INTRODUCTION

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DEVELOPMENTAL CHANGES occur in the mechanisms governing cerebrovascular regulation. Pearce and co-workers (26) have described developmental changes in the composition and reactivity of the cerebral artery of sheep. Hayashi and colleagues (11), using isolated cerebral artery strips from premature, newborn, and adult baboons, reported a maturational decrease in norepinephrine- and acetylcholine-induced constriction and isoproterenol-induced dilation. Others have also shown age-related declines in the sensitivity to adrenergic stimulation in sheep (35) and pigs (34), which appear to be due to the lack of ₁-adrenoceptors in the adult animal. Transmural stimulation and addition of exogenous norepinephrine to isolated porcine cerebral artery strips have been reported to produce vasodilation rather than vasoconstriction and were shown to be mediated by ₁- rather than ₁-adrenoceptors (39). Developmental changes in pulmonary responses also have been reported. Liu and co-workers (19) demonstrated a biphasic developmental augmentation of the response of pig pulmonary artery rings to acetylcholine. Rings from 3- to 10-day-old piglets constricted significantly more than neonatal rings to acetylcholine, but the constrictions began to decrease with maturation to adulthood. An augmented constrictor response to acetylcholine was reported in pig pulmonary rings with age that was not biphasic, but the oldest age group in this study was 30 days postnatal (41).

Considerable information is available concerning the role of nitric oxide (NO) as a regulator of the cerebral circulation. Hypercapnia-induced dilation of pial vessels appears to be NO dependent in various adult animal models, including the rabbit (8), cat (28), and rat (13). In the newborn piglet, however, hypercapnia- and histamine-induced dilations and acetylcholine-induced constrictions of pial arterioles are prostanoid dependent (18). Furthermore, a permissive influence for prostanoids in these dilator and constrictor responses in newborn piglets has been shown (2, 17, 18). Little attention has been given to possible changes in the regulation of cerebral blood flow that may occur with maturation. Zuckerman et al. (42) used the closed cranial window to examine possible maturational effects on the pial arterial responses to acetylcholine and hypercapnia in newborn and juvenile pigs. A biphasic response to acetylcholine was seen in juveniles (immediate short-lived constriction, followed by prolonged dilation), but the later dilation seen in the juveniles was not observed in the newborn. The NO synthase inhibitor N-nitro-L-arginine (L-NNA) had no effect on the acetylcholine response in newborn piglets but was able to block the prolonged dilator response in the juveniles, indicating that at least a portion of the response in the juveniles is NO dependent. Intravenous administration of the cyclooxygenase inhibitor indomethacin, however, was able to completely block constriction in both newborns and juveniles (while augmenting the dilatory response in juveniles), suggesting that acetylcholine-induced constriction in newborns and juveniles is prostanoid dependent. Similarly, hypercapnia-induced dilation of pial arterioles was found to be completely prostanoid dependent in the newborn and to have a prostanoid- and an NO-dependent component in the juvenile. This set of experiments suggests there is a diminishing role for dilator prostanoids and the emergence of a role for NO as the pig matures from neonate to an adult.

Therefore, we hypothesize that the endothelium-derived relaxing factor (EDRF) predominating to provide a permissive dilatory signal to the underlying vascular smooth muscle is altered during maturation from neonate to juvenile. Experiments were designed to 1) determine whether mechanisms involved in histamine- and/or hypercapnia-induced dilations involve prostanoids and/or NO in newborn piglet and/or juvenile pig cerebral circulation, 2) compare and contrast cerebrovascular responses from newborns and juveniles to hypercapnia and histamine, and 3) determine whether the mechanisms by which prostanoids and NO work are permissive or more classical in mediating juvenile dilatory responses that require prostanoids and/or NO.

	METHODS

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The animal protocols used were reviewed and approved by the Animal Care and Use Committee of the University of Tennessee, Memphis. Thirty female juvenile (4-5 mo of age, 52.2 ± 1.3 kg) and twenty-six newborn piglets (1-3 days old, 1.9 ± 0.1 kg) were used in this series of experiments. Animals were anesthetized with ketamine and acepromazine, and anesthesia was maintained with -chloralose (30-40 mg/kg initially, supplemented with 7 mg · kg¹ · h¹). A catheter was placed in the femoral artery to record systemic blood pressure and to sample for arterial blood gas and pH. Another catheter was placed in the femoral vein to permit administration of anesthesia and experimental drugs. Animals underwent a tracheotomy with an endotracheal tube inserted and were mechanically ventilated with room air. Core temperature was monitored with a rectal probe and maintained between 37.5 and 38.0°C.

Cranial window placement. After the catheter placement and tracheotomy was completed, the scalp was surgically removed, and a 2-cm-diameter hole was cut in the skull over the parietal cortex. The dura was cut and reflected over the cut bone edge. Care was taken to avoid contact between the brain surface and the cut edges of the dura. A stainless steel and glass cranial window was placed in the cut hole and cemented in place with dental acrylic. Windows were placed such that the same vascular region was examined in both newborn and juvenile preparations, and vessels studied had initial control diameters that were similar between newborns and juveniles. The space under the window was filled with artificial cerebrospinal fluid (aCSF; in mg/l: 220 KCl, 1,132 MgCl₂, 221 CaCl₂, 7,710 NaCl, 401 urea, 665 dextrose, and 2,066 NaHCO₃). aCSF was warmed in a water bath to 37°C and bubbled with CO₂ (pH 7.33, PCO₂ 46 mmHg, PO₂ 43 mmHg).

Pial arterioles (~40-120 µm) were directly observed via a dissecting microscope with a mounted video camera. Vessel diameter was measured with a video microscaler (model VPA-1000, For-A-Corp., Los Angeles, CA).

Experimental design. After we completed an initial observation period of 20 min, serial measurements of pial arteriolar diameters were made at 5 and 10 min during control conditions. With each measurement taken, mean arterial pressure (MAP) and core temperature were also recorded. At the end of the 10 min, an arterial blood gas sample was also drawn. All tested responses lasted 5 min with serial measurements of vessel diameter taken at 1, 3, and 5 min. At the end of each tested response, the area under the window was gently flushed with fresh aCSF to remove the previous stimulus and to allow the vessels to return to baseline diameters.

Pial arteriolar responses to hypercapnia. Newborn and juvenile pigs were mechanically ventilated with room air plus supplemental CO₂ to achieve PCO₂ values in the ranges of 55-65 and 65-80 mmHg. To determine prostanoid involvement in mediating dilation to hypercapnia, responses of pial arterioles to PCO₂ values of 55-65 and 65-80 mmHg were recorded in newborn and juvenile pigs during a control period, after administration of the cyclooxygenase inhibitor indomethacin trihydrate (5 mg/kg iv; gift from Merck Sharp & Dohme Research Laboratories), and in the combined presence of indomethacin and a subdilator concentration of the prostacyclin agonist iloprost (usually 10¹² M topically; gift from Schering Pharmaceutical Research). Low end dose-response curves were run to determine dilator threshold, and concentrations used were approximately one-tenth of that concentration. Another set of experiments was performed to elucidate the role of NO in mediating dilation to hypercapnia. In this set of experiments, responses to hypercapnia were recorded under control conditions, in the presence of the NO synthase inhibitor L-NNA (topical 10³ M; Sigma Chemical), and in the combined presence of the NO synthase inhibitor and the NO donor sodium nitroprusside (usually 10⁷ M). Low end dose-response curves were run to determine dilator threshold, and concentrations used were approximately one-tenth of that concentration. Subdilator concentrations of iloprost and sodium nitroprusside were used to determine whether prostanoids and NO provide a permissive influence in the dilator responses studied. By permissive, we mean that production of NO or prostanoids may not produce vasodilation directly but may allow or enhance function of another mechanism (18).

Dilation of pial arterioles to isoproterenol (topical 10⁶ M, Sigma Chemical) was recorded at the beginning and end of the experiment to ensure stability of the preparation throughout the experiment. Preparations that demonstrated more than a 15% decline in response to isoproterenol from beginning to end were discarded. Where restoration of responses with agonists was not observed, isoproterenol responses for beginning and end of experiment were reported to demonstrate conservation of vessel reactivity.

Pial arteriolar responses to topical histamine. To determine the role of prostanoids in mediating dilatory responses to histamine, responses to topical histamine (10⁹, 10⁶, and 10⁵ M; Sigma Chemical) were recorded alone, in the presence of indomethacin, and in the presence of indomethacin plus iloprost. To minimize deleterious effects of repeated administration of histamine on the preparation, the experiment was divided into low doses (10⁹ and 10⁶ M) and a high dose (10⁵ M) of histamine in separate pigs. To investigate NO involvement in mediating responses to histamine, responses to histamine were recorded alone, in the presence of L-NNA, and in the presence of L-NNA plus sodium nitroprusside.

Statistical analysis. Data are expressed as means ± SE. Comparisons among populations used analysis of variance with repeated measures. Fisher's protected least significant differences test was used to determine differences between groups. Significant responses to stimuli (i.e., comparisons with zero change) used t-tests. P < 0.05 was considered significant.

	RESULTS

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Initial diameters of pial arterioles measured were similar between newborns and juveniles (newborn: small, 47 ± 2 µm; large, 81 ± 3 µm n = 47; juvenile: small, 47 ± 2 µm; large, 82 ± 2 µm n = 37). Furthermore, diameter ranges of vessels on the pial surface were virtually identical when newborns and juveniles were compared. Smaller and larger vessels responded similarly in all experiments. Therefore, only responses recorded from larger vessels are reported to simplify the paper.

Hypercapnia and prostanoids. Arterial PCO₂ values for hypercapnic experiments are shown in Table 1. PCO₂ values were not significantly different between newborns and juveniles for any controls or treatments and were reproducible when comparing repeated hypercapnic challenges. MAP, PO₂, and pH values were within normal ranges and are not reported.

Control dilatory responses to hypercapnia from all animals in hypercapnia experiments are listed in Table 2 with corresponding PCO₂ values. Under control conditions, newborn and juvenile pial arterioles dilated similarly and in a dose-dependent manner to hypercapnia (Table 2). Figure 1 depicts hypercapnic responses (A, newborns; B, juveniles) during control, after indomethacin, and after indomethacin with iloprost. After indomethacin treatment, newborn arterioles failed to dilate in response to hypercapnia. Indomethacin treatment significantly attenuated the dilatory response to hypercapnia of juvenile arterioles but did not completely prevent the dilatory response (Fig. 1B). When iloprost was placed in the aCSF under the cranial window at a dose that produced no residual dilation, newborn vessels dilated in response to hypercapnia after indomethacin treatment in a manner similar to that before indomethacin treatment (Fig. 1A). The administration of iloprost of the same concentration did not change the dilation of juvenile vessels in response to hypercapnia following indomethacin treatment (Fig. 1B). Dilations of juvenile vessels in response to 10⁶ M isoproterenol were similar at the beginning and end of the experiment (28.6 ± 5.0 and 34.6 ± 4.4% at the beginning and end of experiment, respectively; n = 6).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effect of hypercapnia on dilation of newborn (n = 7 piglets; A) and juvenile (n = 6 pigs; B) pial arterioles during control, after indomethacin (5 mg/kg iv), and after both indomethacin and topical iloprost (1012 to 109 M). Values are means ± SE. * Statistical significance compared with baseline (zero change), P < 0.05.  Statistical significance compared with previous identical hypercapnic challenge, P < 0.05.

Hypercapnia and NO. Pial arteriolar dilation to hypercapnia during control, after topical L-NNA, and in the combined presence of L-NNA and topical sodium nitroprusside (10⁷ M) are shown in Fig. 2, A (newborn) and B (juvenile). Addition of L-NNA to the aCSF under the cranial window had no effect on the dilation of newborn arterioles in response to hypercapnia. Conversely, dilation of juvenile arterioles in response to hypercapnia was significantly attenuated following L-NNA, but restoration of the response was not seen when sodium nitroprusside was coadministered with L-NNA. Isoproterenol responses were not reduced at the end of the experiment (25.3 ± 2.1%) compared with those responses at the beginning of the experiment (13.8 ± 1.7%) (n = 6).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of hypercapnia on dilation of newborn (n = 7 piglets; A) and juvenile (n = 6 pigs; B) pial arterioles during control, after topical N-nitro-L-arginine (L-NNA, 103 M), and after both L-NNA and topical sodium nitroprusside (SNP, 107 M). Values are means ± SE. * Statistical significance compared with baseline (zero change), P < 0.05.  Statistical significance compared with previous identical hypercapnic challenge, P < 0.05.

Histamine and prostanoids. Blood gas, pH, and MAP values were within normal limits for all histamine experiments and therefore not reported.

Control responses of newborn and juvenile pial arterioles to topical histamine (10⁹ to 10⁵ M) from all histamine experiments are listed in Table 3. Figure 3, A (newborn) and B (juvenile), demonstrates pial arteriolar dilator responses to 10⁹ and 10⁶ M histamine during control, indomethacin treatment, and indomethacin plus iloprost. Topical histamine resulted in significant dilations of both newborn and juvenile pial vessels and occurred in dose-dependent manners that were not significantly different between age groups (Table 3). Indomethacin treatment significantly attenuated the dilator response to 10⁶ M histamine in newborn and appeared to reduce the dilation to 10⁹ M histamine. Dilations of pial vessels to both histamine concentrations in juveniles were likewise attenuated but to a lesser degree compared with those of newborns. Coadministration of iloprost completely restored the dilator responses to both 10⁹ and 10⁶ M histamine in newborns (Fig. 3A). In fact, the responses to histamine were enhanced in indomethacin- and iloprost-treated piglets compared with the responses before indomethacin treatment. Conversely, inhibition of the dilator response to histamine was not reversed with iloprost in juvenile arterioles. Dilations of juvenile vessels in response to isoproterenol were not reduced at the end of the experiment (37.7 ± 0.5%) compared with the beginning of the experiment (17.3 ± 1.7%) (n = 4).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of topical histamine (His, 109 and 106 M) on dilation of newborn (n = 9 piglets; A) and juvenile (n = 4 pigs; B) pial arterioles during control, after indomethacin (5 mg/kg iv), and after both indomethacin and topical iloprost (1012 to 109 M). Values are means ± SE. * Statistical significance compared with baseline (zero change), P < 0.05.  Statistical significance compared with previous identical His challenge, P < 0.05.

Figure 4 graphically represents pial arteriolar responses to 10⁵ M histamine under control conditions in the presence of indomethacin and with coadministration of indomethacin and iloprost. Newborn and juvenile vessels significantly dilated to 10⁵ M topical histamine under control conditions (Table 3, Fig. 4). Dilations were significantly attenuated after indomethacin treatment in both newborns and juveniles. Addition of 10¹² M iloprost in the continued presence of indomethacin was unable to restore dilation of juvenile vessels in response to 10⁵ M histamine but completely restored and even augmented the dilatory response in newborn pial arterioles. Again, juvenile isoproterenol responses were not different at the beginning and end of the experiment (20.9 ± 2.8 vs. 25.9 ± 5.0% at beginning vs. end of experimental protocol; n = 5).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of topical His (105 M) on dilation of newborn (n = 7 piglets; A) and juvenile (n = 5 pigs; B) pial arterioles during control, after indomethacin (5 mg/kg iv), and after both indomethacin and topical iloprost (1012 to 109 M). Values are means ± SE. * Statistical significance compared with baseline (zero change), P < 0.05.  Statistical significance compared with previous identical His challenge, P < 0.05.

Histamine and NO. Dilator responses of pial vessels to 10⁹ and 10⁶ M histamine in the absence of inhibitors, in the presence of the NO synthase inhibitor L-NNA, and in the combined presence of L-NNA and sodium nitroprusside are depicted in Fig. 5, A (newborn) and B (juvenile). Cerebral arterioles dilated significantly to both concentrations of histamine in dose-dependent manners that were not significantly different between age groups (Table 3). NO synthase inhibition was unable to block the dilation of newborn arterioles to either dose of histamine, and addition of sodium nitroprusside to the aCSF under the cranial window did not alter the response to either dose of histamine (data not shown). Similar to the responses reported from newborns, juvenile arteriolar responses to these concentrations of histamine were unaffected by NO synthase inhibition (Fig. 5B). Addition of 10⁷ M sodium nitroprusside to aCSF had no effect on the dilation of these vessels in response to these histamine concentrations.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of His (109 and 106 M) on dilation of newborn (n = 6 piglets; A) and juvenile (n = 8 pigs; B) pial arterioles during control and after topical L-NNA (103 M). Values are means ± SE. * Statistical significance compared with baseline (zero change), P < 0.05.

Recorded responses of pial arterioles to 10⁵ M histamine are presented in Fig. 6. Similarly to the lower concentrations of histamine, newborn arterioles dilated significantly with no statistically significant differences before and after NO synthase inhibition with topical L-NNA. Treatment with sodium nitroprusside did not alter the dilator response to a subsequent histamine. NO synthase inhibition had no effect on the dilator response of juvenile vessels in response to 10⁵ M histamine either, and further treatment with sodium nitroprusside resulted in no significant differences in the response.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of His (105 M) on dilation of newborn (n = 7 piglets; A) and juvenile (n = 8 pigs; B) pial arterioles during control and after topical L-NNA (103 M). Values are means ± SE. * Statistical significance compared with baseline (zero change), P < 0.05.

	DISCUSSION

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The new findings of this study are the following points. 1) No differences were observed between newborns and juveniles in the magnitude of the cerebral arteriolar dilatory responses to hypercapnia or topical histamine. 2) However, the mechanisms involved in these responses are not the same, namely, in juveniles, prostanoids contribute to hypercapnic- and histamine-induced dilations, although the mechanism of action appears to be direct, rather than permissive, in contrast to newborns where prostanoids act permissively and alone are sufficient for dilation to occur. 3) Furthermore, NO contributes to hypercapnic-mediated but not histamine-mediated dilations of pial arterioles in juveniles, but in the hypercapnic response, again the mechanism of action seems to be conventional, rather than permissive. Therefore, as the pig matures from neonate to juvenile, mechanisms involved in critical cerebral circulatory responses are modified with the prostanoid contribution to dilatory responses being diminished and less permissive in nature and NO emerging as a significant EDRF mediating cerebral vasodilatory responses.

As previously reported (18), prostanoids work in a permissive fashion to permit dilation of pial arterioles in response to hypercapnia in the newborn piglet, and indomethacin totally abolishes dilation to PCO₂ elevation. In contrast, dilation of cerebral vessels to hypercapnia from older pigs (5-6 mo) only was attenuated by ~50% with indomethacin treatment. Similar results have been obtained in adults of several other species. Iadecola et al. (13) found partial attenuation of cerebral blood flow response to hypercapnic challenge in adult rats pretreated with indomethacin. Similarly Wang et al. (38) report significantly reduced elevations of cerebral blood flow in hypercapnic rats following indomethacin treatment or NO synthase inhibition with N^G-nitro-L-arginine. Complete block of the hypercapnic response required both inhibitors. Decreased resting cerebral blood flow (28-40%) in baboons (30) and decreased resting cerebral blood velocities in healthy human subjects following indomethacin treatment have also been reported (21). In the present study, in contrast to newborns, a subthreshold dose of iloprost was unable to restore dilation of cerebral vessels to hypercapnia in the juvenile animals, suggesting that in the older animal prostacyclin may work conventionally to produce dilation in response to hypercapnia by inducing cAMP elevation.

NO from (endothelial and/or neuronal) NO synthase has recently received considerable attention as a possible mediator of cerebrovascular responses to hypercapnia in adult models (12, 38, 42). Treatment of juvenile pigs (42) and adult rats (12, 13) with nonselective NO synthase inhibitors attenuates vasodilation to hypercapnia, and in rats NO acts permissively in the hypercapnic response (13). Selective neuronal NO synthase inhibitors reduce cerebral vasodilation during parasympathetic stimulation in cats (9), and when administered to adult rats these inhibitors attenuate cerebral dilation to hypercapnia (23, 37). Neuronal NO synthase-derived NO was found to act permissively in mediating this hypercapnic response (23).

Few data exist concerning age-dependent changes in the role of NO (3, 42). Zuckerman et al. (42), comparing hypercapnic pial arteriolar responses in newborn and juvenile pigs, were able to attenuate (~50%) the hypercapnic response in juveniles following NO synthase inhibition with topical L-NNA (42), similar to our results in juvenile pigs. The residual response to hypercapnia could be eliminated when indomethacin was coadministered with L-NNA, suggesting prostanoids and NO contribute to hypercapnic cerebrovascular dilation in the juvenile pig. These findings contrast with data taken from newborn piglets, where inhibition of cyclooxygenases, but not NO synthase(s), attenuates the hypercapnic response (6, 18, 42).

Does NO emerge as the predominant EDRF mediating hypercapnic dilation of cerebral vessels as the piglet matures from neonate to juvenile? Is the action of NO a permissive or a direct influence? Newborn vessels dilated similarly in response to hypercapnia before and after NO synthase inhibition, whereas juvenile pig pial arterioles did show an attenuated response to hypercapnia following NO synthase inhibition with topical L-NNA, although significant dilation was still observed (Fig. 2). In contrast to hypercapnic studies performed in rats (13, 23), where NO was shown to work in a permissive fashion (addition of NO restored hypercapnic dilation in NO synthase inhibited animals), addition of exogenous NO via sodium nitroprusside in our experiments had no effect on the hypercapnic dilatory response of L-NNA-pretreated juvenile pial vessels (Fig. 2). Possible explanations for the differences seen in newborns and juveniles may include 1) the absence of NO production in response to hypercapnia in newborns and 2) the inability of neonatal smooth muscle to respond to NO due to the absence of stimulation of a second messenger system coupled to relaxation of vascular smooth muscle. Evidence suggests the perinatal piglet cerebral vasculature does contain the necessary machinery to respond to NO. Newborn cerebral vessels dilate when provided with an exogenous source of NO such as sodium nitroprusside (3) or monomethyl-L-arginine-containing compounds (6). It is generally accepted that NO-mediated dilation is via activation of a soluble guanylyl cyclase, resulting in cGMP elevation in vascular smooth muscle cells. In addition to dilation, topical sodium nitroprusside elevates cGMP in CSF of newborn piglets (3). Parfenova et al. (25) report a 2.5- and 2-fold increase in periarachnoid CSF concentrations of cAMP and cGMP, respectively, with concomitant dilation of newborn piglet pial arterioles in response to hypercapnia, although absolute levels of cGMP were low. Furthermore, dilation could still be achieved by topical application of dibutyryl-cAMP or -cGMP alone. Others have shown age-related differences in cGMP turnover rates (27) and basal NO release (22) that may affect sensitivity of the vasculature to NO/cGMP-mediated relaxations (5, 32) that may contribute to the absence of NO involvement in hypercapnic and histamine dilations of the newborn cerebral vasculature. Therefore, possible explanations accounting for the inability of NO synthase inhibition to block dilation of newborn cerebral vessels in response to hypercapnia are 1) the absence of stimulation of NO synthase activity in the newborn and 2) altered sensitivity of NO synthase/cGMP-related dilator mechanisms.

NO and prostanoids could interact to produce dilation in response to hypercapnia. Dilations of newborn piglet pial arterioles to prostaglandin I₂ have been shown to be inhibited after NO synthase inhibition (1). In addition, elevations in periarachnoid CSF, cGMP levels were reduced after treatment with L-NNA. Thus prostaglandin I₂ may require the presence/production of NO and possibly a basal "tone" of cGMP to permit dilation. However, the permissive action of prostacyclin only requires very low concentrations of the prostanoid to permit dilation to hypercapnia, much lower than concentrations needed to increase cAMP and cause dilation of cerebral vessels directly.

NO synthase inhibition significantly attenuated the hypercapnic dilation of juvenile vessels. Thus it appears that NO is involved in this dilatory response, albeit our results indicate NO does not appear to be the sole contributor. Perivascular pH may directly contribute to this response in juvenile pigs in conjunction with NO but was not studied in this set of experiments. Perivascular pH has been reported to mediate cerebrovascular responses to hypercapnia in some species (40), without involvement of NO. Conversely, similarly to the present study, others (13, 23) report in adult rats that NO plays a permissive role in hypercapnia-induced cerebrovascular dilation. Therefore, species differences, experimental conditions, and/or age may account for the differences between our results and those reported by others.

Our results suggest that NO contributes to hypercapnic relaxations of the juvenile but not the neonatal cerebral vasculature, and in juveniles, NO appears to exert a direct rather than a permissive influence.

Another potential influence on the cerebral vasculature is histamine. Histamine, as a neurotransmitter from perivascular neurons (10) and from mast cells situated in the cerebral vascular wall (7), may be released in sufficient quantities to affect vascular tone. Depending on the species and the vascular bed examined, histamine has been shown to cause either dilation or constriction. In the majority of species, including rats (4), cats (36), humans (14, 24), and newborn piglets (17, 20), histamine results in dilation of cerebral vessels. Conversely, constriction in response to histamine has been reported in the dog (33) and rabbit (15, 31) cerebral circulation.

In various vascular beds, histamine-induced dilation appears to be at least partially endothelium dependent and also may require the presence of dilator prostanoids, specifically prostaglandin I₂ (prostacyclin). Leffler et al. (17) demonstrated that dilations of newborn piglet pial arterioles in response to topical histamine are abolished following functional removal of the endothelium with light/dye injury. Furthermore, the dilatory response to histamine in endothelium-damaged arterioles could be restored when a subdilator concentration of the prostacyclin analog iloprost was administered to the cortical surface, suggesting that 1) the histamine response requires a functional endothelium, and 2) prostanoids released from endothelial cells exert a permissive influence to allow dilation of piglet pial arterioles in response to histamine. Similarly, Toda et al. (33) report involvement of prostanoids in the dilatory vascular response to histamine from canine extracerebral vessels. In addition, constriction of canine cerebral vessels in response to histamine is augmented following treatment with the prostaglandin I₂ synthase inhibitor tranylcypromine, suggesting prostanoids may also mediate an opposing dilatory action of histamine on the dog cerebral vasculature.

In the present experiments, newborn piglet pial arterioles significantly dilated in a dose-dependent manner to topical histamine (10⁹ to 10⁵ M). Indomethacin treatment significantly attenuated the response to histamine, but in contrast to Leffler et al. (20), complete block of the histamine response was not seen. However, in agreement with the above studies, the attenuation seen with indomethacin treatment was completely reversed when iloprost was coadministered with the cyclooxygenase inhibitor. In fact, histamine-mediated dilations of newborn pial arterioles appeared augmented with iloprost treatment, compared with control responses (Figs. 3 and 4). Incomplete block of the histamine response in this set of experiments may be due to inadequate cyclooxygenase inhibition with indomethacin with residual prostanoid production sufficient to permit dilation to histamine. This is unlikely because Leffler et al. (16) have previously reported that indomethacin at a dose of 5 mg/kg is sufficient to significantly reduce control concentrations of cortical subarachnoid prostanoids and inhibit conversion of exogenous arachidonate to prostanoids by >90%. Evidence that permissive actions may be graded rather than all or none have not been observed. It is possible that there are H₁/H₂ receptors on the vascular smooth muscle that are non-prostanoid, non-endothelium dependent, which would directly result in dilations seen in newborn cerebral vessels following cyclooxygenase inhibition. Lending support to this explanation, guinea pig pulmonary (29), rabbit middle cerebral (31), and human cerebral arteries (14) exhibit endothelium-dependent and -independent actions of histamine. Histamine responses seen in endothelium-denuded vessels were hypothesized to result from activation of non-endothelium-dependent histamine receptors located on the underlying vascular smooth muscle. Furthermore, Jansen-Olesen et al. (14) detected both H₁- and H₂-receptor mRNA in endothelium-denuded and -intact human cerebral arteries. In addition, Toda et al. (33) and Kim et al. (15) have demonstrated prostanoid-dependent and -independent actions of histamine in the canine cerebral and gastric circulations and in the rabbit middle cerebral artery. Activation of non-endothelium-, non-prostanoid-dependent receptors on vascular smooth muscle would therefore be expected to produce dilation/constriction even in the presence of indomethacin.

Similarly to those of newborns, juvenile pial vessels dilated to topical histamine and dilations could be attenuated following indomethacin treatment. However, pial dilatory responses to histamine of juvenile arterioles were unchanged when iloprost was added to aCSF under the window, suggesting that prostanoids do not work in a permissive fashion as they appear to in the newborn but rather produce dilation directly. Again, the residual dilation of juvenile pial arterioles may be attributed to direct activation of histamine receptors located on the vascular smooth muscle. An alternate explanation would include a possible role for a different EDRF, possibly NO. In support of an alternate EDRF mediating histamine-induced dilations of cerebral vessels, Benedito et al. (4) report a rightward shift of the histamine concentration-response curve following treatment of cat middle cerebral arteries with the soluble guanylyl cyclase inhibitor methylene blue, suggesting that NO may contribute to dilations of cerebral vessels in response to histamine. However, we could detect no effect of NO synthase inhibition on histamine-induced dilations of either newborn or juvenile cerebral vasculatures.

Our results lend support to previous studies that show prostanoid influence is necessary for hypercapnia- and histamine-induced dilations in newborn pigs and that prostanoids act permissively to permit dilations of cerebral vessels to these dilatory stimuli. We found no evidence to indicate permissive actions of prostanoids or NO in juvenile dilatory responses to histamine and hypercapnia. However, NO does appear to directly contribute to hypercapnia- but not histamine-induced dilations of juvenile pial arterioles. Therefore, as the pig matures from neonate to juvenile, the prostanoid contribution to dilatory cerebrovascular responses appear to be diminished, and NO emerges as a significant EDRF mediating cerebral vasodilatory response. Results from this study contribute to understanding paracrine/autocrine mechanisms contributing to regulation of the cerebral vasculature during development from neonate to juvenile and may provide insight into the pathophysiology of various cerebral insults (intracranial hemorrhage, cerebral ischemia) and disease processes. With this understanding, more effective treatment protocols for managing cerebrovascular insults/diseases from neonatal to mature patients may be developed.