric artery was a function of the initial vessel diameter, and no attempt was made to regulate this rate to a preset level. Flow was maintained until artery diameter reached a new steady-state level, after which pressures across the terminal mesenteric artery were equalized, returning the flow rate to 0 µl/min. Creation and elimination of the 10-mmHg pressure gradient was repeated twice in each artery. In a second set of animals, NG-monomethyl-L-arginine (L-NMMA) was added to the suffusion buffer (10-4 M) before initial creation of the pressure gradient, and L-arginine (10-3 M) was coadministered to the suffusion buffer before creation of the pressure gradient for the second time, to assess the role of nitric oxide (NO) in mediating the dilatory response to flow.

Protocol III. Incremental rise in pressure in the presence of flow. This protocol was designed to set into opposition myogenic vasoconstriction and flow-induced vasodilation. To this end, the protocol consisted of two phases. First, P1 and P2 were simultaneously raised from 0 to 80 mmHg in a manner identical to that described for protocol I, i.e., maintaining P1 = P2 and flow rate 0 µl/min to determine the response to a pure pressure stimulus. Second, the same series of incremental pressure steps was carried out, but this time in the presence of flow. To this end, P1 and P2 were initially set at 20 mmHg (flow rate 0 µl/min); thereafter, a 10-mmHg pressure gradient and thus flow were established across the vessel by increasing P1 and decreasing P2 by 5 mmHg. P1 and P2 were then simultaneously raised by 20-mmHg increments three times, which maintained the 10 mmHg pressure gradient but increased the net intravascular pressure from 20 to 40, 60, and 80 mmHg. To test this assumption, a 0.2-mm ID plastic tube with a T-branch at its midpoint was mounted in the vessel chamber, as described above, and the changes in P1 and P2 were carried out. As shown in Table 1, the assumptions proved valid.

Protocol IV. Pressure-flow ramp. In this protocol, P1 was increased from 20 to 100 mmHg without concomitant adjustment of P2. Inflow and outflow pressures were initially set at 20 mmHg, and vessel diameter was allowed to reach steady state. P2 was then reduced to 0 mmHg, establishing flow across the terminal mesenteric artery; thereafter, P1 was increased to 100 mmHg in progressive increments of 20 mmHg. No adjustment in the outflow circuit pressure was made during adjustment of inflow pressure, after P2 was initially reduced to 0 mmHg, so that P2 rose slightly as P1 and flow were increased. This circumstance was achieved by disabling the pressure-servo device in the outflow circuit, thus eliminating outflow pressure control.

Statistical Analysis

All data are presented as means ± SE. Statistical significance within each data set was determined by two-way analysis of variance (ANOVA), which utilized age group (3- vs. 35-day-old swine) and time (i.e., independently regulated pressure or flow variables) as main effects. If the overall F statistic for ANOVA was significant (P < 0.05), then post hoc t-tests were carried out to determine the sites of significance.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Pressure and flow (protocol III)

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

In vivo pressure and diameter measurements. Mean systemic pressure was age dependent, as expected, and this initial difference was reflected in all pressures within the mesenteric arterial system (Table 2). Both age groups demonstrated a significant pressure gradient within the terminal mesenteric artery; indeed, the proximal and distal terminal arterial pressures were the only pair of adjacent pressures that proved to be significantly different (Table 2). Localization of this pressure gradient to the terminal mesenteric artery qualifies these vessels as part of the resistance vasculature (1). The average arterial pressure, taken as a simple mean between proximal and distal values, was 45 ± 2 and 53 ± 3 mmHg in 3- and 35-day-old swine, respectively. The in vivo diameters of the terminal mesenteric arteries were also age dependent. In 3-day-old swine, the diameter at the midpoint between the arterial plexus and gut wall insertion site was 182 ± 12 µm, whereas in 35-day-old swine this diameter was 301 ± 17 µm. Finally, the length of the terminal mesenteric arteries was substantially greater in older (4.8 ± 0.3 cm) than in younger swine (2.7 ± 0.2 cm).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Intravascular pressures within the mesenteric arterial tree in 3- and 35-day-old anesthetized, ventilated swine

Protocol I

Active tone in response to a pure pressure stimulus (i.e., intravascular pressure in the absence of flow) was observed in arteries from both age groups, as evidenced by the difference between the pressure-diameter curves generated in standard Krebs buffer vs. those noted in Ca2+-free buffer (Fig. 2). The latter curves define the passive characteristics of these vessels. In both age groups, the maximal passive diameter was reached at intravascular pressures <60 mmHg; however, the maximal passive diameter was significantly greater in arteries from 35-day-old swine (221 ± 12 vs. 332 ± 21 µm in 3- vs. 35-day-old swine). The degree of active tone noted in response to an incremental rise in pressure was age dependent. Terminal mesenteric arteries from younger subjects demonstrated a reduction of diameter at pressures of 60 and 80 mmHg, i.e., they manifested a myogenic vasoconstriction. This effect was not noted in older subjects; consequently, the difference between active and passive tone was less in arteries from 35-day-old swine. Stated otherwise, terminal mesenteric arteries from younger swine demonstrated a greater vasodilator reserve when pressurized in the absence of flow.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of step increases in intravascular pressure, delivered in absence of flow, on diameter of terminal mesenteric arteries from 3-day-old (open symbols) and 35-day-old swine (solid symbols). These incremental step increases in pressure were run in standard Krebs buffer to determine the development of intrinsic myogenic tone (circles) and in a Ca2+-free physiological salt solution to determine the passive characteristics of the vessels (squares). Data are means ± SE; n = 7 for both age groups. F statistic for 2-way analysis of variance (ANOVA) was significant (P = 0.011); thereafter, within-group post hoc t-tests were carried out: * P < 0.05 vs. active; dagger  P < 0.05 vs. diameter at pressure = 40 mmHg.

Protocol II

The effect of flow rate on the diameter of terminal mesenteric arteries was also age dependent (Fig. 3). A pressure gradient of 10 mmHg was established by increasing P1 by 5 mmHg and decreasing P2 by 5 mmHg from the initial starting pressures of 45 and 53 mmHg in 3- and 35-day-old swine, respectively. As expected from the greater initial diameter of arteries from older swine, the flow rate established consequent to this pressure gradient was age dependent: 56 ± 4 vs. 97 ± 6 µl/min in 3- vs. 35-day-old swine. The ratio of flow rate to diameter, which in the presence of a cell-free perfusate provides an estimate of wall shear stress (13), was similar in both groups (0.32 vs. 0.34 in 3- vs. 35-day-old swine); hence, the net stimulus for flow-induced dilation, i.e., wall shear stress, was similar in both groups. Despite this similarity, the degree of flow-induced dilation, expressed as a percent change or in absolute terms, was greater in terminal mesenteric arteries from younger swine (27%, ~45 µm in 3-day-old vs. 7%, ~17 µm in 35-day-old swine). NO blockade studies carried out in a second group of swine with L-NMMA and L-arginine confirmed the important role of NO in mediating the dilation in response to flow (Table 3). The addition of L-NMMA (10-4 M) to the suffusion buffer caused a modest decrease in resting diameter when the vessels were perfused at age-appropriate pressures, in the absence of flow: -11 ± 3 vs. -6 ± 2% in 3- vs. 35-day-old swine, respectively. L-NMMA significantly attenuated the increase in arterial diameter in response to flow in both age groups. This effect was reversed by coadministration of L-arginine (10-3 M) to the suffusion buffer.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of initiation of flow rate on diameter of terminal mesenteric arteries from 3-day-old (open circle ) and 35-day-old swine (bullet ). Pressure and flow noted at each experimental time point are shown below each data point. Data are means ± SE; n = 7 for both age groups. F-statistic for 2-way ANOVA was significant (P = 0.034). Subsequent within-group analysis revealed a significant difference only within younger arterioles (* P < 0.05 vs. no flow).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Effects of L-NMMA and L-arginine on the response to an increased flow rate in terminal mesenteric arteries from 3- and 35-day-old swine

Protocol III

The response of terminal mesenteric arteries to the combined stimuli of pressure and flow was age dependent (Fig. 4). Both age groups demonstrated responses to pure pressure stimuli similar to those noted in protocol I (i.e., Fig. 2); thus, arteries from younger subjects demonstrated vasoconstriction in response to intravascular pressures >60 mmHg, whereas arterioles from older subjects did not. Repeating the pressure ramp in the presence of flow significantly changed the configuration of the pressure-diameter curve in arteries from 3-day-old swine, as progressive dilation was noted in place of constriction in response to all pressure increments when flow was present. In contrast, the presence of flow did not significantly alter the pressure-diameter relationship within arteries from 35-day-old swine. Although the pressure gradient across the terminal mesenteric arteries was maintained at 10 mmHg at each step increase in net intravascular pressure, the flow rate increased to a modest degree at higher pressures (Table 4). This change was somewhat more evident in arteries from younger subjects and was most likely a consequence of the effects of flow-induced dilation on net resistance across the terminal mesenteric artery.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Response of terminal mesenteric arteries from 3-day-old (open symbols) and 35-day-old swine (solid symbols) to step increases in pressure, delivered in absence (circles) or presence (squares) of flow. Although the pressure gradient across the artery was held constant at 10 mmHg throughout the 2nd phase of the protocol, flow rate did not remain static (see Table 4). Data are means ± SE; n = 6 for both age groups. F-statistic for 2-way ANOVA was significant (P = 0.021); thereafter, post hoc t-tests were carried out within group: * P < 0.05 vs. no flow.

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Pressure gradients and flow rates across terminal mesenteric arteries during protocol III

Protocol IV

Pressure-flow curves generated over the pressure range 20-80 mmHg were different in arteries from 3- and 35-day-old swine (Fig. 5). The curve generated for the younger group was concave with respect to flow (the y-axis), indicating that progressive vasodilation occurred as pressure was increased. This observation was readily confirmed by calculation of vascular resistance and Gf, a gain factor that relates pressure and flow variables (21), for each step increase in pressure (Table 5). Thus arterial resistance progressively decreased with each step increase in pressure, so that the rate of change of flow exceeded that of pressure. This circumstance defines the absence of autoregulation. In contrast, the pressure-flow curve generated in terminal mesenteric arteries of 35-day-old swine shows a modest convexity with respect to flow (shown on the y-axis), suggesting the opposite relationship between these variables. Vascular resistance across these older arteries was essentially static at each step increase in pressure, whereas the Gf data revealed a very modest degree of autoregulation, i.e., that the change in flow rate was less than the corresponding change in pressure.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 5.   Relation between pressure and flow within terminal mesenteric arteries from 3-day-old (open circle ) and 35-day-old swine (bullet ). Data were obtained by raising inflow pressure delivered to the artery without concomitant manipulation of outflow pressure. Pressure, resistance, and Gf (flow-controlling gain factor) generated during this protocol are shown in Table 5. Data are means ± SE, presented as mean flow rate noted for each group at each of 4 applied inflow pressures; n = 5 for both age groups. Although curves appear different (i.e., one is slightly convex to the y-axis, and the other is slightly concave), statistical analysis (i.e., ANOVA) failed to demonstrate any significant difference. This similarity was confirmed by regression analysis of individual data points. Both groups were best represented by a linear equation; for 3-day-old swine, flow = (3.34)(pressure) - 58.5, r2 = 0.95; for 35-day-old swine, flow = (2.92)(pressure) + 9.6, r2 = 0.98. These regression equations were not statistically different (ANOVA).

                              
View this table:
[in this window]
[in a new window]
 
Table 5.   Vascular pressures, vascular resistance, and Gf noted in terminal mesenteric arteries from 3and 35-day-old swine during protocol IV

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The principal new observation of these experiments is that the distalmost portion of the mesenteric arterial tree, i.e., the terminal mesenteric arteries, demonstrates significant age-dependent differences in resting dimension and intravascular pressure in vivo and also displays unique responses to pressure and flow stimuli in vitro. In discussing these data, three important caveats must be acknowledged. First, it may not be appropriate to compare the responses of mesenteric arterioles from 3- and 35-day-old swine because of the substantial difference in their in vivo diameters. A small part of this difference may reflect the modestly higher in vivo pressure noted within older arterioles. A greater role, however, is certainly played by anatomic differences: the maximal passive diameter (i.e., pressurized in vitro arteries suffused with Ca2+-free EGTA buffer) is 100 µm greater in 35-day-old than in 3-day-old swine. The importance of initial vessel diameter on vascular responses has been clearly noted in other regional circulations (8, 12). Second, these observations are limited to the responses of terminal mesenteric arteries; more specifically, they do not include the true intestinal microvasculature within the gut wall. The in vivo observations certainly suggest that terminal mesenteric arteries studied herein function as resistance vessels, as evidenced by the significant pressure drop noted across these vessels (1). It is most probable, however, that additional sites of resistance regulation exist within the intramural microvasculature, as has been demonstrated in rats by Gore and Bohlen (9). Third, these experimental protocols applied incremental increases, but not decreases, in pressure and flow. This approach was selected to maximize the probability of inducing significant vascular responses; thus myogenic vasoconstriction in response to increased pressure and flow-mediated dilation in response to increased flow tend to be of greater magnitude than their more subtle counterparts, i.e., myogenic-based dilation after pressure reduction and vasoconstriction in response to diminished flow. It may not be appropriate to predict the effects of pressure or flow reduction based solely on the observations presented in this report. These caveats affirmed, how might these data add to our current understanding of the developmental physiology of the intestinal circulation?

First, it is clear that the development of intrinsic or myogenic tone in response to intravascular pressure is age dependent within terminal mesenteric arteries, as evidenced by the greater difference between passive and active diameters and the presence of vasoconstriction in response to pressures >60 mmHg in younger subjects. This difference does not necessarily predict in vivo mesenteric vascular resistance, because numerous other stimuli, such as adrenergic innervation (24), clearly contribute to basal vascular tone in vivo. It is interesting, however, that the in vivo arteriolar diameters were quite similar to those noted in vitro, when age-appropriate pressures were applied in the absence of flow. One interpretation of this observation is that extrinsic vasoactive stimuli may not play a dominant role in setting basal vascular tone in postnatal terminal mesenteric arteries. Supporting this possibility are the data of Buckley et al. (2), who demonstrated only a minimal change in intestinal vascular resistance after acute denervation in very young swine. On the other hand, the age-dependent difference noted within in vitro terminal mesenteric arteries clearly fails to predict the net response of the postnatal gut circulation to rapid pressure elevation in vivo. Thus Crissinger et al. (5) have demonstrated an equivalent degree of myogenic vasoconstriction in response to rapid elevation of venous pressure in 3- and 35-day-old swine. The disparity between the present in vitro data and previous in vivo observations underscores the fact that intestinal vascular regulation occurs at sites other than the terminal mesenteric artery.

The fact remains, however, that the response of these vessels to a pure pressure stimulus was age dependent, a circumstance which suggests that the mechanism(s) responsible for intrinsic myogenic tone may undergo postnatal change, at least within terminal mesenteric arteries. Several mechanisms have been proposed as mediators of intrinsic myogenic tone, including pressure-sensitive ion channels (6) and endothelium-derived stimuli (11). Clearly, it is beyond the scope of this report to discuss the probability that postnatal change in these or other mechanisms participated in the observed age-dependent differences. A question more reasonably pondered is not how but why the difference is present. What physiological advantage is served by reducing intrinsic myogenic tone in older arteries? Clearly, maintenance of terminal mesenteric arterial diameter close to its maximal level would enhance its vascular conductance, thus limiting the pressure drop down the vessel. This circumstance might serve to counterbalance the loss of pressure expected consequent to the significantly greater length of the terminal mesenteric artery from its origin at the arterial plexus to its insertion site at the gut wall noted in older subjects. This circumstance might assure that vascular pressure at the onset of the intramural microvasculature is adequate, an important condition in light of the substantially greater size (i.e., diameter and wall thickness) of the intestine in older subjects. This advantage would be especially important in arteries in older subjects, in which the degree of flow-mediated dilation is minimal.

The second age-dependent difference noted between terminal mesenteric arteries from 3- and 35-day-old swine is their response to an increase in flow rate. Flow-mediated dilation has been recognized in large and small arteries (25) and arterioles (13) and appears to be mediated by wall shear-stress induced augmentation of NO release by the endothelium (3, 4). The purpose of this vascular response appears to be maintenance and extension of vasodilation, and hence augmentation of oxygen delivery in response to tissue work. According to this model, vasodilation is initiated downstream within the true microvasculature, possibly by metabolic factors (25), a circumstance that increases flow rate through upstream vessels; these vessels then dilate in response to flow, thus enhancing the net increase in oxygen delivery to the parenchyma. In this context, it might be anticipated that terminal mesenteric arteries from newborn swine would demonstrate a greater magnitude of flow-mediated dilation, because intestinal oxygen delivery and tissue uptake are significantly greater at this postnatal age (20, 21). This age-dependent difference might also have been predicted on the basis of basal and stimulated NO production by the postnatal gut endothelium; thus, Nankervis and Nowicki (19) have demonstrated a greater degree of relaxation in precontracted mesenteric artery rings from 3- than from 35-day-old swine in response to NO-dependent dilator agents. The essential role of NO causing flow-mediated dilation was confirmed in these experiments, as evidenced by loss of the response during NO production blockade with L-NMMA and the return of the response after coapplication of L-arginine.

Application of a combined increase of intravascular pressure and flow rate completely eliminated myogenic vasoconstriction within terminal mesenteric arteries from 3-day-old swine, supplanting this constriction with substantial vasodilation. The ability of flow-mediated dilation to offset myogenic vasoconstriction has been observed previously (13-17, 22, 23, 25) and is particularly strong in tissues demonstrating large oxygen demand, such as the heart (16, 17). The capacity of newborn terminal mesenteric arteries to override myogenic constriction may explain, in part, why these vessels could afford the luxury of substantial active tone when pressurized in the absence of flow. The presence of myogenic vasoconstriction would reduce flow and thus oxygen delivery, a circumstance seemingly unfavorable to a tissue with substantial oxidative demand. However, under physiological conditions of combined pressure and flow, dilation supervenes. Although this logic seems tenable, it is important to remember that the present observations were made within buffer-perfused vessels, i.e., that hemoglobin was absent in the system. Hemoglobin acts as an important scavenger of NO; thus, under true in vivo conditions, the extent of NO-mediated, flow-induced dilation might be somewhat less than that noted in these experiments. In contrast to the newborn condition, older subjects demonstrate significantly lower rates of intestinal oxygen delivery and uptake (20, 21). Thus they might not require the same degree of gain in the flow-mediated dilatory apparatus.

The third difference between 3- and 35-day-old terminal mesenteric arteries was their pressure-flow relationships. The pressure-flow curve generated in newborn arteries was very much reminiscent of that noted in blood-perfused in vitro intestinal loops, i.e., in both instances Gf values for each pressure step were substantially negative, indicating the absence of autoregulation (21). We previously suggested that the lack of autoregulation in newborn intestine represented the absence of an effective metabolic mechanism at this early postnatal age (20, 21). This mechanism, however, cannot be invoked to explain the behavior of isolated, buffer-perfused arteries, because it requires parenchymal elaboration of a dilatory metabolic feedback signal (24). Instead, it would appear that the pressure-flow curve of newborn terminal mesenteric arteries represents the balance between active tone, which dominates at low flow rates, and flow-mediated dilation, which dominates at high flow rates. Such a circumstance suggests that the newborn intestine is specifically designed to exist at relatively high flow rates and that it is relatively incapable of preserving perfusion at low pressures and flow rates. The pressure-flow curve generated in terminal mesenteric arteries from 35-day-old swine is also suggestive of that noted in blood-perfused in vitro gut loops; the curve is convex with respect to the flow, or y-axis (21). Furthermore, both gut loops and mesenteric terminal arteries from older subjects demonstrated a modest degree of autoregulation, as evidenced by positive Gf values. However, instead of this circumstance reflecting more effective metabolic vascular regulation, as previously suggested for gut loops (21), the terminal mesenteric arterial data can be explained by the presence of modest active tone in conjunction with a limited flow-mediated dilation.

    ACKNOWLEDGEMENTS

We thank David Dunaway for expert technical assistance and Cecilia Batten for secretarial support.

    FOOTNOTES

Address for reprint requests: K. Reber, Section of Neonatology, Children's Hospital, 700 Children's Dr., Columbus, OH 43205.

Received 19 June 1997; accepted in final form 20 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bevan, J., W. Halpern, and M. Mulvany. The Resistance Vasculature. Totowa, NJ: Humana, 1991.

2.   Buckley, N., P. Brazeau, I. Frasier, and P. Gootman. Circulatory effects of splanchnic nerve stimulation in developing swine. Am. J. Physiol. 248 (Heart Circ. Physiol. 17): H69-H74, 1985[Medline].

3.   Buga, G., M. Gold, J. Fukuto, and L. Ignarro. Shear stress induced release of nitric oxide from endothelial cells grown on beads. Hypertension 17: 187-192, 1991[Abstract].

4.   Cooke, J., J. Stamler, N. Andon, P. Davies, G. McKinley, and J. Loscalzo. Flow stimulates endothelial cells to release a nitrovasodilator that is potentiated by reduced thiol. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H804-H812, 1990[Abstract/Free Full Text].

5.   Crissinger, K., P. R. Kvietys, and D. N. Granger. Developmental intestinal vascular responses to venous pressure elevation. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G658-G663, 1988[Abstract/Free Full Text].

6.   Davis, M., J. Donovitz, and J. Hood. Stretch-activated single-channel and whole cell currents in vascular smooth muscle cells. Am. J. Physiol. 262 (Cell Physiol. 31): C1083-C1088, 1992[Abstract/Free Full Text].

7.   Edelstone, D., and I. Holzman. Oxygen consumption by the gastrointestinal tract and liver in conscious newborn lambs. Am. J. Physiol. 240 (Gastrointest. Liver Physiol. 3): G297-G304, 1981[Abstract/Free Full Text].

8.   Faraci, F. Role of endothelium-derived relaxing factor in cerebral circulation: larger arteries vs. microcirculation. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1038-H1042, 1991[Abstract/Free Full Text].

9.   Gore, R., and H. G. Bohlen. Microvascular pressures in rat intestinal muscle and mucosal villi. Am. J. Physiol. 233 (Heart Circ. Physiol. 2): H685-H693, 1977[Free Full Text].

10.   Halpern, W., G. Osol, and G. Coy. Mechanical behavior of pressurized in vitro prearteriolar vessels determined with a video system. Ann. Biomed. Eng. 12: 463-479, 1984[Medline].

11.   Harder, D. Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium. Circ. Res. 60: 102-107, 1987[Abstract].

12.   Hwa, J., L. Ghibaudi, P. Williams, and M. Chatterjee. Comparison of acetylcholine-dependent relaxation in large and small arteries of rat mesenteric vascular bed. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H952-H958, 1994[Abstract/Free Full Text].

13.   Koller, A., and G. Kaley. Endothelial regulation of wall shear stress and blood flow in skeletal muscle microcirculation. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H862-H868, 1991[Abstract/Free Full Text].

14.   Koller, A., and G. Kaley. Endothelium regulates skeletal muscle microcirculation by a blood flow sensing mechanism. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H916-H921, 1990[Abstract/Free Full Text].

15.   Koller, A., D. Sun, and G. Kaley. Role of shear stress and endothelial prostaglandins in flow- and viscosity-induced dilation of arterioles in vitro. Circ. Res. 72: 1276-1284, 1993[Abstract].

16.   Kuo, L., W. Chilian, and M. Davis. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1706-H1715, 1991[Abstract/Free Full Text].

17.   Kuo, L., M. Davis, and W. Chilian. Endothelium-dependent, flow-induced dilation of isolated coronary arterioles. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1063-H1070, 1990[Abstract/Free Full Text].

18.   Lebenthal, E. Human Gastrointestinal Development. New York: Raven, 1989.

19.   Nankervis, C., and P. Nowicki. Role of nitric oxide in regulation of vascular resistance in postnatal intestine. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G949-G958, 1995[Abstract/Free Full Text].

20.   Nowicki, P., and C. Miller. Effect of O2 availability on intrinsic vascular response to venous pressure elevation in postnatal swine intestine. Am. J. Physiol. 258 (Gastrointest. Liver Physiol. 21): G873-G877, 1990[Abstract/Free Full Text].

21.   Nowicki, P., and C. Miller. Autoregulation in the developing postnatal intestinal circulation. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G189-G193, 1988[Abstract/Free Full Text].

22.   Pohl, U., K. Herlan, A. Huang, and E. Bassenge. EDRF-mediated shear-induced dilation opposes myogenic vasoconstriction in small rabbit arteries. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H2016-H2023, 1991[Abstract/Free Full Text].

23.   Pohl, U., J. Holtz, J. Busse, and E. Bassenge. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 8: 37-42, 1986[Abstract].

24.   Shepherd, A. P., and D. N. Granger. Physiology of the Intestinal Circulation. New York: Raven, 1984.

25.   Smiesko, V., D. Lang, and P. C. Johnson. Dilator response of rat mesenteric aracading arterioles to increased blood flow velocity. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1958-H1965, 1989[Abstract/Free Full Text].


AJP Gastroint Liver Physiol 274(2):G290-G298
0193-1857/98 $5.00 Copyright © 1998 the American Physiological Society