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Am J Physiol Regul Integr Comp Physiol 284: R734-R741, 2003. First published December 5, 2002; doi:10.1152/ajpregu.00510.2002
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Vol. 284, Issue 3, R734-R741, March 2003

Maturation depresses mouse cerebrovascular tone through endothelium-dependent mechanisms

Greg G. Geary, John N. Buchholz, and William J. Pearce

Departments of Physiology and Pharmacology, Center for Perinatal Biology, School of Medicine, Loma Linda University, Loma Linda, California 92354


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In light of previous observations that the range of arterial pressures over which cerebral blood flow is autoregulated differs dramatically in neonates and adults, the present experiments explored the hypothesis that pressure-induced intrinsic arterial tone is regulated differently in neonatal and adult cerebral arteries. In cannulated and pressurized endothelium-intact mouse cerebral arteries <150 µm in diameter, active intrinsic tone was evident at intraluminal pressures as low as 10 mmHg in neonatal arteries, but only at pressures of 60 mmHg or greater in adult arteries. Administration of 10 µM indomethacin produced no significant effect on tone at any pressure in either neonatal or adult arteries, but subsequent addition of 100 µM nitroarginine methyl ester (NAME) significantly vasoconstricted both neonatal and adult arteries at all pressures. Conversely, administration of 100 µM NAME alone significantly vasoconstricted adult arteries only, and subsequent addition of 10 µM indomethacin produced a significant additional vasoconstriction in adult arteries only, indicating an important interaction between the nitric oxide synthase and cyclooxygenase pathways, at least in adult arteries. In the presence of both indomethacin and NAME, intrinsic tone was significantly greater in neonatal than adult arteries, but when the endothelium was removed, tone was similar in neonatal and adult arteries at all pressures. Together, these results suggest that pressure-induced myogenic tone is regulated similarly in neonatal and adult mouse cerebral arteries but that the contribution of endothelial vasoactive factors to intrinsic tone is highly age dependent.

neonatal mouse; myogenic tone; pressure; cerebral artery


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NOT LONG AFTER the establishment of the nitrous oxide method for measurement of human cerebral blood flow in the late 1950s, it was widely recognized that cerebral perfusion was regulated independent of arterial pressure between pressures of approx 60 and approx 160 mmHg (22, 25, 37). This pattern of regulation, termed "cerebral autoregulation," has since been the focus of more than 2,600 studies that together indicate involvement of multiple different mechanisms in this response. Most important among these are the myogenic mechanisms that transduce changes in intraluminal hydraulic pressure and/or vascular wall stresses into changes in vascular smooth muscle contractility (10, 16). Release of vasoactive substances from the vascular endothelium in response to changes in shear stress also undoubtedly contributes to cerebrovascular autoregulatory adjustments (13). In addition, autonomic neurovascular mechanisms can potently influence the arterial pressure range over which cerebral autoregulation is effective (6).

Owing to the central role of cerebral autoregulation for maintenance of cerebrovascular homeostasis, insults that compromise cerebral autoregulation are associated with numerous patterns of neurological morbidity. This relation appears to be particularly important in the neonate, where autoregulatory failure is associated with multiple cerebral pathologies including periventricular leukomalacia and germinal matrix hemorrhage (4, 35). Despite the obvious importance of cerebral autoregulation in the neonate, however, the mechanisms involved remain poorly understood, due in large part to the fact that most studies of autoregulation have focused on mature, and not immature, experimental models. From what little is known with certainty about neonatal cerebral autoregulation, it is clear that it operates over a much narrower and lower range of arterial pressures (30 to 50 mmHg) and thus may be governed by a different combination of mechanisms than are involved in the adult cerebral circulation (3). For example, both perivascular adrenergic nerves (28) and the vascular endothelium (27) are less capable of influencing cerebrovascular tone in immature, than in mature, cerebral arteries.

The present studies explore two of the main categories of the mechanisms potentially involved in determination of intrinsic tone in immature cerebral arteries. Because the relation between arterial pressure and intrinsic tone in small immature cerebral arteries remains largely unstudied, we examined the relations between perfusion pressure and artery diameter in cannulated cerebral arteries <150 µm in diameter taken from neonatal and adult mice. In light of the potent ability of the vascular endothelium to influence vascular tone, we also examined the effects of endothelium denudation, as well as inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX), on the relations between pressure and artery diameter. Together, these approaches suggest that both myogenic and endothelial influences on intrinsic tone change dramatically during postnatal maturation in mouse cerebral arteries.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General Preparation

The Animal Research Committee of Loma Linda University approved all procedures. Male neonatal (4-8 days old) and adult (6-8 wk old) C57 Black mice (Jackson Laboratory, Bar Harbor, ME) were housed under a 12:12-h light-dark cycle with food and water available ad libitum. Brains were rapidly removed from decapitated mice and placed in cold physiological salt solution (PSS) containing (in mM) 130 NaCl, 10.0 HEPES, 6.0 glucose, 4.0 KCl, 4.0 NaHCO3, 1.8 CaCl2, 1.8 KH2PO4, 1.2 MgSO4, and 0.025 EDTA at pH 7.4. Middle cerebral artery segments 1 mm in length were dissected, cannulated, and mounted in an organ chamber (Living Systems, Burlington, VT) positioned on an inverted microscope. Internal diameters of all arteries were measured by videomicroscopy (Ionoptix, Milton, MA).

The proximal cannula was connected to a pressure transducer and a windkessel reservoir of PSS whose pressure was controlled by a servo system used to set transmural pressures. The distal cannula was connected to a luer-lock valve that was open to gently flush the lumen during the initial equilibration. After equilibration, the valve was closed and all measurements were conducted under no-flow conditions.

Endothelium Removal

In arteries from both adult and neonatal mice, the endothelium was removed by gently rubbing the artery lumen with a glass cannula. Successful removal of the endothelium was verified by the absence of a vasodilator response to 10 µM ADP, an endothelium-dependent vasodilator in this preparation.

Experimental Protocols

In all protocols, artery diameters were measured in response to a series of 10-mmHg pressure steps from 10 to 80 mmHg. Where indicated, NG-nitro-L-arginine-methyl ester (L-NAME), EDTA, and indomethacin were added to the organ chamber 20 min before commencing a series of pressure steps, and each pressure step was maintained for 5-10 min to allow vessel diameter to stabilize before measurement. All drugs were purchased from Sigma Chemical (St. Louis, MO) and were added, individually or in combination, to the organ chamber in their final optimally effective concentrations (31, 34). Three separate protocols were conducted.

Protocol 1. This protocol was designed to determine pressure-diameter relationships in the absence and presence of NOS inhibitors in endothelium-intact arteries from both neonates and adults. As previously described (15), the first series of pressure steps was completed in PSS, and a second series was completed in the presence of NAME (100 µM). To assess the additivity of NOS and COX inhibition, a third series of pressure steps was completed in the presence of 100 µM NAME plus 10 µM indomethacin. A final pressure-diameter relationship was conducted in Ca2+-free PSS with EDTA (3 mM) to determine the maximum passive diameters.

Protocol 2. This protocol was designed to determine pressure-diameter relationships in the absence and presence of COX inhibitors in endothelium-intact arteries from both neonates and adults. As described for protocol 1, the first series of pressure steps was completed in PSS, after which a second series was completed in the presence of 10 µM indomethacin. To assess the additivity of NOS and COX inhibition, a third series of pressure steps was completed in the presence of 10 µM indomethacin plus 100 µM NAME. A final pressure-diameter relationship was conducted in Ca2+-free PSS with EDTA (3 mM) to determine the maximum passive diameters.

Protocol 3. This protocol determined the pressure-diameter relationship in the absence of the endothelium and thus assessed myogenic tone in adult and neonatal arteries. Endothelium-denuded arteries from both neonates and adults were continuously exposed to 100 µM NAME plus 10 µM indomethacin throughout these experiments. After development of a stable diameter, a series of pressure steps was completed to determine the pressure-diameter relationship. After this run, a second series of pressure steps was completed in Ca2+-free PSS with EDTA (3 mM) to determine the maximum passive diameters.

Data Analysis and Statistics

Contractile effects of indomethacin or NAME were determined by subtracting artery diameter after drug treatment from artery diameter before drug treatment at any given pressure. Percent intrinsic tone was determined in endothelium-intact arteries by subtracting the diameter at any given pressure from the maximum passive diameter (obtained in zero calcium with 3 mM EDTA at 80 mmHg) and dividing the difference by the maximum passive diameter. Similarly, percent myogenic tone was determined in endothelium-denuded arteries by subtracting the diameter at any given pressure from the maximum passive diameter (obtained in zero calcium with 3 mM EDTA at 80 mmHg) and dividing the difference by the maximum passive diameter. Because the conditions for the first series of pressure steps were identical in protocols 1 and 2 (PSS only), the data from these measurements were pooled within each age group. Similarly, because the conditions for the third series of pressure steps were identical in protocols 1 and 2 (NAME + indomethacin), the data from these measurements were also pooled within each age group. Data are expressed as means ± SE. Statistical significance was determined using ANOVA with Scheffé's test for post hoc comparisons. Statistical significance implies P < 0.05 unless otherwise stated.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A total of 21 neonatal and 24 adult mice was used in these experiments. Animal ages averaged 45.5 ± 2.0 and 5.3 ± 0.3 days for adult and neonatal mice, respectively. Corresponding values for body weights averaged 21.3 ± 0.6 and 2.8 ± 0.2 g, respectively. Adult and neonatal brain weights averaged 536 ± 9 and 249 ± 13 mg. Control diameters in PSS (10 mmHg) for arteries from neonatal and adult mice were 87 ± 5.9 and 117 ± 2.7 µm, respectively.

Time Control Responses to Repeated Pressure Challenges

In intact adult arteries equilibrated at 40 mmHg in PSS, a single step increase in pressure to 80 mmHg yielded average diameter values of 129 ± 3.5 µm (n = 3). When the arteries were returned to 40 mmHg, equilibrated, and then exposed to a second single step increase in pressure to 80 mmHg, the final diameters averaged 124 ± 4.9 µm (n = 3). Similarly, a third single step increase in pressure to 80 mmHg yielded average diameters of 133 ± 2.6 µm (n = 3). Corresponding responses of neonatal arteries to three consecutive single step pressure increases from 20 to 50 mmHg yielded average diameters of 81 ± 5.8, 88 ± 4.1, and 89 ± 7.1 µm (n = 3). Thus, diameter responses to step changes in pressure were highly reproducible and fully reversible in both adult and neonatal arteries.

Passive Diameters and Intrinsic Tone in Intact Neonatal and Adult Arteries

Passive endothelium-intact artery diameters, measured in PSS without Ca2+ and with 3 mM EDTA, were significantly larger in adult than in neonatal arteries at all pressures examined (Fig. 1A). In the presence of 1.8 mM Ca2+, adult arteries developed significant active intrinsic tone, but only at pressures at or above 60 mmHg. In contrast, Ca2+-replete neonatal arteries developed significant active intrinsic tone at all pressures between 10 and 70 mmHg. When the contractile effects of pressure were expressed as percent intrinsic tone (Fig. 1B), the magnitudes of intrinsic tone observed in endothelium-intact arteries were significantly less in adult than in neonatal arteries at all pressures except 80 mmHg, where forced dilation was observed in the immature arteries.


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  		Fig. 1.   Effects of intraluminal pressure on cerebral arteries from neonatal (5 days old) and adult (45 days old) mice. A: arterial diameters of endothelium-intact arteries in physiological salt solution (PSS; open symbols) are compared with passive diameters in 0 Ca2+ + 3 mM EDTA (filled symbols). B: percent intrinsic tone in PSS is compared between endothelium-intact cerebral arteries from neonatal and adult mice. Percent intrinsic tone was calculated as a percent difference between maximum passive diameter and endothelium-intact diameters in PSS. In all experiments, arteries were exposed to EDTA solution for 20 min before determining passive responses to pressure. Values are means ± SE, n = 9-10. *P < 0.05 compared with respective passive response by ANOVA. #P < 0.05 compared with respective adult response by ANOVA.

Effects of NOS Inhibition in Intact Neonatal and Adult Arteries

The addition of 100 µM L-NAME to adult arteries equilibrated in PSS significantly reduced the diameters observed at all pressures between 20 and 80 mmHg (Fig. 2A). The absolute magnitudes of these changes ranged from 20.0 to 23.7 µm. In contrast, L-NAME had no significant effects on the diameters of neonatal arteries at any pressure between 20 and 80 mmHg; the magnitudes of the L-NAME-induced decreases in diameter ranged from only 6.1 to 12.3 µm (Fig. 2B).


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  		Fig. 2.   Effects of maturation on constriction to indomethacin (Indo) in the presence of NG-nitro-L-arginine-methyl ester (NAME). Diameter responses to increasing transmural pressures of endothelium-intact cerebral arteries treated with 100 µM NAME alone, or 10 µM Indo in the presence of NAME, are shown in A for 45-day-old adult and in B for 5-day-old neonatal mice. C: averaged constrictions in micrometers to Indo in the presence of NAME at different transmural pressures. Arteries were exposed to drug solutions for 20 min before step increases in transmural pressure were applied. Values indicate means ± SE, n = 9. *Significant (P < 0.05) differences between treatments as determined by repeated-measures ANOVA. #Significant (P < 0.05) differences between corresponding adult and neonatal values, as determined by a factorial ANOVA.

In adult arteries equilibrated with 10 µM indomethacin, the addition of 100 µM L-NAME again significantly reduced the diameters observed at all pressures between 20 and 80 mmHg (Fig. 3A). The absolute magnitudes of these decreases ranged from 31 ± 3 to 35 ± 2 µm (Fig. 3C). As indicated by repeated-measures ANOVA, pretreatment with indomethacin significantly enhanced the magnitudes of the L-NAME-induced decreases in diameters at all pressures (P < 0.001) (Figs. 2A and 3A).


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  		Fig. 3.   Effects of maturation on constriction to NAME in the presence of Indo. Diameter responses to increasing transmural pressures of endothelium-intact cerebral arteries treated with 10 µM Indo alone or 100 µM NAME in the presence of Indo are in A for 45-day-old adult and in B for 5-day-old neonatal mice. C: averaged constrictions in micrometers to NAME in the presence of Indo at different transmural pressures. Arteries were exposed to drug solutions for 20 min before step increases in transmural pressure were applied. Values indicate means ± SE, n = 9. *Significant (P < 0.05) differences between treatments as determined by repeated-measures ANOVA. #Significant (P < 0.05) differences between corresponding adult and neonatal values, as determined by a factorial ANOVA.

In neonatal arteries equilibrated with 10 µM indomethacin, the addition of 100 µM L-NAME significantly decreased the diameters observed at all pressures (Fig. 3B). The absolute magnitudes of these decreases ranged from 7 ± 2 to 18 ± 2 µm and were significantly less than observed in adult arteries at all pressures (Fig. 3C).

Effects of COX Inhibition in Intact Neonatal and Adult Arteries

The addition of 10 µM indomethacin to adult arteries equilibrated in PSS had no significant effect on artery diameter (Fig. 3A). Similarly, indomethacin had no significant effects on the diameters of neonatal arteries at any pressure between 20 and 80 mmHg (Fig. 3B).

In adult arteries equilibrated with 100 µM L-NAME, the addition of 10 µM indomethacin significantly reduced the diameters observed at all pressures between 20 and 80 mmHg compared with diameters in L-NAME (Fig. 2A). The absolute magnitudes of these decreases ranged from 24 ± 1 to 26 ± 1 µm (Fig. 2C). Repeated-measures ANOVA showed that pretreatment with L-NAME revealed a significant contractile effect of indomethacin, which did not appear with indomethacin alone (P < 0.001; Figs. 2A and 3A). Artery diameters from adult mice in L-NAME plus indomethacin were significantly smaller than those diameters in PSS (Fig. 2A).

In neonatal arteries equilibrated with 100 µM L-NAME, the addition of 10 µM indomethacin tended to decrease artery diameters observed at all pressures, but none of these decreases were significant from diameters in L-NAME alone (P > 0.05; Fig. 2B). The absolute magnitudes of these decreases ranged from 12 ± 1 to 14 ± 2 µm, and were significantly less than observed in adult arteries at all pressures (Fig. 2C). Artery diameters from neonatal mice in L-NAME plus indomethacin were significantly smaller than those diameters in PSS (Figure 2B).

NOS- and COX-Independent Intrinsic Tone

After treatment with 10 µM indomethacin plus 100 µM L-NAME or L-NAME plus indomethacin, intrinsic tone was determined from adult and neonatal arteries (Fig. 4). Because final drug combination L-NAME plus indomethacin (Fig. 2, A and B) or indomethacin plus L-NAME (Fig. 3, A and B) did not affect calculation of NOS- and COX-independent intrinsic tone, data were combined from these experiments. In adult arteries, intrinsic tone ranged from 32 ± 3 to 35 ± 3% at all pressures. In neonatal arteries, intrinsic tone ranged from 42 ± 4 to 43 ± 3% between 20 and 60 mmHg. Between 20 and 60 mmHg, intrinsic tone was significantly less in adult than in neonatal arteries.


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  		Fig. 4.   Effects of maturation on intrinsic tone in the presence of combined nitric oxide synthase and cyclooxygenase inhibition. Intrinsic tone was calculated as a percentage difference between maximum passive diameter and diameter in the presence of both 100 µM NAME and 10 µM Indo in endothelium-intact cerebral arteries from 45-day-old adult and 5-day-old neonatal mice. Error bars indicate SE for n = 9. #Significant (P < 0.05) differences between corresponding adult and neonatal values, as determined by a factorial ANOVA.

Endothelium-Independent Myogenic Tone

To verify efficient endothelium denudation, we determined responses to the endothelium-dependent dilator ADP (10 µM). In endothelium-intact arteries, the magnitudes of vasodilation to ADP were significantly greater in adult (66 ± 5%) than in neonatal (43 ± 2%) arteries. After endothelium removal, ADP did not produce a significant dilation in either adult or neonatal arteries. Despite the absence of a functional endothelium, both adult and neonatal arteries exhibited little change in artery diameters as pressure was increased in 20-mmHg steps from 20 to 80 mmHg (Fig. 5), indicating preserved myogenic reactivity. After endothelial denudation, however, age-related differences in myogenic tone were not significant (P > 0.05).


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  		Fig. 5.   Effects of intraluminal pressure on myogenic tone in endothelium-denuded cerebral arteries from 45-day-old adult and 5-day-old neonatal mice. Myogenic tone was calculated as a percentage difference between maximum passive diameters and diameters in PSS alone in endothelium-denuded arteries. Error bars indicate SE for n = 9.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Postnatal maturation dramatically influences smooth muscle contractility through gradual changes in many different vascular characteristics. Some postnatal changes involve shifts in artery hydration, protein content, and structure, whereas others affect receptor density, calcium metabolism, or endothelial function (1, 29, 30, 33). Much of what is known about the vascular consequences of postnatal maturation, however, has been learned through studies of large resistance-sized cerebral arteries with diameters greater than 350 µm. Arteries smaller than 150 µm in diameter also contribute to vascular resistance and blood flow distribution but have yet to be examined in vitro from neonatal animals. For these reasons, very little is currently known of the contractile properties of immature arteries smaller than 150 µm. The present study addresses this deficit and demonstrates that endothelial contributions to intrinsic tone in small cerebral arteries are markedly different in mature and immature mice.

Intrinsic Tone in the Immature Cerebral Microcirculation

Although previous studies of microvascular contractility focused heavily on responses to specific vasoconstrictor or vasodilator agonists and blockers, the present study focuses on the mechanisms governing pressure-induced intrinsic tone. Intrinsic tone is responsible for the basal level of vascular resistance against which vasodilator metabolites can act, and in many respects it may be more important physiologically than the efficacy or potency of any single contractile or relaxant agonist. Intrinsic tone is determined in large part by pressure-sensitive myogenic tone, which, in turn, is determined solely by the physical effects of pressure and deformation on vascular smooth muscle cells (10). Intrinsic tone also includes the influences of both vasoconstrictor and vasodilator molecules released from the vascular endothelium. In the cerebral and coronary circulations, the ability to autoregulate blood flow over a broad range of perfusion pressures is almost completely dependent on regulation of intrinsic tone (10, 14). Because cerebral autoregulation in the neonate is vulnerable to numerous different insults, and loss of cerebral autoregulation is closely associated with neurological morbidity, regulation of intrinsic tone is immediately relevant to strategies for clinical management of neonates surviving from, or at risk for, cerebrovascular insults.

As indicated in Fig. 1, neonatal mouse cerebral arteries developed significant intrinsic tone at transmural pressures as low as 10 mmHg, and the magnitude of this tone remained constant between pressure 20 and 60 mmHg. In contrast, adult mouse cerebral arteries developed significant intrinsic tone only at transmural pressures of 60 mmHg or greater, and even at 80 mmHg the tone developed in adult arteries was less than in neonatal arteries at 10 mmHg. These results reveal that both the sensitivity to pressure and the magnitude of intrinsic tone were significantly greater in neonatal than in adult mouse cerebral arteries. From a teleological perspective, these results reflect a matching between the pressure range over which intrinsic tone is developed and mean arterial pressures that average 80 mmHg in adults but only 30 mmHg in neonates (18, 19). From a mechanistic perspective, the observed age-related differences in pressure sensitivity could be explained by corresponding differences in the contributions of either myogenic tone or endothelial factors to the development of intrinsic tone.

Effects of NOS and COX on Intrinsic Tone

Two important vasoactive factors released from the endothelium, NO and prostaglandins, are synthesized by the enzymes endothelial NOS (eNOS) and COX, respectively (13). Within blood vessels, eNOS is found predominantly in endothelial cells but can also be expressed in vascular smooth muscle cells (8). Similarly, COX activity is distributed mainly within endothelial cells but may also be expressed within smooth muscle (2, 17), particularly in the neonate (5). Owing to these heterogeneous patterns of distribution, it is advantageous to eliminate the contributions of eNOS and COX to pressure-dependent responses through the use of enzyme inhibitors. At concentrations previously shown to be effective in our preparation (15), indomethacin alone had no significant effect on the relations between pressure and diameter in either adult or neonatal arteries (Fig. 2). However, when NAME was added in the presence of indomethacin, the relation between pressure and diameter was shifted to significantly smaller diameters in both age groups. Consistent with reports of reduced endothelium-dependent vasodilator capacity (39) and reduced endothelial expression of eNOS (27) in immature compared with mature cerebral arteries, the vasoconstrictor effects of NAME were significantly greater in adult than in neonatal arteries (Fig. 2C). Prima fascia, these results suggest that NO, but not COX products, modulates resting and pressure-dependent intrinsic tone in mouse cerebral arteries and that the magnitude of this contribution increases with postnatal age.

In light of numerous reports of possible interactions between NO and prostaglandin-dependent pathways (11, 38), we carried out an additional series of experiments in which the order of addition of the inhibitors was reversed. When NAME was administered first, the relation between pressure and diameter was shifted to significantly smaller diameters but only in adult arteries (Fig. 3). Subsequent addition of indomethacin produced further reductions in artery diameters, but these effects were significant only in adult arteries. Alone, these results suggest that both NO and COX products modulate resting and pressure-dependent intrinsic tone, but only in adult arteries. Together with the results shown in Fig. 2, these results strongly indicate a significant interaction between the NO and prostaglandin-dependent pathways present in mouse cerebral arteries.

On the basis of previous findings, one potential mechanism of interaction between the eNOS and COX pathways is the possible binding of NO to the heme iron of COX (20). Binding to heme iron is a common mechanism whereby NO alters the activity of many enzymes (9, 26) including the soluble guanylate cyclase that in large part mediates endothelium-dependent vasodilatation (12). If basal NO release were able to bind the iron in COX and thereby inhibit its activity, then application of indomethacin might not be expected to have any further effect, as indicated in Fig. 3. On the other hand, NAME addition alone could eliminate NO production and relieve inhibition of COX, in which case subsequent addition of indomethacin might yield vasoconstriction, as indicated in Fig. 2. Consistent with this scheme, NO has been shown to inhibit prostaglandin synthesis in macrophages (32). Although plausible, this hypothesized interaction between NO and COX is highly speculative and must be carefully validated through numerous additional experiments.

Another important result of the NAME and indomethacin (Fig. 2) and indomethacin and NAME (Fig. 3) experiments was that, following combined drug treatment, intrinsic tone remained significantly greater in arteries from neonatal compared with adult mice (Fig. 4). This finding not only indicates the reproducibility of the methods employed but also the presence of an important additional influence on intrinsic tone that is independent of both eNOS and COX. Although it is possible that this age-dependent influence derived mainly from myogenic mechanisms, it is also possible that endothelial factors other than NO or prostanoids were involved because the endothelium was intact in these arteries.

Endothelial Contributions to Intrinsic Tone

To separate endothelial from myogenic contributions to intrinsic tone, we determined pressure-diameter relations in neonatal and adult arteries denuded of endothelium. In the absence of endothelium, significant myogenic tone was observed at all pressures examined in both age groups (Fig. 5). More importantly, the relations between myogenic tone and pressure were not significantly different in neonatal and adult arteries. This finding suggests that the basic mechanisms governing myogenic tone are probably quite similar in neonatal and adult arteries and that the age-dependent differences observed in intrinsic tone are most likely due to maturational differences in the contributions of endothelial vasoactive factors. In view of the results shown in Fig. 4, there must be at least one vasoconstrictor factor independent of eNOS and COX whose release is greater in neonatal than adult arteries. Alternatively, there must be at least one vasodilator factor independent of eNOS and COX whose release is significantly less in neonatal than adult arteries. In either case, it is clear that neonatal and adult cerebral arteries produce significant and similar levels of myogenic tone but that this tone is modulated in a highly age-dependent manner by at least three different vasoactive factors released from the vascular endothelium. The identity of the NOS- and COX-independent factor(s) remains a subject for future experimentation but could possibly be either endothelin or endothelium-derived hyperpolarizing factor (21, 23, 24).

The current study is the first to show that maturation increased endothelial dependent modulation of pressure-induced intrinsic tone without an apparent effect on the smooth muscle contractile machinery. Increasing endothelial function with maturation has been described in pig and human models. For example, prostanoid-dependent dilation mediated nearly all the endothelium-dependent relaxation in newborn pigs while NO-dependent dilatory mechanisms increased in arteries from juvenile pig (36). Vasodilatory prostanoids also appeared to be the primary substances regulating cerebrovascular tone in the human neonate. With maturation, the relative importance of prostanoid-dependent modulation declined, which corresponded with increased NOS-sensitive modulation of tone in adult human cerebral arteries (7).

Overall, the present experiments indicate that neonatal and mouse cerebral arteries develop significant but similar levels of myogenic tone. This tone is modulated in a pressure- and age-dependent manner by the release of at least three different endothelial vasoactive factors. Endothelial release of NO appears to be significant in both age groups, but it is greater in adult than in neonatal arteries. Endothelial release of COX products also appears to be significantly greater in adult than in neonatal arteries. The relative magnitudes of the effects of NO and indomethacin-sensitive products, however, may be difficult to estimate due to significant interactions between these two pathways. In addition, the endothelium also appears to release at least one additional factor that either preferentially increases intrinsic tone in immature arteries or preferentially decreases intrinsic tone in adult arteries. The identity of this third endothelial factor awaits future experimentation, as does the mechanism mediating NO-COX interactions. Owing to the fact that the arteries used in the present studies were not continuously perfused and thus had little or no endothelial shear stress, it is possible that the contributions of endothelial factors to intrinsic tone may be greater in vivo than observed in the present in vitro experiments. Aside from these uncertainties, it is clear that intrinsic tone is actively governed in immature cerebral arteries by multiple independent mechanisms that should constitute a fruitful substrate for future investigations of both physiological and pathophysiological cerebrovascular regulation in the neonate.


    FOOTNOTES

Address for reprint requests and other correspondence: G. G. Geary, Dept. of Physiology and Pharmacology, School of Medicine, Loma Linda Univ., Loma Linda, CA 92354 (E-mail: ggeary{at}som.llu.edu).

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. Section 1734 solely to indicate this fact.

First published December 5, 2002;10.1152/ajpregu.00510.2002

Received 23 August 2002; accepted in final form 28 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 284(3):R734-R741
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