Expression and localization of Inter-alpha Inhibitors in rodent brain.

Inter-alpha Inhibitor Proteins (IAIPs) are a family of related serine protease inhibitors. IAIPs are important components of the systemic innate immune system. We have identified endogenous IAIPs in the central nervous system (CNS) of sheep during development and shown that treatment with IAIPs reduces neuronal cell death and improves behavioral outcomes in neonatal rats after hypoxic-ischemic brain injury. The presence of IAIPs in CNS along with their exogenous neuroprotective properties suggests that endogenous IAIPs could be part of the innate immune system in CNS. The purpose of this study was to characterize expression and localization of IAIPs in CNS. We examined cellular expressions of IAIPs in vitro in cultured cortical mouse neurons, in cultured rat neurons, microglia, and astrocytes, and in vivo on brain sections by immunohistochemistry from embryonic (E) day 18 mice and postnatal (P) day 10 rats. Cultured cortical mouse neurons expressed the light chain gene Ambp and heavy chain genes Itih-1, 2, 3, 4, and 5 mRNA transcripts and IAIP proteins. IAIP proteins were detected by immunohistochemistry in cultured cells as well as brain sections from E18 mice and P10 rats. Immunoreactivity was found in neurons, microglia, astrocytes and oligodendroglia in multiple brain regions including cortex and hippocampus, as well as within both the ependyma and choroid plexus. Our findings suggest that IAIPs are endogenous proteins expressed in a wide variety of cell types and regions both in vitro and in vivo in rodent CNS. We speculate that endogenous IAIPs may represent endogenous neuroprotective immunomodulatory proteins within the CNS.

Ontogeny of tight junction protein expression in the ovine cerebral cortex during development.

Tight junctions of the blood-brain barrier are composed of transmembrane and associated cytoplasmic proteins. The transmembrane claudin proteins form the primary seal between endothelial cells and junctional adhesion molecules (JAMs) regulate tight junction formation. We have previously shown that claudin-1, claudin-5, zonula occludens (ZO)-1, and ZO-2 exhibit differential developmental regulation from 60% of gestation up to maturity in adult sheep. The purpose of the current study was to examine developmental changes in claudin-3, -12, and JAM-A protein expression in cerebral cortices of fetuses at 60%, 80%, and 90% gestation, and in newborn and adult sheep. We also examined correlations between changes in endogenous cortisol levels and tight junction protein expression in cerebral cortices of the fetuses. Claudin-3, -12 and JAM-A expressions were determined by Western immunoblot. Claudin-3 and -12 were lower (P<0.01) at 60%, 80%, 90% and in newborns than in adults, and JAM-A was lower in adults than in fetuses at 80% and 90% gestation. Claudin-3 expression demonstrated a direct correlation with increasing plasma cortisol levels (r=0.60, n=15, P<0.02) in the fetuses. We conclude that: claudin-3, -12 and JAM-A are expressed as early as 60% of gestation in ovine cerebral cortices, exhibit differential developmental regulation, and that increasing endogenous glucocorticoids modulate claudin-3 expression in the fetus.

Maternal treatment with glucocorticoids modulates gap junction protein expression in the ovine fetal brain.

Gap junctions facilitate intercellular communication and are important in brain development. Connexins (Cx) comprise a transmembrane protein family that forms gap junctions. Cx-32 is expressed in oligodendrocytes and neurons, Cx-36 in neurons, and Cx-43 in astrocytes. Although single antenatal steroid courses are recommended for fetal lung maturation, multiple courses can be given to women at recurrent risk for premature delivery. We examined the effects of single and multiple glucocorticoid courses on Cx-32, Cx-36, and Cx-43 protein expressions in the fetal cerebral cortex, cerebellum, and spinal cord, and differences in Cx expression among brain regions under basal conditions. In the single-course groups, the ewes received dexamethasone (6 mg) or placebo as four intramuscular injections every 12h over 48 h. In the multiple-course groups, the ewes received the same treatment, once a week for 5 weeks starting at 76-78 days of gestation. Cx were measured by Western immunoblot on brain samples from 105 to 108-day gestation fetuses. A single dexamethasone course was associated with increases (P<0.05) in cerebral cortical and spinal cord Cx-36 and Cx-43 and multiple courses with increases in cerebellar and spinal cord Cx-36, and cerebral cortical and cerebellar Cx-43. Cx-32 did not change. Cx-32 was higher in the cerebellum than cerebral cortex and spinal cord, Cx-36 higher in the spinal cord than cerebellum, and Cx-43 higher in the cerebellum and spinal cord than cerebral cortex during basal conditions. In conclusion, maternal glucocorticoid therapy increases specific Cx, responses to different maternal courses vary among Cx and brain regions, and Cx expression differs among brain regions under basal conditions. Maternal treatment with glucocorticoids differentially modulates Cx in the fetal brain.

In-vitro validation of cytokine neutralizing antibodies by testing with ovine mononuclear splenocytes.

Cytokines have gained increasing attention as therapeutic targets in inflammation-related disorders and inflammatory conditions have been investigated in sheep. Monoclonal antibodies (mAbs) specific for the ovine pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 could be used to study the effects of blocking pro-inflammatory cytokines in sheep. Ovine-specific IL-1ß and IL-6 proteins and mAbs specific for these molecules were produced and the ability of the mAbs to neutralize the proteins was tested in cultures of ovine splenic mononuclear cells. Expression of nuclear factor (NF)-κß and signal transducer and activator of transcription (STAT)-3 was evaluated by western blotting and densitometric quantification. Treatment with purified IL-1ß and IL-6 proteins increased NF-κß (P < 0.001) and STAT-3 (P < 0.01) expression, respectively, in cell culture. Treatment with these proteins that were pre-incubated with IL-1ß and IL-6 mAbs attenuated (P < 0.01) these effects. These results confirm the bioactivity of ovine IL-1ß and IL-6 proteins and the neutralizing capacity of anti-ovine-IL-1ß and -IL-6 mAbs in vitro. These mAbs could be used to investigate anti-inflammatory strategies for attenuation of the effects of these pro-inflammatory cytokines in sheep.

Ischemia-reperfusion impairs blood-brain barrier function and alters tight junction protein expression in the ovine fetus.

The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) and tight junction proteins by Western immunoblot in fetal sheep at 127 days of gestation without ischemia, and 4, 24, or 48 h after ischemia. The largest increase in K(i) (P<0.05) was 4 h after ischemia. Occludin and claudin-5 expressions decreased at 4 h, but returned toward control levels 24 and 48 h after ischemia. Zonula occludens-1 and -2 decreased after ischemia. Inverse correlations between K(i) and tight junction proteins suggest that the decreases in tight junction proteins contribute to impaired blood-brain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (K(i)) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4 h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24 and 48 than 4 h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia.

Effects of phenobarbital on cerebral blood flow velocity after endotracheal suctioning in premature neonates.

OBJECTIVE: To examine the effect of phenobarbital administration on anterior cerebral artery blood flow velocity before and after endotracheal suctioning in premature neonates. DESIGN: Transcutaneous PO(2) (TcPO(2)), heart rate, mean arterial blood pressure (MABP), and Doppler velocimeter blood flow of the left anterior cerebral artery were measured before and immediately after 3 consecutive endotracheal suctioning procedures in premature neonates. Intravenous phenobarbital (20 mg/kg) was administered immediately after the first procedure. SETTING: Neonatal intensive care unit. PATIENTS: Nine neonates with a mean birth weight of 807 g (range, 620-1060 g) and a mean gestational age of 27 weeks (range, 25-30 weeks) were studied at age 8 to 12 hours. RESULTS: Transcutaneous PO(2) decreased in response to endotracheal suctioning at each of the suctioning procedures before and after phenobarbital was given (P<.001). Changes in heart rate were not observed. There were increases in MABP and area under the velocity curve (AUVC) per minute in response to endotracheal suctioning before but not after phenobarbital administration (P=.046). Use of phenobarbital lowered the overall peak systolic blood flow velocity in response to endotracheal suctioning (P =.02, analysis of variance, interactions for the effect of phenobarbital therapy on the response to suctioning). Changes in end-diastolic blood flow velocity were not observed. There were decreases in the differences before and after endotracheal suctioning for MABP at 2 and 4 hours and for AUVC and peak systolic blood flow velocity 4 hours after phenobarbital was given (P =.04). CONCLUSIONS: In very low-birth-weight neonates, endotracheal suctioning is associated with decreases in TcPO(2) and increases in MABP and AUVC. Treatment with phenobarbital attenuates the increases in MABP and AUVC but not the decreases in TcPO(2) after endotracheal suctioning.

Effects of postnatal dexamethasone on blood-brain barrier permeability and brain water content in newborn lambs.

We showed that antenatal corticosteroids reduced blood-brain barrier permeability in fetuses at 60 and 80%, but not 90% of gestation, and decreased brain water content in fetuses. Our objective was to examine the effects of postnatal corticosteroids on regional blood-brain barrier permeability and brain water content in newborn lambs. Three dexamethasone treatment groups were studied in 3- to 5-day-old lambs. A 0.01 mg/kg dose was selected to estimate the amount of dexamethasone that might have reached fetuses via antenatal treatment of ewes in our previous studies. The other doses (0.25 and 0.5 mg/kg) were chosen to approximate those used clinically to treat infants with bronchopulmonary dysplasia. Lambs were randomly assigned to receive four intramuscular injections of dexamethasone or placebo given 12 h apart on days 3 and 4 of age. Blood-brain barrier function was measured with the blood-to-brain transfer constant (K(i)) to alpha-aminoisobutyric acid, brain plasma volume was measured with polyethylene glycol for the calculation of K(i,) and brain water was measured by wet-to-dry tissue weights. Postnatal treatment with corticosteroids did not reduce barrier permeability in newborn lambs. Brain blood volume was higher in the 0.25 and 0.5 mg/kg dose dexamethasone groups than in the placebo group. Brain water content did not differ among the groups. We conclude that postnatal treatment with corticosteroids did not reduce regional blood-brain barrier permeability or brain water content but increased the brain plasma volume in newborn lambs. These findings are consistent with our previous work indicating that barrier permeability is responsive to corticosteroids at 60 and 80% of gestation and brain water regulation at 60% of gestation, but not in near-term fetuses or newborn lambs.

Effect of dexamethasone treatment on maturational changes in the NMDA receptor in sheep brain.

The objective of the present study was to examine the effect of antenatal or postnatal treatment with corticosteroids on the NMDA receptor, one of the mediators of both normal brain development and hypoxic-ischemic injury, by determining the characteristics of the receptor MK-801 binding site in untreated and corticosteroid-treated fetal and newborn lambs. (3)H-MK-801 binding was performed in cerebral cortical cell membranes from fetal sheep at 88, 120, and 136 d gestation (term = 150 d), and from 5-d-old lambs and adult ewes. Animals were randomized to receive dexamethasone [fetuses: 6 mg, i.m. every 12 hr for four doses to mother; lambs: 0.01 mg/kg (low dose) or 0.25 mg/kg (high dose) every 12 hr for four doses] or placebo. During development, B(max) (apparent number of receptors) increased, reaching a maximum in 5-d-old lambs (p < 0.05) and decreasing in the adult brain. K(d) (dissociation constant) did not change, suggesting that receptor affinity was not altered during maturation. Dexamethasone treatment had no effect on MK-801 binding in the fetus or adult, but in lambs was associated with a significant decrease in B(max) from 2.17 +/- 0.18 pmol/mg protein in placebo-treated animals to 1.65 +/- 0.8 and 1.62 +/- 0.07 pmol/mg protein in low-dose and high-dose animals, respectively. Affinity for (3)H-MK-801 decreased 20% after dexamethasone treatment in lambs only (p < 0.05). Thus, dexamethasone treatment appears to modify the NMDA receptor only during a specific period of brain development.

Exogenous and endogenous corticosteroids modulate blood-brain barrier development in the ovine fetus.

We previously reported decreases in blood-brain barrier permeability in the ovine fetus at 80% of gestation after antenatal corticosteroids and shown that permeability is not reduced in newborn lambs after postnatal corticosteroids. We now test the hypotheses that exogenous antenatal corticosteroids decrease blood-brain barrier permeability at 60% but not 90% of gestation in ovine fetuses and that endogenous increases in plasma cortisol concentrations are associated with ontogenic decreases in barrier permeability during gestation. Chronically instrumented ovine fetuses were studied 12 h after the last of four 6-mg dexamethasone or placebo injections were given 12 h apart over 48 h to ewes. Fetuses at 80% of gestation from placebo-treated ewes studied under the same protocol were also included. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) to alpha-aminoisobutyric acid. K(i) values were lower in cerebral cortex, caudate nucleus, hippocampus, superior colliculus, thalamus, medulla, and cervical spinal cord in fetuses of dexamethasone- than placebo-treated ewes at 60% but not 90% of gestation. Regional brain K(i) values demonstrated inverse correlations with increases in gestation and plasma cortisol concentrations in most brain regions. We conclude that maternal treatment with exogenous corticosteroids was associated with decreases in blood-brain barrier permeability at 60% but not 90% of gestation and that increases in gestation and endogenous cortisol concentrations were associated with ontogenic decreases in barrier permeability during fetal development.

Cerebral and intestinal perfusion and metabolism in normocythemic hyperviscous hypoxic newborn pigs.

We studied the effects of hypoxia on cerebral cortical and intestinal perfusion and metabolism in normocythemic hyperviscous newborn pigs. Seven pigs were made hyperviscous by an injection of cryoprecipitate, increasing viscosity from 5.8 +/- 0.9 to 9.0 +/- 1. 2 (SD) cycles/s. Six normoviscous pigs received 0.9% NaCl. Reducing the inspired O(2) decreased the arterial O(2) content (Ca(O(2))) from 9.5 +/- 1.6 to 3.6 +/- 1.3 ml O(2)/100 ml. Increases in brain and decreases in gastrointestinal blood flow at the lower Ca(O(2)) values were similar between the groups. During hypoxia, blood flow to stomach, distal intestinal mucosa, and large intestines was lower (-50, -23, and -28%, respectively) in the hyperviscous than normoviscous group. At the lower Ca(O(2)) values, cerebral cortical vascular resistance decreased in both groups and intestinal vascular resistance increased (+257%) in the hyperviscous but not in the normoviscous group. During hypoxia, systemic oxygen delivery decreased, extraction increased, and uptake did not change; cerebral cortical O(2) delivery, extraction, and uptake did not change; and intestinal O(2) delivery decreased, extraction increased, and uptake did not change in both groups. Our study demonstrated that 1) during hypoxia, increases in systemic O(2) extraction compensated for decreases in delivery and systemic uptake did not change; vasodilation sustained cerebral cortical O(2) delivery and preserved metabolism; increases in intestinal oxygen extraction offset decreases in delivery and uptake was preserved; and 2) nonpolycythemic hyperviscosity did not have a major influence on cardiovascular or metabolic responses to hypoxia, except for modest effects on intestinal resistance and perfusion to certain gastrointestinal regions. We conclude that, under normocythemic conditions, a moderate increase in viscosity does not have a major impact on hemodynamic or metabolic adjustments to hypoxia in newborn pigs.

Effects of duration of positive-pressure ventilation on blood-brain barrier function in premature lambs.

We have been studying the ontogeny of the blood-brain barrier function in ovine fetuses and lambs. During these studies, we have found that the duration of ventilation also influences blood-brain barrier permeability in premature lambs. Chronically instrumented hysterotomy-delivered surfactant-treated premature lambs were studied at 90% or 137 days of gestation (n = 9). Blood-brain barrier function was quantified with the blood-to-brain transfer constant K(i) to alpha-aminoisobutyric acid. Linear regression analysis was used to compare the K(i) values in the brain regions, as the dependent variable, to the duration of ventilation, as the independent variable. There were direct correlations (P < 0.05) between the K(i) values and the duration of ventilation [306 min (mean), 162-474 min (range)] in the cerebral cortex, cerebellum, medulla, caudate nucleus, hippocampus, superior colliculus, inferior colliculus, thalamus, pons, cervical spinal cord, and choroid plexus, but not in the pituitary gland. Ventilatory pressures and rates were established before the onset of the permeability studies. Calculated mean airway pressures [14 cmH(2)O (mean), 7-20 cmH(2)O (range)] from similarly studied premature lambs did not correlate with the duration of positive-pressure ventilation. We conclude that increases in the duration of positive-pressure ventilation predispose premature lambs to increases in regional blood-brain barrier permeability. These alterations in barrier function occur over relatively short time intervals (minutes to hours). In our study, these changes in permeability are most likely not attributable to changes in mean airway pressure.

Postnatal hemodynamic changes in very-low-birthweight infants.

The purpose of this study was to characterize postnatal changes in regional Doppler blood flow velocity (BFV) and cardiac function of very-low-birthweight infants and to examine factors that might influence these hemodynamic changes. Mean and end-diastolic BFV of the middle cerebral and superior mesenteric arteries, cardiac output, stroke volume, and fractional shortening were measured in 20 infants birthweight 1,002 +/- 173 g, gestational age 28 +/- 2 wk) at 6, 30, and 54 h after birth and before and after feedings on days 7 and 14. Postnatal increases in cerebral BFV, mesenteric BFV, and cardiac output were observed that were not associated with changes in blood pressure, hematocrit, pH, arterial PCO(2), or oxygen saturation. The postnatal pattern of relative vascular resistance (RVR) differed between the cerebral and mesenteric vasculatures. RVR decreased in the middle cerebral but not the superior mesenteric artery. Physiological patency of the ductus arteriosus did not alter postnatal hemodynamic changes. In response to feeding, mesenteric BFV and stroke volume increased, and mesenteric RVR and heart rate decreased. Postprandial responses were not affected by postnatal age or the age at which feeding was initiated. However, the initiation of enteral nutrition before 3 days of life was associated with higher preprandial mesenteric BFV and lower mesenteric RVR than was later initiation of feeding. We conclude that in very-low-birthweight infants over the first week of life 1) systemic, cerebral, and mesenteric hemodynamics exhibit region-specific changes; 2) asymptomatic ductus arteriosus patency and early feedings do not significantly influence these postnatal hemodynamic changes; and 3) cardiac function adapts to increase local mesenteric BFV in response to feedings.

Antenatal steroids decrease blood-brain barrier permeability in the ovine fetus.

Antenatal corticosteroid therapy reduces the incidence of intraventricular hemorrhage in premature infants. Enhanced microvascular integrity might provide protection against intraventricular hemorrhage. In the adult, there is evidence to suggest that the blood-brain barrier may be under hormonal control. We hypothesized that antenatal corticosteroids decrease blood-brain barrier permeability in the preterm ovine fetus. Chronically instrumented 120-day-gestation fetuses were studied 12 h after the last of four 6-mg dexamethasone (n = 5) or placebo (n = 6) injections had been given over 48 h to the ewes. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (Ki) for alpha-aminoisobutyric acid (AIB). Ki was significantly lower across brain regions in the fetuses of ewes that received antenatal dexamethasone compared with placebo (ANOVA; interaction, F = 2.54, P < 0.004). In fetuses of dexamethasone- and placebo-treated ewes, Ki (microliter . g brain wt-1. min-1, mean +/- SD) was, respectively, 2.43 +/- 0.27 vs. 3.41 +/- 0.74 in the cortex, 4.46 +/- 0.49 vs. 5.29 +/- 0.85 in the cerebellum, and 3.70 +/- 0.49 vs. 5.11 +/- 0.70 in the medulla. We conclude that antenatal treatment with corticosteroids reduces blood-brain permeability in the ovine fetus.

Comparison of intravenous and endotracheal epinephrine during cardiopulmonary resuscitation in newborn piglets.

OBJECTIVE: To compare the efficacy of intravenous and endotracheal epinephrine administration, and intravenous administration above and below the diaphragm, during cardiopulmonary resuscitation in newborn piglets. DESIGN: Prospective, randomized, experimental laboratory protocol. SETTING: Perinatal cardiovascular research laboratory at a university school of medicine. SUBJECTS: Forty newborn piglets (Sus domesticus). INTERVENTIONS: After cardiac arrest by ventricular fibrillation, cardiopulmonary resuscitation was begun. Radiolabeled epinephrine or placebo (0.9% sodium chloride) was administered into the right atrium, femoral vein, or endotracheal tube. Chest compressions and ventilation were continued for 10 mins. MEASUREMENTS AND MAIN RESULTS: After epinephrine or placebo administration, samples were obtained from the systemic arterial circulation for measurement of radioisotope activity and plasma epinephrine concentrations. Mean carotid arterial blood pressure, right atrial, and inferior vena caval pressures were measured continuously. Epinephrine administration via the right atrium and femoral vein resulted in significant increases in plasma epinephrine concentration, percent of radioisotope recovery, and mean carotid arterial blood pressure, whereas endotracheal epinephrine administration did not. Placebo administered into the femoral vein resulted in a significant increase in percent radioisotope recovery, but not in plasma epinephrine concentration or carotid arterial blood pressure. Endotracheal administration of placebo did not result in significant increases in plasma epinephrine concentration, percent radioisotope recovery, or carotid arterial blood pressure. There were no significant differences between right atrial or inferior vena caval pressures among the groups. CONCLUSIONS: During cardiopulmonary resuscitation in newborn piglets, intravenous administration of epinephrine is more efficacious than endotracheal administration. Furthermore, efficacy is similar between femoral venous and right atrial administration.

Reductions in cardiac output in hypoxic young pigs: systemic and regional perfusion and oxygen metabolism.

We tested the hypotheses that, in hypoxic young pigs, reductions in cardiac output restrict systemic oxygen transport to a greater extent than does hypoxia alone and that compensatory responses to this restriction are more effective in higher than in lower priority vasculatures. To study this, 10- to 14-day-old instrumented awake hypoxic (arterial oxygen tension = 39 Torr) pigs were exposed to reduced venous return by inflation of a right atrial balloon-tipped catheter. Blood flow was measured with radionuclide-labeled microspheres, and oxygen metabolism was determined with arterial and venous oxygen contents from appropriate vessels. Hypoxia resulted in a reduction in oxygen tension; increases in cardiac output and perfusion to brain (72% over baseline), heart, adrenal glands, and liver without reductions to other organs except for the spleen; reductions in systemic and intestinal oxygen delivery; and increases in systemic and intestinal oxygen extraction without changes in systemic, cerebral, or intestinal oxygen uptake. During hypoxia, decreasing venous return was associated with increases in arterial lactic acid concentration and central venous pressure; attenuation of the hypoxia-related increase in cardiac output; sustained increases in brain (72% over baseline) and heart perfusion; reductions in lung (bronchial artery), pancreatic, renal, splenic, and intestinal (-50% below baseline) perfusion; decreases in systemic and gastrointestinal oxygen delivery; sustained increases in systemic and intestinal oxygen extraction; and decreases in intestinal oxygen uptake, without changes in cerebral oxygen metabolism. We conclude that when venous return to the heart is reduced in hypoxic young pigs, the hypoxia-related increase in cardiac output was attenuated and the relative reduction in cardiac output was associated with preserved cerebral oxygen uptake and compromised intestinal oxygen uptake. Regional responses to hypoxia combined with relative reductions in cardiac output differ from that of hypoxia alone, with the greatest effects on lower priority organs such as the gastrointestinal tract.

Effects of prophylactic low-dose indomethacin on hemodynamics in very low birth weight infants.

Indomethacin decreases cerebral and mesenteric blood flow velocities in premature infants with symptomatic patent ductus arteriosus. Low-dose indomethacin is recommended for the prevention of intraventricular hemorrhage in very low birth weight infants. The hemodynamic effects of prophylactic indomethacin have not been previously examined. We hypothesized that prophylactic indomethacin does not change cerebral and mesenteric blood flow velocities and cardiac function in very low birth weight infants. Twenty-one infants (775 to 1245 gm, 24 to 31 weeks' gestation) were studied before and after indomethacin (0.1 mg/kg) administration at 6, 30, and 54 hours of life. Mean and end-diastolic cerebral and mesenteric blood flow velocities decreased (ANOVA, p < 0.05) after prophylactic indomethacin. The 38% increase in cerebral relative vascular resistance was significantly greater than the 18% increase in mesenteric relative vascular resistance (ANOVA, p < 0.05). In five infants who were fed 1 hour after the third indomethacin dose, the postprandial mesenteric blood flow velocity was significantly greater than the mesenteric blood flow velocity before both indomethacin and feeding (ANOVA, p < 0.05). Cardiac output, stroke volume, fractional shortening, and blood pressure did not change after prophylactic indomethacin administration. We conclude that prophylactic indomethacin (1) reduces cerebral and mesenteric blood flow velocity without affecting cardiac function, (2) increases cerebral more than mesenteric relative vascular resistance, and (3) does not prevent postprandial increases in mesenteric blood flow velocity. We speculate that the increase in cerebral relative vascular resistance is a beneficial effect that contributes to protection against intraventricular hemorrhage.

Ischemia-reperfusion injury in the intestines of newborn pigs.

Although the pathogenesis of necrotizing enterocolitis remains uncertain, ischemia appears to be an important contributing factor to the development of this disorder. Reperfusion plays a major role in ischemia-related injury, and oxygen free radicals produced during reperfusion most likely contribute to the injury. These oxidants can be generated during prostanoid metabolism, which increases during reperfusion of ischemic gut in adult subjects. The present study was designed to: 1) examine the effects of superior mesenteric artery occlusion, e.g. ischemia and reperfusion in vivo on the development of histopathologic intestinal injury; 2) determine whether products of arachidonic acid metabolism, e.g. prostanoids are increased during reperfusion of ischemic gut; and 3) determine whether oxygen free radical scavengers attenuate the injury in newborn pigs. Chronically catheterized placebo-pretreated newborn pigs exposed to ischemia-reperfusion, placebo-pretreated nonischemic control pigs, and polyethylene glycol-superoxide dismutase (SOD) plus polyethylene glycol-catalase (CAT)-pretreated, ischemic pigs were studied by examining changes in intestinal circulation, oxygenation, prostanoids, and tissue injury. In the placebo-pretreated pigs, intestinal blood flow decreased to very low levels during superior mesenteric artery occlusion. During reperfusion, blood flow increased, but remained below baseline. After ischemia, oxygen uptake returned to values that were similar to baseline. Intestinal efflux of the vasodilator 6-keto-prostaglandin F1alpha was evident (p < 0.05 versus no or zero efflux) during early reperfusion. Histopathologic scoring of terminal ileal samples showed significant mucosal necrosis, surface epithelial disruption, lamina propria congestion and hemorrhage, submucosal hemorrhage, edema, and increases in cells compared with the placebo-pretreated nonischemic pigs. In the SOD plus CAT-pretreated ischemic pigs, changes in intestinal blood flow, oxygen uptake, 6-keto-prostaglandin F1alpha efflux, and the pattern of the ileal tissue injury did not differ significantly from the placebo-pretreated ischemic pigs. In summary, superior mesenteric artery occlusion for 1 h and reperfusion for 2 h resulted in severe intestinal ischemia, early postocclusive limited increases in intestinal perfusion and oxygen uptake, efflux of vasodilating prostanoids during early reperfusion, and signs of ischemic tissue injury in the placebo- and SOD plus CAT-pretreated pigs. This study demonstrates that, after superior mesenteric artery occlusion and reperfusion, severe intestinal tissue injury is detected in vivo, prostanoid efflux increases, and SOD plus CAT given just before occlusion does not attenuate the extent of injury in newborn pigs.

Effects of nitric oxide synthesis inhibition on mesenteric perfusion in young pigs.

We tested the hypothesis that nitric oxide (NO) modulates postprandial hyperemia in young pigs. To test this hypothesis, we studied five groups of 3-wk-old pigs: group 1: milk fed, N(G)-monomethyl-L-arginine (L-NMMA) and L-arginine (L-Arg) treated (n = 10); group 2: milk fed, placebo treated (n = 8); group 3: water fed, L-NMMA and L-Arg treated (n = 6); group 4: water fed, placebo treated (n = 4); and group 5: fasted sham fed, L-NMMA and L-Arg treated (n = 6). After catheter placement and electromagnetic flow probe instrumentation of the mesenteric artery, systemic blood pressure and mesenteric artery blood flow were measured during preprandial baseline, postprandial, and postprandial intra-arterial L-NMMA- and L-Arg-infused study periods. The same measurements were made in the milk- and water-fed placebo-treated groups except that diluent replaced the L-NMMA and L-Arg infusions. In the milk- and water-fed placebo-treated groups, a significant (analysis of variance, P < 0.05), postprandial hyperemia was observed. The change in blood flow was greater (P < 0.05) in the milk-fed group than in the water-fed group. Inhibition of NO synthesis with L-NMMA diminished (P < 0.05) the hyperemic responses to both water and milk feeding and resulted in a decrease (P < 0.05) in mesenteric artery perfusion in the fasted sham-fed state. We conclude that, in young pigs, NO is a modulator of mesenteric vascular tone in both the postprandial and fasted states.

Magnesium is a cerebrovasodilator in newborn piglets.

We tested the hypothesis that, in newborn piglets, magnesium results in a dose-dependent prostanoid-mediated cerebrovasodilation. Pial arterioles (50-200 microns in diameter) were serially measured, and cortical subarachnoid cerebrospinal fluid (CSF) was collected for radioimmunoassay of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha, hydrolysis product of prostacyclin) and thromboxane B2 (TxB2, metabolite of thromboxane A2) before and after CSF containing 1.2, 2.4, 4.8, and 9.6 mM MgCl2 was suffused over the parietal cortex under a closed cranial window in twelve 2- to 4-day-old piglets. Magnesium suffusion resulted (P < 0.05) in a dose-dependent pial arteriolar vasodilation. The increase in vessel diameter was greater (P < 0.001) with 2.4, 4.8, and 9.6 mM than with 1.2 mM concentration of magnesium. The increase in vessel diameter with 9.6 mM was also greater (P < 0.001) than with the 2.4 mM concentration of magnesium. Magnesium suffusion did not result in changes in cortical CSF prostanoid concentrations. The effect of intravenous indomethacin (5 mg/kg) on cyclooxygenase inhibition in the pial arterioles was confirmed by a 24 +/- 3% decrease in vessel diameter at the baseline (1.2 mM) magnesium concentration. In contrast, cyclooxygenase inhibition with intravenous indomethacin did not attenuate the magnesium-induced cerebrovasodilation. We conclude that in new born piglets magnesium suffusion over the parietal cortex results in a dose-dependent cerebrovasodilation that is most likely not mediated by prostanoids.

Ontogeny of blood-brain barrier function in ovine fetuses, lambs, and adults.

The ontogeny of regional blood-brain barrier function was quantified with the rate constant for influx (Ki) across the blood-brain barrier with the small molecular weight synthetic, inert hydrophilic amino acid alpha-aminoisobutyric acid (AIB) in chronically instrumented early (87 days of gestation, 60% of gestation) and late (137 days of gestation, 90% of gestation) gestation fetal, newborn (3 days of age), older (24 days of age), and adult (3 years of age) sheep. The Ki was significantly (P < 0.05) lower in the brain regions of the adult sheep and in most brain regions of newborn and older lambs compared with fetuses at 60 and 90% of gestation. The Ki exhibited regional brain heterogeneity (P < 0.05) in the five groups. The patterns of regional heterogeneity were accentuated (P < 0.05) in the younger groups. We conclude that ontogenic decreases in blood-brain barrier permeability are observed in ovine fetuses from 60% of gestation to maturity in the adult.