D. W. Busija

Maintenance of cerebral circulation during hemorrhagic hypotension in newborn pigs: role of prostanoids.

The possibility that the prostanoid system contributes to the capability of the newborn piglet to maintain cerebral blood flow and cerebral metabolic rate during hypotension was investigated. The effect of hemorrhage on net (arterial-to-venous) cerebral prostacyclin production and the effects of indomethacin on cerebral hemodynamic response to hemorrhage and on the cerebral oxygen utilization following hemorrhage were determined in chronically Instrumented, unanesthetized newborn pigs. Hemorrhage decreased arterial pressure about 35% but did not affect cerebral blood flow or cerebral O2 consumption. Hemorrhage was accompanied by an increase in net cerebral 6-ket0-PGFlα production from 4.0 ±1.1 to 15.3 ± 4.9 ng/100gmin (mean ± SEM). Indomethacin treatment of piglets following hemorrhage inhibited the net cerebral production of 6-keto-PGFla and caused a decrease in blood flow (= 40%) to all brain regions within 20 minutes. The decrease in cerebral blood flow was the result of an increase in cerebral vascular resistance of 57 and 180%, 20 and 40 minutes post treatment, respectively. Cerebral O2 consumption was reduced from 2.5 ± 0.3 ml/100 g. min to 1.5 ± 0.3 ml/100 gmin 20 minutes following treatment of hemorrhaged piglets with indomethacin and to 1.1 ± 0.3 ml/100 g. min 40 minutes after treatment. Six of 8 piglets for whom the data were recorded that were administered indomethacin following hemorrhage became comatose with cerebral O2 consumption of 0.4 ± 0.1 ml O2/100 g-min by 40 minutes after treatment. These data are consistent with the hypothesis that the prostanoid system contributes to the maintenance of cerebral blood flow and cerebral metabolic rate during hypotension in the newborn.

Cerebral Superoxide Anion Generation during Seizures in Newborn Pigs

Cerebral superoxide anion generation during bicuculline-induced seizures was measured in newborn pigs. Using two closed cranial windows inserted over the parietal cortices, superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was determined during 20 min of seizure activity induced by bicuculline, 5 mg/kg i. v. A modest increase in SOD-inhibitable NBT reduction was observed in piglets subjected to bicuculline-induced seizure activity (2.4 ± 0.6 pmol/mm2 in 20 min) when compared to control piglets (0.4 ± 0.3 pmol/mm2 in 20 min). Pretreatment with indomethacin (5 mg/kg i.v.) reduced SOD-inhibitable NBT reduction during seizures to the control level (0.5 ± 0.4 pmol/mm2 in 20 min). We conclude that small quantities of superoxide anion radical are produced by newborn pig brain during bicuculline-induced seizures and that cyclooxygenase metabolism of arachidonic acid appears to be a major source.

Mono-L-arginine-containing compounds dilate piglet pial arterioles via an endothelium-derived relaxing factor-like substance.

We determined the effects of mono-L-arginine-containing compounds on pial arterioles of anesthetized piglets. A closed cranial window was implanted, and the diameter of one pial arteriole was determined by intravital microscopy. Diameter was determined during application of artificial cerebrospinal fluid containing no drugs and during application of 10(-5), 10(-4), 10(-3), and 10(-2) M L-arginine (ARG), L-arginine ethyl ester (AEE), N alpha-benzoyl-L-arginine (NBA), N alpha-benzoyl-L-arginine ester ethyl (BAEE), and L-citrulline (CIT). Initial diameters were 100-200 microns. All of these compounds dilated arterioles, but the threshold concentration needed to elicit dilation varied: 10(-5) M for NBA (n = 5), 10(-3) M for AEE (n = 9) and BAEE (n = 6), and 10(-2) M for ARG (n = 6) and CIT (n = 4). Maximal responses were 15 +/- 8% for CIT, 17 +/- 4% for ARG, 19 +/- 8% for BAEE, 28 +/- 5% for NBA, and 27 +/- 6% for AEE. Indomethacin pretreatment (5 mg/kg i.v.) did not change arteriolar responses to AEE, NBA, and BAEE. However, coadministration of methylene blue (0.5 x 10(-4) M or 0.5 x 10(-3) M) abolished dilation to 10(-3) M AEE or BAEE and attenuated dilation to 10(-5) M NBA. In addition, coadministration of hemoglobin (0.4 x 10(-4) M) abolished dilation to AEE, BAEE, or NBA. Last, intravenous (5 mg/kg) and coadministration (10(-3) M) of NG-methyl-L-arginine blocked dilation to NBA or AEE. We conclude that mono-L-arginine-containing compounds produce pial arteriolar dilation in piglets, possibly involving an endothelium-derived relaxing factor.

Platelet activating factor: a potent constrictor of cerebral arterioles in newborn pigs.

This study characterized the nature of the response to platelet activating factor (PAF) in the cerebral microcirculation of the newborn pig. Pig arterioles were observed directly using a closed cranial window in chloralose-anesthetized piglets. Topical application of 10-100 ng/ml PAF produced dose-dependent decreases in pial arteriolar diameter; diameters were 193 ± 27 γ for control, 167 ± 25 γ at 10 ng/ml, and 129 ±21 γ at 100 ng/ml. Topical application of 30-300 ng/ml norepinephrine and 3-30 ng/ml U46619, a purported thromboxane A2 receptor agonist, also produced dose-dependent decreases in pial arteriolar diameter. After topical administration of U66985 (1 /mg/ml), a putative PAF antagonist, responses to PAF were attenuated significantly, but responses to norepinephrine and U46619 were unchanged. Moreover, intravenously administered U66985 (0.1 mg/kg) antagonized PAF responses as well. Responses to PAF were unchanged after cyclooxygenase and leukotriene receptor inhibition. Further, PAF did not increase cortical subarachnoid cerebrospinal fluid prostaglandin or leukotriene levels. These data indicate that PAF is a potent constrictor of cerebral arterioles in newborn pigs and that its mechanism of action is independent of formation of cyclooxygenase and lipoxygenase products of arachidonic acid metabolism. These data also suggest that U66985 may be a selective PAF antagonist that crosses the blood-brain barrier. Since PAF is an endogenous lipid released from a variety of tissues and may be an important mediator of inflammation and allergic reaction, PAF could be involved in the pathophysiology of the cerebral circulation in the perinatal period. (Circulation Research 1988;62:1-7)

Eicosanoid synthesis elicited by norepinephrine in piglet parietal cortex

We investigated effects of exogenous norepinephrine and isoproterenol on pial arterial diameter and cerebral eicosanoid synthesis in anesthetized newborn pigs. Norepinephrine in artificial cerebrospinal fluid (CSF) constricted pial arteries from 203 +/- 27 micron (X +/- S.E.M.) to 164 +/- 18 micron (20 +/- 2%) (n = 21 vessels from 16 animals) at 10(-4) M. In the same animals, norepinephrine caused the concentration in CSF of 6-keto-prostaglandin F1 alpha to increase from 768 +/- 91 to 1544 +/- 151 pg/ml, thromboxane B2 to increase from 188 +/- 37 to 269 +/- 38 pg/ml, and prostaglandin E2 to increase from 2067 +/- 448 to 6575 +/- 751 pg/ml. Topical application of prostaglandin E2 in CSF to the cortical surface demonstrated that concentrations as low as 10,000 pg/ml were able to dilate pial arteries substantially. Blockade of cyclo-oxygenase activity by indomethacin (5-10 mg/kg, i.v.) potentiated pial arterial constriction to norepinephrine. Topical isoproterenol dilated pial arteries, but isoproterenol did not affect levels of measured eicosanoids in CSF. We conclude that norepinephrine elicits release of prostanoids from the cortical surface, and that these substances limit cerebrovascular constriction to norepinephrine.

Opioids in cerebrospinal fluid in hypotensive newborn pigs.

This study was designed to determine if opioids were detectable in cerebrospinal fluid (CSF) and if these concentrations were altered by hemorrhagic hypotension. This study was further designed to determine the effects of topically administered opioids on pial arteriolar diameter during normotension and hypotension. Closed cranial windows were used to determine pial arteriolar diameter. Periarachnoid cortical and cisterna magna CSF was collected from piglets during normotension and hypotension (systemic arterial pressure decreased from 63 +/- 1 to 33 +/- 1 mm Hg). Opioid profiles were assessed qualitatively by radioreceptor assay, and individual opioids were measured quantitatively by radioimmunoassay. Periarachnoid cortical and cisterna magna CSF methionine enkephalin-, leucine enkephalin-, dynorphin-, and beta-endorphin-like receptor active values all were increased by hypotension. When quantified by radioimmunoassay, periarachnoid cortical CSF values for methionine enkephalin-like immunoreactivity were 1,167 +/- 58 and 2,975 +/- 139 pg/ml for normotension and hypotension, respectively. Periarachnoid cortical CSF radioimmunoassay values for dynorphin-like immunoreactivity were 15 +/- 2 and 28 +/- 2 pg/ml for normotension and hypotension, respectively. When applied topically to the cortical surface, synthetic methionine enkephalin increased pial arteriolar diameter (134 +/- 4, 158 +/- 4, and 163 +/- 4 microns for control, 574 pg/ml [10(-10) M], and 5,740 pg/ml [10(-9) M], respectively). Similarly, topical synthetic leucine enkephalin and dynorphin elicited pial arteriolar dilation. However, beta-endorphin produced arteriolar constriction. Hypotension attenuated methionine and leucine enkephalin-induced dilation and reversed dynorphin-induced dilation to concentration-dependent constriction. beta-Endorphin-induced constriction was not changed by hypotension. Therefore, opioids could contribute to the control of the cerebral circulation during hypotension.

Cerebral hemodynamics during cortical spreading depression in rabbits

Effects of a single cortical spreading depression (CSD), elicited by KCl microinjection, on diameter of pial arterioles and venules in the parieto-occipital cortex were examined in urethane-anesthetized adult rabbits using a closed cranial window. The velocity of CSD propagation was 2.7 +/- 0.1 mm/min (mean +/- S.E.M.). All arterioles (n = 39) except for those in the retrosplenial region (n = 6) increased their diameter significantly during CSD. The arteriolar dilation lasted for 1.5 +/- 0.1 min. Location of dilating arteriole and propagating CSD showed that they were always closely associated temporally. As a percentage change, diameters of smaller arterioles significantly increased (from 60 +/- 1 to 103 +/- 2 microns, 71%, n = 12) more than those of larger ones (from 82 +/- 2 to 129 +/- 3 microns, 57%, n = 27). While venules with initial diameter of 85 +/- 4 microns (n = 5) did not dilate, those with initial diameter of 49 +/- 3 microns increased to 57 +/- 3 microns (16%, n = 8) for 1.4 +/- 0.2 min during CSD. The majority of the dilated venules started to increase their diameter after nearby arterioles had dilated maximally. Pial arterioles, which dilated during ipsilateral CSD, decreased their diameter significantly from 78 +/- 2 to 72 +/- 3 microns (8%, n = 11) during contralateral CSD for 13.8 +/- 3.6 min with similar onset latencies as those observed for the dilation. Indomethacin pretreatment significantly enhanced arteriolar dilation during CSD (from 73 +/- 4 to 138 +/- 6 microns, 89%, n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular responses to vasopressin are tone-dependent in the cerebral circulation of the newborn pig.

The effects of lysine vasopressin (LVP) on pial arteriolar diameter and cortical periarachnoid fluid prostanoid concentrations were investigated in newborn pigs. Chloralose-anesthetized piglets were equipped with closed cranial windows over the parietal cortex for observation of pial arterioles and collection of cerebrospinal fluid (CSF) passing over the cerebral surface. Prostanoids in the CSF were determined by radioimmunoassay. LVP (10-1,000 μU/ml) elicited concentration-dependent increases in pial arteriolar diameter associated with increased levels of 6-keto-prostaglandin (PG)F1α, PGE2, thromboxane B2, and PGF2α. LVP-induced pial arteriolar dilation was unchanged after intravenous indomethacin (5 mg/kg). Conversely, LVP constricts pial arterioles previously dilated by physiological (hemorrhagic hypotension) and pharmacological (topically applied PGE2 or isoproterenol) intervention. This constriction is potentiated by indomethacin. Vascular and biochemical changes elicited by LVP were blocked by intravenous [l-(β-mercapto-ββ-cyclopentamethylene propionic acid), 2, (O-methyl)-Tyr-AVP] (5 μg/kg), a putative V1 receptor antagonist, whereas vascular effects of norepinephrine and U46619, a thromboxane A2 mimic, were unchanged. Therefore, the degree of vascular tone appears to influence responses of the newborn pig cerebral circulation to LVP.

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.

Brain superoxide anion generation during asphyxia and reventilation in newborn pigs.

ABSTRACT: Superoxide anion generation during severe asphyxia and reventilation was assessed in newborn pigs. Using closed cranial windows over the parietal cortices, superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was determined during asphyxia/ reventilation. Asphyxia was induced by turning off the respirator and occluding the endotracheal tube. In each animal, 2.4 mM NBT dissolved in artificial cerebrospinal fluid was placed under one window and under the opposite window, NBT (2.4 mM) plus SOD (60 U/mL) dissolved in artificial cerebrospinal fluid was used. A significant increase in SOD-inhibitable NBT reduction was observed in asphyxiated piglets (14.67 ± 4.5 pmol/mm2 · 20 min) when compared with control piglets (2.82 ± 1.1 pmol/mm2 · 20 min). In another group in which the animals were treated with indomethacin (5 mg/kg i.v.) before asphyxia/reventilation, there was minimal SOD-inhibitable NBT reduction. Our results indicate that superoxide anion is generated on the cerebral cortex during asphyxia/reventilation via the prostaglandin endoperoxide synthase pathway.