Effects of trigeminal neurotransmitters on piglet pial arterioles.

We investigated effects of calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) on pial arterioles in newborn pigs. Pial arteriolar diameter was determined using a closed cranial window and intravital microscopy. Initial diameters were approximately 100 microns. Calcitonin-gene related peptide dilated pial arterioles by 22 +/- 8% at 10(-9)M and by 34 +/- 6% at 10(-8)M (n = 8), and this response was not significantly altered by prior administration of indomethacin (5mg/kg, iv) (n = 6) or administration of NG-methyl-L-arginine (5mg/kg, iv, and 10(-3)M in CSF) (n = 10). Substance P dilated arterioles at 10(-10)M through 10(-5)M (maximal response = 23 +/- 3%) (n = 6), and this response was unaffected by indomethacin administration (n = 6). In contrast, NG-methyl-L-arginine blocked much of the pial arteriolar dilation to SP. Unlike the other two peptides, NKA did not change pial arteriolar diameter. Radioimmunoassay determinations indicated that cerebrospinal fluid levels of 6-keto-prostaglandin F1 and prostaglandin E2 did not change appreciably during application of CGRP or SP. We conclude that CGRP and SP but not NKA are dilator stimuli in the piglet pial circulation. Dilation by CGRP probably involves direct activation of receptors on vascular smooth muscle, while SP probably partially dilates pial arterioles via release of an endothelium-dependent relaxing factor.     

Development of fetal vascular responses to endothelin-1 and acetylcholine in the sheep

Responses to K(+), endothelin-1 (ET-1), and acetylcholine (ACh) of isolated adrenal, femoral, middle cerebral, and renal arteries from fetal [110--145 days gestational age (dGA, term approximately 148 dGA)] and 0- to 24-h newborn (NB) lambs were evaluated using the technique of wire myography. Responses at distinct developmental ages for each vascular bed were compared. In all arteries sensitivity to K(+)-induced vasoconstriction was similar at all fetal age points examined. In contrast, sensitivity to ET-1 increased with increasing fetal age in arteries from all vascular beds. The magnitude of the maximal vasoconstriction was positively correlated with GA for K(+) in adrenal, femoral, and cerebral arteries and for ET-1 in femoral, cerebral, and renal arteries. Cerebral arteries showed a greater sensitivity when compared with the other systemic arteries to K(+) and ET-1 at all fetal ages and to K(+) in NB. ACh evoked relaxatory responses in fetal and NB femoral and adrenal arteries. However, renal arteries relaxed comparatively less in response to ACh, and no vasodilation was noted in middle cerebral arteries at any age points examined. For femoral arteries ACh-induced vasorelaxation decreased with increasing GA but was restored in arteries from NB lambs. In summary, the responsiveness of isolated resistance arteries varies with developmental age in the fetal and perinatal sheep and these effects are both agonist and vascular bed specific. The augmented sensitivity in response to ET-1 of middle cerebral compared with other systemic arteries may reflect the importance of cerebral blood flow control during this critical developmental period.     

Higher sensitivity of cerebral arteries isolated from premature and newborn baboons to adrenergic and cholinergic stimulation

To find whether effects of adrenergic and cholinergic agents on cerebral artery were dependent on maturity, we examined responses of isolated cerebral artery strips harvested from premature, term newborn and adult baboons. Although cerebral arteries from many species are only mildly sensitive to norepinephrine, we found the perinatal cerebral arteries to be quite responsive to the amine. Cerebral arteries from premature and newborn baboons were significantly (P less than 0.001) more sensitive to norepinephrine than were arteries from adults; medium effective concentration (EC50) for norepinephrine were 3 X 10(-8), 6 X 10(-8) and 32 X 10(-8)M for prematures, newborns and adults, respectively. Arteries showed a similar age-dependence in the sensitivity of the response to phenylephrine, an alpha 1-adrenoceptor agonist. EC50 values for KC1 did not differ among groups, nor did the maximum response to norepinephrine. Arteries from premature and newborn baboons showed marked contractile response to acetylcholine (maximum tensions 5.9 +/- 0.6 and 6.4 +/- 0.8 g/mm2, respectively), whereas arteries from adult baboons showed little response (0.6 +/- 0.1 g/mm2). Arteries from premature and newborn animals showed a more marked relaxation response to isoproterenol than did arteries from adult animals; the degree of relaxation from an induced contraction was 63% (premature), 72% (newborn) and 10% (adult). There was no age-dependence in the relaxation response to sodium nitrite. We conclude that the events coupling alpha 1, beta or muscarinic receptor activation with cerebral arterial contraction or relaxation are more effective in perinatal than in adult baboons.     

Experimental Modelling of the Consequences of Brief Late Gestation Asphyxia on Newborn Lamb Behaviour and Brain Structure

Brief but severe asphyxia in late gestation or at the time of birth may lead to neonatal hypoxic ischemic encephalopathy and is associated with long-term neurodevelopmental impairment. We undertook this study to examine the consequences of transient in utero asphyxia in late gestation fetal sheep, on the newborn lamb after birth. Surgery was undertaken at 125 days gestation for implantation of fetal catheters and placement of a silastic cuff around the umbilical cord. At 132 days gestation (0.89 term), the cuff was inflated to induce umbilical cord occlusion (UCO), or sham (control). Fetal arterial blood samples were collected for assessment of fetal wellbeing and the pregnancy continued until birth. At birth, behavioral milestones for newborn lambs were recorded over 24 h, after which the lambs were euthanased for brain collection and histopathology assessments. After birth, UCO lambs displayed significant latencies to (i) use all four legs, (ii) attain a standing position, (iii) find the udder, and (iv) successfully suckle - compared to control lambs. Brains of UCO lambs showed widespread pathologies including cell death, white matter disruption, intra-parenchymal hemorrhage and inflammation, which were not observed in full term control brains. UCO resulted in some preterm births, but comparison with age-matched preterm non-UCO control lambs showed that prematurity per se was not responsible for the behavioral delays and brain structural abnormalities resulting from the in utero asphyxia. These results demonstrate that a single, brief fetal asphyxic episode in late gestation results in significant grey and white matter disruption in the developing brain, and causes significant behavioral delay in newborn lambs. These data are consistent with clinical observations that antenatal asphyxia is causal in the development of neonatal encephalopathy and provide an experimental model to advance our understanding of neuroprotective therapies.     

Maturational modulation of endothelium-dependent vasodilatation in ovine cerebral arteries

To address the hypothesis that maturation enhances endothelial vasodilator function in cerebral arteries, relaxant responses to ADP and A-23187 were determined in ovine carotid and cerebral arteries harvested from 25 newborn lambs (3-7 days) and 23 adult sheep. Maturation significantly increased pD(2) values for A-23187 (newborn range: 4.9 +/- 0.3 to 5.4 +/- 0.3; adult range: 6.0 +/- 0.2 to 7.1 +/- 0.2) and the maximal vasodilator response to A-23187 by 10-18%. In contrast, maturation decreased maximum responses to ADP by 5-25% with no change in pD(2). The magnitudes of endothelium-dependent relaxation were not affected by 10 microM indomethacin but were virtually abolished by 100 microM N(G)-nitro-L-arginine methyl ester/L-nitro arginine, indicating that nitric oxide (NO) is the primary endothelium-dependent vasodilator in these arteries. Maturation also modestly decreased endothelial NO synthase (eNOS) abundance in both carotid (32%) and cerebral (26%) arteries. Together, these findings reinforce the view that receptor coupling to endothelial activation is tightly regulated and may offset underlying changes in maximal endothelial vasodilator capacity. This capacity, in turn, appears to increase with postnatal age despite major growth and expansion of endothelial cell size and vascular wall volume. In ovine cerebral arteries, endothelial vasodilator capacity appears completely dependent on eNOS activity but not on cyclooxygenase activity. In turn, eNOS activity appears to be postnatally regulated by mechanisms independent of changes in eNOS abundance alone.     

Contributions of astrocytes and CO to pial arteriolar dilation to glutamate in newborn pigs

Astrocytes can act as intermediaries between neurons and cerebral arterioles to regulate vascular tone in response to neuronal activity. Release of glutamate from presynaptic neurons increases blood flow to match metabolic demands. CO is a gasotransmitter that can be related to neural function and blood flow regulation in the brain. The present study addresses the hypothesis that glutamatergic stimulation promotes perivascular astrocyte CO production and pial arteriolar dilation in the newborn brain. Experiments used anesthetized newborn pigs with closed cranial windows, piglet astrocytes, and cerebrovascular endothelial cells in primary culture and immunocytochemical visualization of astrocytic markers. Pial arterioles and arteries of newborn pigs are ensheathed by astrocytes visualized by glial fibrillary acidic protein staining. Treatment (2 h) of astrocytes in culture with L-2-alpha-aminoadipic acid (L-AAA), followed by 14 h in toxin free medium, dose-dependently increased cell detachment, suggesting injury. Conversely, 16 h of continuous exposure to L-AAA caused no decrease in endothelial cell attachment. In vivo, topical L-AAA (2 mM, 5 h) disrupted the cortical glia limitans histologically. Such treatment also eliminated pial arteriolar dilation to the astrocyte-dependent dilator ADP and to glutamate but not to isoproterenol or CO. Glutamate stimulated CO production by the brain surface that also was abolished following L-AAA. In contrast, tetrodotoxin blocked dilation to N-methyl-D-aspartate but not to glutamate, isoproterenol, or CO or the glutamate-induced increase in CO. The concurrent loss of CO production and pial arteriolar dilation to glutamate following astrocyte injury suggests astrocytes may employ CO as a gasotransmitter for glutamatergic cerebrovascular dilation.     

Blood-brain barrier permeability during dopamine-induced hypertension in fetal sheep.

Dopamine is often used as a pressor agent in sick newborn infants, but an increase in arterial blood pressure could disrupt the blood-brain barrier (BBB), especially in the preterm newborn. Using time-dated pregnant sheep, we tested the hypothesis that dopamine-induced hypertension increases fetal BBB permeability and cerebral water content. Barrier permeability was assessed in nine brain regions, including cerebral cortex, caudate, thalamus, brain stem, cerebellum, and spinal cord, by intravenous injection of the small tracer molecule [(14)C]aminoisobutyric acid at 10 min after the start of dopamine or saline infusion. We studied 23 chronically catheterized fetal sheep at 0.6 (93 days, n = 10) and 0.9 (132 days, n = 13) gestation. Intravenous infusion of dopamine increased mean arterial pressure from 38 +/- 3 to 53 +/- 5 mmHg in 93-day fetuses and from 55 +/- 5 to 77 +/- 8 mmHg in 132-day fetuses without a decrease in arterial O(2) content. These 40% increases in arterial pressure are close to the maximum hypertension reported for physiological stresses at these ages in fetal sheep. No significant increases in the brain transfer coefficient of aminoisobutyric acid were detected in any brain region in dopamine-treated fetuses compared with saline controls at 0.6 or 0.9 gestation. There was also no significant increase in cortical water content with dopamine infusion at either age. We conclude that a 40% increase in mean arterial pressure during dopamine infusion in normoxic fetal sheep does not produce substantial BBB disruption or cerebral edema even as early as 0.6 gestation.     

Carbon monoxide as an attenuator of vasoconstriction in piglet cerebral arterioles

Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral circulation. The present study addressed the hypothesis that endogenous CO attenuates pial arteriolar vasoconstriction caused by hypocapnia, platelet activating factor, and elevated blood pressure. Experiments used anesthetized piglets with implanted, closed cranial windows. Topical application of a metal porphyrin inhibitor of heme oxygenase was used to inhibit production of CO. Chromium mesopophyrin increased vasoconstriction in response to hypocapnia. The constrictor response to a topical stimulus, platelet activating factor, was also increased by application of chromium mesoporphyrin. Inhibition of heme oxygenase did not constrict pial arterioles in normotensive newborn pigs (mean arterial pressure of about 70 mmHg), but did constrict pial arterioles of piglets with experimentally induced increases in arterial pressure (mean arterial pressure greater than 90 mmHg). In fact, pial arterioles of normotensive piglets transiently dilated to chromium mesoporphyrin, whereas those of hypertensive piglets progressively constricted during 10 min of chromium mesoporphyrin treatment. Therefore, inhibition of heme oxygenase augments cerebral vasoconstriction in response to several very different constrictor stimuli. These data suggest endogenous CO attenuates vasoconstrictor responses in the newborn cerebral circulation.     

Regulation of brain water during acute hyperosmolality in ovine fetuses, lambs, and adults.

In adult rats, when plasma osmolality increases, water flows across the blood-brain barrier down its concentration gradient from brain to plasma, and brain volume deceases. The brain responds to this stress by gaining osmotically active solutes, which limit water loss. This phenomenon is termed brain volume (water) regulation. We tested the hypothesis that brain volume regulation is more effective in young lambs and adult sheep than in fetuses, premature lambs, and newborn lambs. Brain water responses to acute hyperosmolality were measured in the cerebral cortex, cerebellum, and medulla of fetuses at 60 and 90% of gestation, premature ventilated lambs at 90% of gestation, newborn lambs, young lambs at 20-30 days of age, and adult sheep. After exposure of the sheep to increases in systemic osmolality with mannitol plus NaCl, brain water content and electrolytes were quantified. The ideal osmometer is a system in which impermeable solutes do not enter or leave in response to an osmotic stress. There were significant differences from an ideal osmometer in the cerebral cortex of fetuses at 90% of gestation, cerebral cortex, and cerebellum of newborn lambs, and cerebral cortex, cerebellum, and medulla of young lambs and adult sheep; however, there were no differences in the brain regions of fetuses at 60% of gestation and premature lambs, cerebellum and medulla of fetuses at 90% of gestation, and medulla of newborn lambs. We conclude that 1) brain water loss is maximal and brain volume regulation impaired in most brain regions of fetuses at 60 and 90% of gestation and premature lambs; 2) brain volume regulation develops first in the cerebral cortex of the fetuses at 90% of gestation and in the cerebral cortex and cerebellum of newborn lambs, and then it develops in the medulla of the lambs at 20-30 days of age; 3) brain water loss is limited and volume regulation present in the brain regions of young lambs and adult sheep; and 4) the ability of the brain to exhibit volume regulation develops in a region- and age-related fashion.     

Glucagon-like peptide 2 has limited efficacy to increase nutrient absorption in fetal and preterm pigs

Exogenous glucagon-like peptide 2 (GLP-2) prevents intestinal atrophy and increases nutrient absorption in term newborn pigs receiving total parenteral nutrition (TPN). We tested the hypothesis that the immature intestine of fetuses and preterm neonates has a diminished nutrient absorption response to exogenous GLP-2. This was accomplished using catheterized fetal pigs infused for 6 days (87-91% of gestation) with GLP-2 (25 nmol.kg(-1).day(-1) iv; n = 7) or saline (n = 7), and cesarean-delivered preterm pigs (92% of gestation) that received TPN with GLP-2 (25 nmol.kg(-1).day(-1) iv; n = 8) or saline (n = 7) for 6 days after birth. Responses to GLP-2 were assessed by measuring intestinal dimensions, absorption of nutrients (glucose, leucine, lysine, proline) by intact tissues and brush border membrane vesicles, and abundance of sodium-glucose cotransporter mRNA. Infusion of GLP-2 increased circulating GLP-2 levels in fetuses, but did not increase intestinal mass or absorption of nutrients by intact tissues and brush border membrane vesicles, except for lysine. Administration of exogenous GLP-2 to preterm TPN-fed pigs similarly did not increase rates of nutrient absorption, yet nutrient absorption capacities of the entire small intestine tended to increase (+10-20%, P < 0.10) compared with TPN alone due to increased intestinal mass (+30%, P < 0.05). GLP-2 infusion did not increase sodium-glucose cotransporter-1 mRNA abundance in fetuses or postnatal preterm pigs. Hence, the efficacy of exogenous GLP-2 to improve nutrient absorption by the intestine of fetal and preterm pigs is limited compared with term pigs and more mature animals and humans.     

Chronic exposure to nicotine alters endothelium-dependent arteriolar dilatation: effect of superoxide dismutase.

The first goal of this study was to determine whether chronic injection of nicotine alters endothelium-dependent arteriolar dilatation. We measured the diameter of cheek pouch resistance arterioles (approximately 50 microm in diameter) in response to endothelium-dependent (acetylcholine and ADP) and -independent (nitroglycerin) agonists in control hamsters and hamsters treated with nicotine (2 microg. kg-1. day-1 for 2-3 wk). In control hamsters, acetylcholine (0.1 and 1.0 microM) dilated arterioles by 13 +/- 2 and 31 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by 18 +/- 1 and 30 +/- 1%, respectively. In contrast, acetylcholine (0.1 and 1.0 microM) dilated arterioles by only 5 +/- 2 and 12 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by only 7 +/- 2 and 13 +/- 3%, respectively, in animals treated with nicotine (P < 0.05 vs. response in control hamsters). Nitroglycerin produced similar dose-related dilatation of cheek pouch arterioles in control and nicotine-treated hamsters. Our second goal was to examine a possible mechanism for impaired endothelium-dependent arteriolar dilatation during chronic treatment with nicotine. We found that superfusion of the cheek pouch microcirculation with superoxide dismutase (150 U/ml) restored impaired endothelium-dependent, but did not alter endothelium-independent, arteriolar dilatation in hamsters treated with nicotine. Superfusion with superoxide dismutase did not alter endothelium-dependent or -independent arteriolar dilatation in control hamsters. We suggest that chronic exposure to nicotine produces selective impairment of endothelium-dependent arteriolar dilatation via a mechanism related to the synthesis/release of oxygen-derived free radicals.     

Chronic administration of indomethacin increases role of nitric oxide in hypercapnic cerebrovasodilation in piglets.

Hypercapnia-induced cerebral vasodilation is associated with prostanoids in the piglet, but is a primarily nitric oxide (NO) associated response in many adult models. Hypercapnia-induced cerebral vasodilation is both NO and prostanoid associated in the juvenile pig. We hypothesized that with chronic administration of indomethacin the piglet would advance the role of the NO system in cerebrovascular responses. The closed cranial window technique was used in piglets to determine pial arteriolar response. Chronically indomethacin treated newborn animals dilated in response to CO2 similarly to control newborns (40.9+/-4.4% vs 48.4+/-4.1%). Topical n-nitro L-arginine (L-NA, 10(-3) M), attenuated CO2 induced dilation in the chronically indomethacin treated animals (11.7+/-3.3% vs 40.9+/-4.4%; p < 0.001), but had no effect on the response to hypercapnia of piglets not treated with indomethacin. Neither indomethacin nor L-NA altered response to topical isoproterenol (10(-6) M). We conclude that with chronic indomethacin administration there develops a significant hypercapnia-induced cerebral vasodilation in which NO has an important role. The chronic inhibition of the newborn's principal dilator system appears to increase the role of NO in newborn cerebral hemodynamics.     

Contribution of adenosine A2A and A2B receptors and heme oxygenase to AMPA-induced dilation of pial arterioles in rats

Nitric oxide (NO) has been implicated in mediation of cerebral vasodilation during neuronal activation and, specifically, in pharmacological activation of N-methyl-d-aspartate (NMDA) and kainate receptors. Possible mediators of cerebral vasodilation to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have not been well studied in mature brain, although heme oxygenase (HO) activity has been implicated in newborn pigs. In anesthetized rats, 5 min of topical superfusion of 30 and 100 microM AMPA on the cortical surface through a closed cranial window resulted in increases in pial arteriolar diameter. The dilatory response to AMPA was not inhibited by superfusion of an NO synthase inhibitor, a cyclooxygenase-2 inhibitor, or a cytochrome P-450 epoxygenase inhibitor, all of which have been shown to inhibit the cortical blood flow response to sensory activation. However, the 48 +/- 13% dilation to 100 microM AMPA was attenuated 56-71% by superfusion of the adenosine A(2A) receptor antagonist ZM-241385, the A(2B) receptor antagonist alloxazine, and the HO inhibitor chromium mesoporphyrin. Combination of the latter three inhibitors did not attenuate the dilator response more than the individual inhibitors, whereas an AMPA receptor antagonist fully blocked the vasodilation to AMPA. These results indicate that cortical pial arteriolar dilation to AMPA does not require activation of NO synthase, cyclooxygenase-2, or cytochrome P-450 epoxygenase but does depend on activation of adenosine A(2A) and A(2B) receptors. In addition, CO derived from HO appears to play a role in the vascular response to AMPA receptor activation in mature brain by a mechanism that is not additive with that of adenosine receptor activation.     

Metabolic and hormonal responses to cooling the fetal sheep in utero

The metabolic and hormonal effects of cooling 10 fetal sheep in utero (115-142 days of gestation) for 2h were studied. The fetal core temperature fell by 2.81 +/- 0.14 degrees C while the maternal temperature fell 0.86 +/- 0.15 degrees C. This hypothermia caused a significant rise in the fetal and maternal plasma glucose concentrations (P less than 0.001) and a fall in the fetal insulin concentrations (P less than 0.01). The fetal plasma lactate and cortisol concentrations rose rapidly (P less than 0.01) while the growth hormone fell (P less than 0.01) and remained low until cooling ceased when a rapid rebound occurred. There was no significant change in any of the fetal iodothyronines and no elevation of nonesterified free fatty acid concentrations, in contrast to the rapid rise (P less than 0.01) which occurred when newborn lambs were cooled. These observations demonstrate that appropriate glucose, insulin, lactate and cortisol responses to hypothermia have differentiated by 120 days of gestation. However, neither a thyroid hormone response nor an elevation in free fatty acid levels was observed. Thus not all components of the thermogenic response to hypothermia can be demonstrated in the late gestation fetail sheep in utero.     

High-altitude chronic hypoxia during gestation and after birth modifies cardiovascular responses in newborn sheep

Perinatal exposure to chronic hypoxia induces sustained pulmonary hypertension and structural and functional changes in both pulmonary and systemic vascular beds. The aim of this study was to analyze consequences of high-altitude chronic hypoxia during gestation and early after birth in pulmonary and femoral vascular responses in newborn sheep. Lowland (LLNB; 580 m) and highland (HLNB; 3,600 m) newborn lambs were cathetherized under general anesthesia and submitted to acute sustained or stepwise hypoxic episodes. Contractile and dilator responses of isolated pulmonary and femoral small arteries were analyzed in a wire myograph. Under basal conditions, HLNB had a higher pulmonary arterial pressure (PAP; 20.2 +/- 2.4 vs. 13.6 +/- 0.5 mmHg, P < 0.05) and cardiac output (342 +/- 23 vs. 279 +/- 13 ml x min(-1) x kg(-1), P < 0.05) compared with LLNB. In small pulmonary arteries, HLNB showed greater contractile capacity and higher sensitivity to nitric oxide. In small femoral arteries, HLNB had lower maximal contraction than LLNB with higher maximal response and sensitivity to noradrenaline and phenylephrine. In acute superimposed hypoxia, HLNB reached higher PAP and femoral vascular resistance than LLNB. Graded hypoxia showed that average PAP was always higher in HLNB compared with LLNB at any Po2. Newborn lambs from pregnancies at high altitude have stronger pulmonary vascular responses to acute hypoxia associated with higher arterial contractile status. In addition, systemic vascular response to acute hypoxia is increased in high-altitude newborns, associated with higher arterial adrenergic responses. These responses determined in intrauterine life and early after birth could be adaptive to chronic hypoxia in the Andean altiplano.     

Endothelin-1-induced pulmonary arterial dilation is reduced by N omega-nitro-L-arginine in fetal lambs.

Endothelin-1 (ET-1) is a pulmonary vasodilator in the unventilated fetal lamb. The site and mechanism of this vasodilator response were investigated in isolated blood-perfused lungs from nine fetal lambs delivered at 127-140 days gestation. The vascular occlusion technique was used to partition the total pulmonary pressure gradient into pressure gradients across large and small arteries (delta PLA and delta PSA, respectively) and veins (delta PV). Injection of ET-1 (74 ng/kg) into the pulmonary artery significantly decreased delta PLA from 12.4 +/- 2.1 to 5.2 +/- 1.1 mmHg and delta PSA from 49.2 +/- 2.7 to 31.3 +/- 4.9 mmHg. The pressure measured by double occlusion, an estimate of pulmonary capillary pressure, was not altered by ET-1 (15.5 +/- 1.0 vs. 14.8 +/- 1.0 mmHg), indicating that ET-1 had no effect on pulmonary veins. Addition of N omega-nitro-L-arginine (estimated perfusate concentration 2-6 mM), an analogue of L-arginine that inhibits the production of endothelium-derived relaxing factor (EDRF), significantly attenuated the dilator responses to acetylcholine (10 micrograms) and ET-1 (74 ng/kg) by 35 and 56%, respectively. These results in unventilated fetal lungs indicate that 1) ET-1 dilates both large and small pulmonary arteries with no effect on pulmonary veins, and 2) this effect is mediated in part through the action of the EDRF pathway.     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid synthesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3, 6, or 30 micrograms/ml) was placed under cranial windows in newborn pigs that had been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentrations, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dose-dependent increases of PGE2 in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF1 alpha and TXB2 only increased at the highest concentration of arachidonic acid (30 micrograms/ml). Cerebral ischemia did not decrease the conversion of any concentration of arachidonic acid to PGE2, 6-keto-PGF1 alpha, or TXB2. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Early gestation dexamethasone programs enhanced postnatal ovine coronary artery vascular reactivity

Excessive exposure of the fetus to maternally derived corticosteroids has been linked to the development of adult-onset diseases. To determine if early gestation corticosteroid exposure alters subsequent coronary artery reactivity, we administered dexamethasone (0.28 mg.kg(-1).day(-1)) to pregnant ewes at 27-28 days gestation (term being 145 days). Vascular responsiveness was assessed in endothelium-intact coronary and mesenteric arteries isolated from steroid-exposed and age-matched control fetal sheep at 123-126 days gestation and lambs at 4 mo of age. Lambs exposed to maternal dexamethasone had higher mean arterial blood pressures than the age-matched controls (93 +/- 3 vs. 83 +/- 5 mmHg, P < 0.05). Mesenteric arteries from the steroid-exposed fetuses displayed diminished responses to ANG II, relative to controls. In 4-mo-old lambs, prenatal dexamethasone exposure significantly increased coronary artery vasoconstriction to ANG II, ACh, and U-46619, but not KCl. In contrast, postnatal mesenteric artery reactivity was unaltered by steroid exposure. Compared with fetal mesenteric reactivity, postnatal mesenteric reactivity to ANG II, phenylephrine, and U-46619 was diminished, whereas the response to 120 mmol/l KCl was heightened. Coronary artery ANG II receptor protein expression was not significantly altered by steroid exposure in either age group. These findings demonstrate that early-gestation glucocorticoid exposure programs postnatal elevations in blood pressure and selectively enhances coronary artery responsiveness to second messenger-dependent vasoconstrictors. Glucocorticoid-induced alterations in coronary vascular smooth muscle structure or function may provide a mechanistic link between an adverse intrauterine environment and later cardiovascular disease.     

Intrauterine hypertension decreases lung VEGF expression and VEGF inhibition causes pulmonary hypertension in the ovine fetus

Although vascular endothelial growth factor (VEGF) plays a vital role in lung vascular growth in the embryo, its role in maintaining endothelial function and modulating vascular structure during late fetal life has not been studied. We hypothesized that impaired lung VEGF signaling causes pulmonary hypertension, endothelial dysfunction, and structural remodeling before birth. To determine whether lung VEGF expression is decreased in an experimental model of persistent pulmonary hypertension of the newborn (PPHN), we measured lung VEGF and VEGF receptor protein content from fetal lambs 7-10 days after ductus arteriosus ligation (132-140 days gestation; term = 147 days). In contrast with the surge in lung VEGF expression during late gestation in controls, chronic intrauterine pulmonary hypertension reduced lung VEGF expression by 78%. To determine whether VEGF inhibition during late gestation causes pulmonary hypertension, we treated fetal lambs with EYE001, an aptamer that specifically inhibits VEGF(165). Compared with vehicle controls, EYE001 treatment elevated pulmonary artery pressure and pulmonary vascular resistance by 22 and 50%, respectively, caused right ventricular hypertrophy, and increased wall thickness of small pulmonary arteries. EYE001 treatment reduced lung endothelial nitric oxide synthase protein content by 50% and preferentially impaired the pulmonary vasodilator response to ACh, an endothelium-dependent agent. We conclude that chronic intrauterine pulmonary hypertension markedly decreases lung VEGF expression and that selective inhibition of VEGF(165) mimics the structural and physiological changes of experimental PPHN. We speculate that hypertension downregulates VEGF expression in the developing lung and that impaired VEGF signaling may contribute to the pathogenesis of PPHN.     

Age and species dependence of pial arteriolar responses to topical carbon monoxide in vivo

In newborn pigs, carbon monoxide (CO) contributes to regulation of cerebrovascular circulation. Results from isolated adult cerebral arteries suggest CO may have less dilatory potential in mature animals. However, few data are available on the direct effects of CO on cerebrovascular circulation in vivo except for those from newborn pigs. Therefore, we tested the hypothesis that i) rat cerebral arterioles dilate to CO in vivo and ii) CO-induced cerebrovascular dilatory responses are age dependent in pigs. Also, we examined whether the permissive role of nitric oxide in CO-induced dilation observed in piglets is present in older pigs and rats. Experiments used anesthetized newborn, 7-week-old, and juvenile (3- to 4-month-old) pigs and 3- to 4-month-old rats with closed cranial windows and topical applications of CO and sodium nitroprusside (SNP). Dilations to SNP were not different at different ages in pigs or between pigs and rats. CO produced pial arteriolar dilations in all groups. Dilation to 10(-5) M CO was reduced in juvenile pigs as compared to newborn and 7-week-old pigs, and tended to less at 10(-6) M CO. Dilations of rat pial arterioles to all concentrations were less than those of newborn and 7-week-old pigs, but not different from those of juvenile pig pial arterioles. In newborn and 7-week-old pigs, l-nitro-arginine (LNA) inhibited the dilation to CO, an effect reversed by a constant background of SNP. In contrast, LNA did not reduce dilation to CO in juvenile pigs or rats. In conclusion, rat pial arterioles like those in piglets dilate to CO in vivo, but there are age and species differences with regard to reactivity and interaction with NO.