Main

It is generally accepted that adult human and animal cerebral circulations autoregulate(1) and that intrinsic myogenic tone plays an important role in this process(2). Autoregulation has been demonstrated in the fetal lamb(3) and the newborn dog(4, 5), and there is evidence of active regulation of cerebral blood flow in the preterm newborn(6) and infant(7), although the effectiveness of this control is debated. An increasing number of studies show changes in the mechanisms involved in the control of the cerebral circulation during maturation and growth(8–11). In the mature animal cerebrovascular tone has been shown to be influenced by perivascular nerve activity(12–14), endogenous vasoactive substances(15, 16), and by intraluminal flow(17, 18).

We assessed the stretch-dependent myogenic tone that developed in isolated segments of the basilar and middle cerebral arteries of eight infants who at birth ranged from 23 wk of gestation to term and whose postnatal survival ranged from 1 h to 34 d. Segments were studied in two experimental systems, the wire myograph(19, 20) and a pressure perfusion arteriograph(21). The main conclusion is that infant cerebral arteries exhibit high levels of spontaneous tone. The tone can be reduced by increases in intravascular flow, acetylcholine, β-adrenoceptor activation, histamine, papaverine, and an indomethacin-sensitive mechanism.

METHODS

This study was approved by the Ethics Committee of the University of Vermont. After consent from the parents upon the decease of the infant, the cerebral arteries were obtained during autopsy. The cranial contents were removed according to the method of Valdes-Dapena and Huff(22). In all cases the removal of the blood vessels was completed within 2 h of death. The leptomeninges at the base of the brain were carefully dissected to expose the major arteries. Portions of the middle cerebral and basilar arteries were removed and placed in oxygenated, cold PSS[composition (in mM): NaCl 130, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, NaHCO3 14.9, EDTA 0.026, dextrose 11.0] containing deferoxamine (100 μM), heparin (10 U/mL), penicillin (50 U/mL), and streptomycin (50 μg/mL). They were transferred to the laboratory and prepared for in vitro study. Only segments free of adherent blood were studied. They were either examined on the same day of removal or after storage for 24 h in PSS at 4°C.

Isometric myograph technique. Arteries were cut into rings 3-4 mm long, suspended on wires in a resistance artery myograph(19, 20), and maintained in PSS at 37°C, pH 7.4± 0.1, continuously gassed with 95% O2/5% CO2. Each ring was connected to a force transducer (Grass FT03, Quincy, MA), and changes in isometric force were recorded on a strip chart recorder. Internal diameter was measured using a video camera (Colorado video micrometer). The myograph-mounting wires were slowly separated until a just significant change in the force record was observed. Wire separation was taken to be half the unstretched circumference of the artery segment.

Because segments from different infants varied in their internal diameter and the time required for determination of the optimum length of the muscle cells from studies of the active length-tension relationship in each case would compromise the subsequent experimentation, the rings were stretched to an internal diameter obtained from the relationship between diameter and optimum length determined in prior experiments on a large series (n> 40) of adult human pial arteries (J. A. Bevan, unpublished data). The rings were then allowed to equilibrate for 1 h at this preload optimum before the experimental protocol was initiated.

The majority of the segments spontaneously developed tone, and the size and characteristics were noted. The effect on this tone of a number of drugs added to the tissue bath was studied. In addition, PSS was infused into the vessel lumen by a Harvard syringe infusion pump (model 22) through a glass micropipette the tip of which was positioned just within 0.3 mm of one end of the segment. Flow rates were those that had produced medium effective responses in adult pial vessels of similar diameter(18). If no response was obtained, the effect of increases and decreases in flow rates was explored(23).

Pressure-perfusion arteriograph. Segments 1.5-3.0 mm in length were mounted in a pressure-perfusion system arteriograph(21). The proximal end of the artery was attached to a cannula, and the segment flushed with PSS by raising the proximal pressure to 5-10 mm Hg. The free end of the artery was then attached to the distal cannula, and the artery was pressurized to 60 mm Hg after measuring the internal diameter (see below). The segment was then superfused with PSS at 2-3.5 mL/min and warmed up from room temperature to 37°C. The wall diameter was imaged using a video-dimension analyzer (Living Systems Instrumentation, Burlington, VT) linked to a chart recorder. This is identical to the technique used to study human adult pial arteries(18). In the majority of segments myogenic tone began to develop after 0.5-1 h and progressed to a steady state over the subsequent 1-2 h. The effects of drugs added to the superfusate were observed. In a few segments an attempt was made to establish the relationship between pressure and diameter by changing transmural pressure in increments of 10-20 mm Hg in random sequence over the range of 30-90 mm Hg. Only in two segments was this technically successful. The steady state diameter that was achieved several minutes after the change in pressure was considered the equilibrium response. During the course of the series of pressure changes, intravascular pressure was occasionally returned to one or more standard levels between 30 and 90 mm Hg to determine the consistency of response. Then the segment was superfused with K+ (127 mM) PSS to produce a maximum contraction, and a sequential order of pressure steps to 120 mm Hg at increments of 10 or 20 mm Hg was instituted, with diameter measurements made at each step. This provided the relationship between pressure and diameter during maximum wall force. Finally, to maximally relax the smooth muscle preparatory to determining the passive pressure-diameter relationship of the vessel, the superfusate was changed to Ca2+-free PSS containing EDTA (2 mM). When dilatation was maximal, indicating that all stretch-induced tone was lost, the intravascular pressure was changed in increments as before, spanning the entire range used in the study and lumen diameter measured at each pressure step.

Statistics. Some data are presented as means ± SE of n segments.

RESULTS

Observations were made on cerebral artery segments obtained from eight infants aged at birth between 23 wk of gestation and term. In Table 1 the diagnosis of each infant is noted and if postnatal survival was longer than 1 d. The majority of segments were studied within 24 h of removal, but a significant number were held at 4°C to the 2nd d when more segments were available than could be examined at 24 h. This is a common practice in our laboratory when studying adult pial arteries. No distinction is made between the results obtained on the 1st and 2nd d as they were quantitatively and qualitatively similar. The results of studies on the basilar and middle cerebral arteries are pooled.

Table 1 Clinical diagnosis of infants
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A summary of some of the data from the study of the infants is presented in Table 2.

Table 2 Spontaneous vascular tone and its modification in human infant cerebral arteries
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Manifestations of spontaneous vascular tone.Myographmounted segments. Some manifestation of spontaneous tone development was seen in arterial segments from all infants. Of the 55 segments studied, 47 showed some evidence of such activity. Because of the extreme variability in size and time course and its inconsistency during an experiment, it is difficult to quantitate the myogenic tone that occurs in myographmounted segments (Fig. 1), whereas that which develops in perfused segments (see below) is usually maintained and can be measured. Many segments spontaneously developed a maintained level of tone, often shortly after they had been stretched in the myograph to their optimum length. This spontaneous tone lasted for a variable length of time up to 1 h. Such tone also appeared spontaneously during the course of the experiment, unconnected to stretch or other experimental manipulation. Frequently the tone plateau showed superimposed, sometimes regular oscillatory behavior. Occasionally, rhythmic, fairly regular waves starting from baseline tone levels were observed that varied widely in size, frequency, and persistence(for example, patterns similar to those shown in Figs. 2 and 3). In two instances it was demonstrated that spontaneous tone was uninfluenced by tetrodotoxin (3 × 10-7 M), an Na+ channel blocker. In two segments the addition of prazosin (10-6 M) did not influence the level of maintained tone.

Figure 1
figure 1

Infant 3. Spontaneous period of increased tone in the middle cerebral artery of the infant. Gestational age was 30 wk. Dilatation to acetylcholine (10-5 M). W, wash.

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Figure 2
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Infant 5. Period of raised tone with regular and irregular oscillations of tone occurring after a small stretch (20 mg). Middle cerebral artery from infant of 32 wk of gestation.

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Figure 3
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Infant 5. Periodic rhythmic, complex increases in wall force in middle cerebral artery from infant of 32 wk of gestation after exposure to indomethacin (10-5 M).

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Pressure perfused segments. Seven out of 10 segments subjected to an intraluminal pressure of 60 mm Hg developed maintained tone. The three that did not show this tone increase also failed to respond to vasocontrictor and vasodilator drugs. The mean level of tone was 38.5 ± 8.6% (n= 7) of the maximum possible constriction seen with PSS containing 127 mM K+. This is a level not significantly different from that found in human adult pial arteries of similar lumen diameter exposed to this pressure(18).

The relationship between intramural pressure and equilibrium tone for a segment from infant no. 7 (37 wk of gestation) is shown in Figure 4. Like adult pial arteries of similar lumen size(18), infant artery segments tend to maintain their diameter over the pressure range 30-80 mm Hg. As passive diameter in calcium-free PSS increases with pressure rise, this observation indicates an increase in force development as the pressure increases within this range.

Figure 4
figure 4

Infant 6. Pressure-diameter relationship of human infant middle cerebral artery aged 36.5 wk of gestation plus 13 d. The artery was subjected to changes in transmural pressure in random steps between 30 and 80 mm Hg. Pressure steps were repeated in the presence of PSS containing 127 mmol/L of KCl (circle symbols) to obtain the maximum pressure-diameter relationships and finally repeated over a wider pressure range of 5-100 mm Hg in the presence of Ca2+-free PSS containing 2 mmol/L EGTA (triangle symbols) to obtain the maximum passive diameter at each pressure value.

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Effect of indomethacin. An early observation in this study that greater increases in tone occurred after the inhibition of PG synthesis with indomethacin in the infant, compared with adult pial artery segments (R. D. Bevan and J. A. Bevan, unpublished data), led to an emphasis on this characteristic. Indomethacin (10-5 M) added to isometrically mounted segments from six of seven infants studied caused an increase in tone that in five was maintained indefinitely. The mean level was 31.9 ± 8.3%(n = 6). In several segments, this tone increase was close to the maximum level of force attainable. Only one segment of infant no. 4 was tested, and indomethacin caused no change in basal tone. Other segments from this infant failed to develop tone in the pressure-perfusion system. These segments had outer clotted blood removed before study.

Indomethacin (10-5 M) was added to pressure-perfused segments with maintained tone from three infants. In all instances it caused further constriction.

Effect of various pharmacologic agents.Acetylcholine. In normal adult human pial artery segments with intact endothelium, acetylcholine causes relaxation of intrinsic and agonist-induced tone. In all infant segments at the beginning of each experiment, the effectiveness of acetylcholine (10-7 to 10-5 M) to reverse norepinephrine-induced tone (3 × 10-6 M) was used as part of a preliminary assessment of their reactivity. Mean values for acetylcholine-induced dilatation of segments from each infant was 87.3± 5.2%(9) of preaddition tone. In perfused infant vessels, acetylcholine (10-6 M) reduced spontaneous tone in the three segments in which it was tested by 88.0 ± 3.8% of the preaddition level. Early attempts to remove the endothelium from infant arteries by mechanical rubbing or by the air-blowing technique were unsuccessful without some damage to the muscle layer. Because other functional features were considered of higher priority, this aspect was not pursued. No segments were used in which the acetylcholine reversal of agonist-induced tone was less than 50%.

Isopropylnorepinephrine (10-8 to 10-5 M). In three infants (nos. 3, 4, and 8), isopropylnorepinephrine caused relaxation of spontaneous myogenic tone of 67.3 ± 7.0%(3). This effect was reversed by drug removal, and it was also blocked by prior exposure to propranolol (10-5 M) (Fig. 5).

Figure 5
figure 5

Infant 6. (A) Effect of indomethacin on active force development of segment of middle cerebral artery of infant, 36.5 wk of gestation plus 13 postnatal days. (B) Dilatation to increasing concentrations of isoproterenol. (C) After the addition of propranolol (10-5 M), dilatation to isoproterenol is reduced.

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Papaverine (10-5 M). Papaverine was tested in segments of infants nos. 3 and 8, in which high levels of spontaneous tone developed, and it caused a maximum dilatation of 99.3 ± 0.7%(3). The effect was reversible.

Histamine. Histamine was a potent dilator of the cerebral artery segments, the maximal effective concentration was 3 × 10-6 M(93.3 ± 5.3%)(7) (Fig. 6). In several instances higher concentrations reversed the dilatation to contraction as is seen in adult pial arteries of similar size (our unpublished observations).

Figure 6
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Infant 4. Effect of histamine on spontaneously developed tone of middle cerebral artery of infant of 30 wk of gestation.

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L-Nω-Nitro-L-arginine methyl ester (3 × 10-4 M). L-Nω-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthesis, had a variable effect on resting tone in isometrically mounted segments from the three infants studied. In these segments the endothelium was pressumed to be functional as maximal acetylcholine dilatation was 87.1 ± 7.2%. Basal tone was increased(mean 16.7 ± 7.0%) and maintained in infants nos. 6 and 7, and also responses to norepinephrine were potentiated. No change in basal tone was seen in the basilar artery segment of infant no. 5.

Effect of intraluminal flow. The effect of changing the flow of PSS through the lumen of isometrically mounted segments of arteries appears to be quantitatively similar to that in adult human pial and rabbit cerebral arteries. Flow-induced contraction was demonstrated in segments from four infants when the level of tone was low (see for example Fig. 7), and in several instances this was repeatable. Flow-induced dilatation was demonstrated in spontaneously contracted segments and also when the level of tone was augmented by indomethacin (infant no. 3). It was also seen in perfused arteries exhibiting tone (infant no. 4). As the level of tone preceding flow was increased, the response to flow reversed from contraction to dilatation. This is an important feature of the flow response in the adult also. The variability in the flow-induced change in the isometrically mounted vessels precluded quantitative analysis.

Figure 7
figure 7

Infant 5. Effect of intraluminal flow of PSS in the middle cerebral artery of infant, 32 wk of gestation.

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Passive pressure/diameter curves. At the end of a perfusion experiment PSS was replaced by a solution in which the calcium was omitted. These perfused segments do not develop myogenic tone. The relationship between artery diameter and intraluminal pressure was determined by changing the pressure in increments of 10-20 mm Hg in random sequence over the range 5-100 mm Hg. These data provide the passive pressure/diameter curves shown in Figure 8. They reflect the distensibility or elastic characteristics of the artery segment.

Figure 8
figure 8

Passive pressure/diameter curves from infant middle cerebral and basilar (2 and 5) arteries. Vessels were placed in Ca2+-free PSS containing 2 mM EGTA to prevent active tone generation.

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DISCUSSION

Observations on stretch-initiated and/or spontaneous vascular smooth muscle tone have been made on segments of human basilar and middle cerebral arteries of eight infants whose ages at birth ranged from 23 wk of gestation to term, with postnatal survival from 1 h to 34 d. The tone was a consistent, pronounced feature of myograph-mounted and cannulated segments. Using the latter technique, a tone level equivalent to that seen in human adult arteries of similar diameter was observed(18). The small number of infants studied and the varied reasons for limited postnatal survival precludes making definitive conclusions. In our experience this type of tone is seldom demonstrated in small segments of myograph-mounted adult human arteries of similar size, which gives some confidence to the conclusion that it is an important characteristic of infant cerebral arteries. The tone was extracellular calcium-dependent, and because it was not influenced by tetrodotoxin (3 × 10-7 M) or prazosin (10-6 M), was not due to activation of perivascular catecholamine-containing nerves. The tone was inhibited by vasodilator agents with differing modes of action: acetylcholine, histamine, isopropylnorepinephrine, and papaverine. In several segments it was reduced by intraluminal flow. Because the addition of indomethacin was seen to initiate tone, or potentiate preexisting tone in segments of both basilar and middle cerebral arteries, it can also be inhibited by an endogenous indomethacin-sensitive mechanism.

In general, the main features of myogenic tone in the infant resemble that seen in adult animals and human pial arteries obtained during surgery(18, 24–26). The level of myogenic tone in the perfused segments was not significantly different from that of arteries of similar diameter in the adult. At 60 mm Hg intraluminal pressure, the mean level of myogenic tone of responding infant pial arteries of 400-μm average segment diameter was approximately 60%(24). There are, however, differences between the human infant and adult arteries that we have studied. Maintained tone of the dimension seen consistently in the infant occurs only occasionally in myographmounted adult human pial artery segments and is rarely seen in vessels from other species(27). Human adult pial arteries frequently exhibited periodic spontaneous contractions, which were associated with vascular smooth muscle membrane depolarization and the generation of action potentials(28). Similar patterns of spontaneous tone were observed in the infant arteries (see Figs. 1–3).

In a study of the myogenic tone of a 100-μm inside diameter branch of the posterior cerebral artery of 4-7-d neonatal rats, myogenic responses to transmural pressure changes were effective in a range of pressures appropriate to the age and were accompanied by spontaneous rhythmic vasomotion(29). Elevation of intravascular pressure induced greater increases of myogenic tone than those observed in adults.

Indomethacin is an inhibitor of cyclo-oxygenase and hence the synthesis of PGs, and is effective in the medical treatment of patent ductus arteriosus in premature infants. It has been reported to reduce cerebral blood flow by a mean of 24% in premature infants(30). Van Bel et al.(31) used Doppler ultrasound to study blood flow velocity changes in the superior mesenteric and anterior cerebral arteries of 15 preterm infants with symptomatic patient ductus arteriosus before and after indomethacin administration. They found a similar decrease in mean blood flow in both arteries and reasoned that the changes were not secondary to ductus closure. In a study by Saliba et al.(32) of mechanically ventilated premature infants, indomethacin caused a significant decrease in Doppler mean frequency in the anterior cerebral artery within minutes of starting the infusion. The authors point out that this could be not only due to vasoconstriction of the cerebral arteries distal to the recording site, but to decreases in cardiac output or an effect on the patent ductus. They concluded that it was most likely that indomethacin modified cerebral hemodynamics.

PGs may participate in the regulation of fetal hemodynamics as well as the patency of the ductus and in the circulatory changes that occur during development. There are significant age-related differences in the responses of arteries to PGs in premature, newborn and adult baboons(33–36). The vasodilator response to low concentrations of PGE, PGE2, and PGF2α decrease during development although PGI2 dilator effectiveness is not age-related. The constrictor responses to PGs increase with age. The sheep fetus has a higher concentration of PGE in its plasma than does the pregnant ewe, but it falls rapidly after birth(37). Prostanoids promote pial arteriolar dilatation and mask constriction to oxytocin in piglets(38). This latter observation is consistent with the prostanoid masking of myogenic tone in our studies and indomethacin potentiation of agonist-induced tone in the same vessels (R. D. Bevan and J. A. Bevan, unpublished data).

In adult human pial vessels and various arteries from animals, myogenic tone is inhibited by papaverine, which acts by inhibiting phosphodiesterases and prolonging the activity of cAMP and cGMP. Stretch-induced tone is reversed by acetylcholine, which is usually considered to act by stimulating the endothelial cell production of nitric oxide, but which can also hyperpolarize the smooth muscle membrane by other mechanisms(39). Selective endothelium removal was not achieved in these studies. Histamine is an extremely powerful inhibitor of human infant and adult pial artery tone (R. D. Bevan and J. A. Bevan, unpublished data). The very effective reversal of spontaneous tone by isopropylnorepinephrine, mediated by β-adrenoceptors, contrasts with most adult pial artery segments, which are unresponsive(40). Considerable changes take place in adrenergic mechanisms with maturation and growth including a decrease inα-adrenoceptor-mediated responses(41). Hayashi et al.(42) found that in the baboon maximum dilatation to isopropylnorepinephrine was 63% in the premature, 72% in the newborn, and 10% in the adult.

The passive pressure/diameter curve of the infant arteries has the same general shape as those obtained with adult arteries(43). Pearce et al.(44) carried out a detailed study of the common carotid and cerebral arteries from near-term and newborn lambs and adult sheep. They found a progressive increase in stiffness with increasing age, although the changes were least in the cerebral arteries. This change in compliance has been attributed to an increase in collagen-to-elastin ratio or increase of collagen cross-linkage(45).

In summary, we have found that myogenic tone is a prominent feature of isolated segments of human basilar and middle cerebral arteries of premature newborn infants. This tone is markedly inhibited by an endogenous indomethacin-sensitive mechanism, and this tone can also be inhibited by acetylcholine, histamine, papaverine, and isopropylnorepinephrine and modified by intraluminal flow. As the tone can be induced or potentiated by indomethacin, this indicates that in vivo the vascular tone can be modulated by an indomethacin-sensitive dilator system.