PROSTAGLANDINS
PROSTANOID SYNTHESIS AND VASCULAR RESPONSES TO EXOGENOUS ARACRIDONIC ACID FOLLOWING CEBEBRU ISCHEMIA IN PIGLETS C.W. Leffler, R. Mirro, W.M. Armstead, D.W. Busija, and 0. Thelin Laboratory Departments of
for Research in Neonatal Physiology c Biophysics, Obstetrics P Gynecology, University of Tennessee, Meraphis, Tennessee 38163
Physiology, Pediatrics,
and
ABSTRACT In newborn pigs, cerebral ischemiaabolishes both increasedcerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuationof prostaglandinendoperoxidesynthaseactivitycould accountfor thefailureto increase prostanoid synthesis and loss of responses to these stimuli. To test this
possibility, arachidonic acid (3,6, or 30 &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 dosedependent increases of PGEz in cortical periarachnoid cerebral spinal fluid. 6keto-PGFI, and TXB2 only increased at the highest concentration of arachidonic acid (30 &ml). Cerebral ischemia did not decrease the conversion of any concentration of arachidonic acid to PGE2, 6-keto_PGFt,, 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 ti-ee arachidonic acid. INTRODUCTION Cerebral hemodynamic responses of newborn pigs to hypercapnia and hypotension are dependent upon products of prostaglandin endoperoxide synthase (“prostanoid dependent”) (1). Increases in cortical periarachnoid pmstanoids in response to hypercapnia and hypotension occur concomitantly with increases in pial arteriolar diameter. In addition, treatment with indomethacin abolishes increases in cerebral prostanoid concentrations and markedly attenuates or abolishes pial arteriolar dilation in response to arterial hypercapnia and hypotension. Following total cerebral ischemia, these “prostanoid dependent” responses are lost, while prostanoid independent constrictor (norepinephrine) and dilator (isoproterenol) responses are not altered (2-4). Further, exogenous PGE;! dilates pial arterioles similarly both before and after ischemia, indicating that failure of the “prostanoid dependent” responses is not due to an inability of the vessels to respond to prostanoids (2). The
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PROSTAGLANDINS responses to other products of prostaglandin endoperoxide synthase have not been examined following ischemia. The altered responses appear to result from failure of the stimuli to increase arachidonic acid metabolism via the prostaglandin endoperoxide synthase pathway following ischemia (2-4). The mechanisms responsible for these changes are unclear. Loss of arachidonic acid metabolism via the prostaglandin endoperoxide synthase pathway could result from failure to generate free arachidonic acid or from inactivation of prostaglandin endoperoxide synthase. Therefore, the present study tests the hypothesis that cerebral ischemia reduces prostaglandin endoperoxide synthase activity in the brain. We examine effects of cerebral ischemia on cerebral conversion of exogenous arachidonic acid to prostanoids and on pial arteriolar responses to topical arachidonic acid.
METHODS The animal protocols used were reviewed and approved by the Animal Care and Use Committee of the University of Tennessee, Memphis. Cranial Window Imnlantation. Newborn pigs (I-3 days old) were anesthetized with ketamine hydrochlol~de (33 mg/kg i.m.) and acepromazine (3.3 mg/kg i.m.) and maintained on ct-chloralose (50 mg/kg i.v. initially, plus 5 mg-kg-l,h-1). The animals were intubated and ventilated with air. Catheters were inserted in the femoral vein for maintenance of anesthesia and blood withdrawal and in the femoral artery to record blood pressure and draw samples for blood gas and pH analysis. Body temperature was maintained between 37-38°C. The scalp was retracted and a hole 2 cm in diameter was made in the skull over the parietal cortex. The dura was cut without touching the brain and all cut edges were retracted over the bone so that the periarachnoid space was not exposed to damaged bone or damaged membranes. A stainless steel and glass cranial window was placed in the hole and cemented into place with dental acrylic. The space under the window was filled with artificial CSF (150 mEq Na+/1, 3 mEq K+/1, 2.5 mEq Ca++/1, 1.2 rnEq Mg++/1,132 mEq CI-/1, 3.7 mM glucose, 6 mM urea, 25 mEq HC03-/1, pH, 7.33; PC02, 46 mm Hg; P02, 43 mm Hg) through needles incorporated into the sides of the window. The volume of fluid directly under the window was 500 pl and was contiguous with the periarachnoid space. At the same time that the windows were implanted, at a site remote from the window, a hollow stainless steel bolt was implanted in the skull of each piglet without damaging the dura. The bolt also was secured with dental acrylic. After implantation of the window and the hollow bolt, 20 min. were allowed before experimentation was begun. Pial arterioles were observed with a stereomicroscope. Pial arteriolar diameter was measured with a television camera mounted on the microscope, a video monitor, and a video mieroscaler. Cerebral surface CSF (300 i11)was collected by placing a syringe on an injection port of the cranial window. Fresh artificial CSF was placed under the cranial window and collection made 10 rain. later. CSF was collected by slowly infusing artificial CSF into one side of the window and allowing the CSF under the window to drip freely into a collection tube on the opposite side. To determine the loss of exogenously placed prostanoids, we measured the recovery of PGE2 placed under the cranial window for 10
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PROSTAGLANDINS rain in indomethacin pretreated piglets. The recovery was 63 + 4% (n = 5) indicating that some relatively consistent dispersion, uptake, brain-to-blood transport, or dilution occurs. Exoerimental Desien. Piglets were divided into 3 no ischemia groups and 3 ischemia groups, given different concentrations of arachidonic acid. In the no ischemia groups, the hollow bolt was implanted, but intracranial pressure was not altered. In the ischemia groups, 20 rain of total brain ischemia was produced by increasing the intracranial pressure. Artificial CSF (37°C) was infused into the hollow bolt in the skull in order to maintain intracranial pressure (measured at a cranial window needle port) 15 rnm Hg above mean arterial pressure. Blood was withdrawn as necessary to maintain the mean arterial pressure no higher than I00 mm Hg. We found that this procedure results in reduction of blood flow throughout the brain and spinal cord to a level that is not detectable using radioactively labeled microspheres (5). At the end of the 20-rain ischemia period, the intracranial pressure was returned to atmospheric pressure, the infusion tube removed from the hollow bolt, and the bolt sealed with bone wax. After 1 hr of reperfusion, measurements of pial arteriolar diameter and arterial pressure were made before and during application of arachidonic acid. Two collections of cortical periarachnoid CSF were made in each piglet: 60 rain following ischemia (or similar timing in no ischemia group) and during the next 10 min with arachidonic acid (3, 6, or 30 ~tg/rnl in artificial CSF) under the cranial window. P r 0 ~ a n o i d Analvsis. Prostanoids (6-keto-prostaglandin F l a [6-keto-PGFlct], thromboxane B2 [;rXB2], and prostaglandin E 2 [PGE2]), in cortical periarachnoid CSF were analyzed by radioimmunoassay against an artificial CSF matrix as described previously (6). All unknowns were processed at 3 dilutions, with parallelism between the unknown dilution curve and the standard curve required before the result was used. Sample dilutions used in the present study allowed analysis of prostanoid concentrations between 100-100,000 pg/ml. Previously, we demonstrated large proportional increases in prostanoids examined after topical application of arachidonic acid and greater than 90% decreases in concentrations of all prostanoids examined in the cortical periarachnoid fluid following treatment with indomethacin (10 mg/kg i.v.) under basal conditions and when stimulated with exogenous arachidonic acid (6). Our antibodies cross-react minimally (less than 1%) with other prostanoids studied. Further, target ligands are not displaced from the antibodies by arachidonic acid (30 l~g/ml); 5-HETE, 12-HETE, or 15-HETE (l~tg/ml); LTB4, LTC4, LTD4, or LTE4 (5 lag/ml); or lipoxin A4, or lipoxin B4 (10 ~tg/ml). Statistical Analvses. All values are presented as means + SEM. Comparisons between two populations were made using t-tests for planned comparisons (paired or unpaired, as appropriate). P < 0.05 was required for inference that populations were different. RESULTS The blood pressures of the piglets in the different groups are shown in Table 1. Ischemia did not alter the mean arterial pressure. The blood pressure in the ischen~ia group given 30 ~tg/ml of arachidonic acid was lower than those in the other groups before any procedure. However, we see no way that this difference would affect the interpretation of the data. Ischemia had no effect on pial arteriolar diameter at 60 minutes of reperfusion. Further, the diameters of pial arterioles before application of arachidonic acid in the different groups were not different: no ischemia, 3 ~tg arachidonic acid/ml, 129 + 15 tzm; no ischemia, 6 ~tg ar~/chidonic acid/ml, 105 + 11 ~tm; no ischemia, 30 ~tg arachidonic acid/ml, 130 + 16 I~m; ischemia, 3 ~tg arachidonic acid/ml, 132 + 17 ~tm; ischemia, 6 I~g arachidonic acid/rnl, 125 + 19 lwn; ischemia, 30 ~tg arachidonic acid/ml, 143 + 9 Wn.
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PROSTAGLANDINS T A B L E 1.
Mean arterial pressures (ram Hg) of newborn pigs. Preischemia
2ug2mLml
Postischemia
Arachidonic Acid (Min.~* 1 5 9
NO ISCHEMIA (n = 5) ISCHEMIA (n = 5)
72+4
NA
72+4
71+5
72+4
68 5:8
57 + 4
57 + 4
57 + 4
57 + 4
NO ISCHEMIA (n = 5) ISCHEMIA (n = 5)
68 + 5
NA
73 + 9
68 + 5
69 + 5
74 + 5
63 + 1
63 + 2
61 -t- 1
62 + 1
68 + 6
NA
74 + 6
66 + 7
65 + 7
50 + 1
50 + 2
49 + 2
49 + 2
49 + 2
NO ISCHEMIA (n = 6) ISCHEMIA (n = 5)
*Time after arachidonic acid at 3, 6, or 30 gg/ml was placed under the cranial window. NA = not applicable Mean + SEM Topical application of arachidonic acid caused dilation of pial arterioles (Fig. 1). No dose-response relationships could be detected in the range of arachidonic acid concentrations examined (3-30 ~tghnl). The time to the maximum increase in diameter was similar among groups (no significant differences): no ischemia, 4 + I rain, 5 + 2 rain, 6 + 1 rain, with 3, 6, or 30 ~tg arachidonic acid/ml respectively; postischemia, 4 + 2 rain, 6 + 2 min, and 4 + 1 rain with 3, 6, 30 ~tg arachidonic acid/ml, respectively. Topical arachidonic acid caused dose dependem increases in cortical periarachnoid PGE2 concentrations in both no ischemia and ischemia piglets (Table 2). These increases were similar in no ischemia and ischemia piglets. Apparent dose-dependent increases were seen in 6-keto-PGFlct as well, but these reached significance only at 30 gg arachidonic acid/ml. The changes were the same in no ischemia and ischemia piglets. The increases in TXB2 in no ischemia and ischemia piglets did not show consistent dose-response relationships to arachidonic acid.
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PROSTAGLANDINS 20
e~ llu i11 IE
e~ < ..1
IO ISCHEMIA 1 ISCHEMIA ~
A
10
J~ o v
m
a.
3
6
30
ARACHIDONATE CONC. ( ~. glm I) Figure 1. Effect of topical arachidonic acid on pial arteriolar diameters of piglets not exposed to cerebral ischemia (no ischemia) and 1 hr following cerebral ischemia (ischemia). Values are the maximum increase in diameter during the 10 rain. period of exposure to arachidonic acid. *P < 0.05 compared to zero. N = 5 for all groups except no ischemia, 30 ~tg arachidonic acid/ml, where n = 6. T A B L E 2.
Effect of topical arachidonate (AA) on cortical CSF prostanoids CSF Prostanoid Concentration (pg/ml)
NO ISCHEMIA
6-keto-PGFlct
N o A A (n = 5) 3 ~tg AA/ml
847 ± 141
955 4- 301
TXB2
PGE2
258 + 155 376 + 228
1403 ± 205 4106± 1475"
141 + 58 375 4- 105"
4822± 1996 85664- 2754*
No A A (n = 5) 6 rtg AA/ml
1323 ± 336
No A A (n = 6) 30 mg AMml
1545 ± 234 5356 4- 1860"
354 4- 114 1030 + 244*
29594- 352 401664- 13407*
No A A (n = 5) 3 ~tg AA/rnl
2487 4- 1214 29454-1605
726 + 362 1217+889
2908 4- 920 46954-801"
No A A (n = 5) 6 txg AMml
2331 + 930
5012 + 2226
284 + 165 1049 -I- 465
3060 + 834 17850 + 5635*
No A A (n = 5) 30 Ilg AA/ml
2072 + 601 6231 4- 616"
129 + 49 1383 + 418"
2327 + 744 50575 + 12627*
834 5 : 1 2 4
I~O-mMIA
*P < 0.05 compared to no AA. Mean + SEM
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PROSTAGLANDINS
The present studies demonstrate that ischemia and subsequent reperfusion do not inhibit prostaglandin endoperoxide synthase in the newborn pig brain, suggesting that the mechanism for the decreased stimulation-induced prostanoid synthesis following ischemia, indicating decreased arachidonic acid metabolism via prostaglandin endoperoxide synthase, involves inhibition of release of free arachidonic acid. These results are surprising as various known mechanisms could inhibit prostaglandin endoperoxide synthase following ischemia. For example, ischemia causes an increase in free arachidonic acid (7, 8) with rapid conversion to prostanoids upon reperfusion (9). Such an increase in prostanoid endoperoxide synthase activity could result in a decrease in prostaglandin endoperoxide synthase activity because this enzyme is autodestructive (10). Also, during postischemie reperfusion, superoxide anion is generated in large quantities on the piglet brain surface as a result of arachidonic acid metabolism by prostaglandin endoperoxide synthase (I I). Free radicals, in particular hydroxyl radical, could inhibit prostaglandin endoperoxide synthase. Another potential inhibitor is 15HETE, which may be synthesized in large quantities during postischemic reperfusion and is an endogenous inhibitor of prostaglandin endoperoxide synthase (12). However, exogenous 15-HETE does not appear to inhibit prostanoid synthesis by the newborn pig cerebral cortical surface (13). None of these mechanisms appear to significantly inhibit prostaglandin endoperoxide synthase following cerebral ischemia in newborn pigs. Therefore, the mechanism preventing the increase in prostanoid synthesis in response to appropriate stimuli following ischemia appears to be prevention of release of free arachidonic acid. However, it remains possible that the increase in prostanoid synthesis triggered by hypercapnia and hypotension is isolated to a specific cell type, for example, endothelium, and that increased arachidonic acid metabolism only in this cell type is necessary for dilation in response to hypercapnia and hypotension. If ischemia/reperfusion inhibited prostaglandin endoperoxide synthase in this cell type only, conversion of topical arachidonic acid to prostanoids could appear normal. This possibility cannot be excluded. Arachidonic acid release from membrane phospholipids occurs via specific phospholipases (14). Therefore, phospholipase inhibition by events occurring during or following ischemia could account for a reduction in prostanoid synthesis in response to stimuli following ischemia. For example, lysophosphatidylcholine and palmitoylcamitine, which increase during ischemia (16), inhibit phospholipase A2 (15). After 20 min of ischemia, phospholipase A2 activity is decreased in homogenates and mitochondria fractions of ischemic rat myocardium (15). Oxygen centered free radical damage to phospholipases is another possibility. Oxygen centered free radicals generated in the immediate postischemia period cause lipid peroxidation in membranes (17). It is possible that peroxidation of membrane lipids, including arachidonic acid, prevents release or metabolism to prostanoids. Finally, the mechanism(s) by which hypercapnia and hypotension stimulate prostanoid synthesis by the newborn pig brain are unknown. The ability of such stimuli to activate phospholipase(s) may be lost following ischemia. The vasodilator products of prostaglandin endoperoxide synthase involved in arachidonic acid induced dilation were not determined in the present experiments. Prostaglandin endoperoxide synthase catalyzes conversion of arachidonic acid to a variety of both dilators and constrictors. The predominant stable prostanoids produced dilate pial arterioles (I). However, the clear dose-response relationship between prostanoid production and araehidonie acid concentrations coupled with the absence of a dose-response relationship between pial arteriolar dilation and arachidonic acid concentxation suggests that other products of arachidonic acid must be involved in the effects on pial arteriolar tone.
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PROSTAGLANDINS One group of arachidonic acid metabolic products are the activated oxygen species. These compounds also predominantly dilate pial arterioles of newborn pigs (18, 19) and of adults of other species (20, 21). By contrast, the cyclic endoperoxide intermediates in prostanoid synthesis cause constriction of newborn pig pial arterioles via thromboxane receptor activation (22). PGG2/PGH2 probably are the main pial arteriolar constrictor metabolites of topical arachidonic acid. Increasing concentrations of cyclic endoperoxides during very rapid metabolism of arachidonic acid at the higher concentrations could partially counteract the effects of higher prostanoid concentrations. Such a circumstance could account for the poor dose-response relationship between pial arteriolar dilation and arachidonic acid concentration even though concentrations of stable prostanoids increase progressively with increasing arachidonic acid concentration. Dilator products of prostaglandin endopemxide synthase are important mediators of cerebral vasodilation in response to hypercapnia and hypotension in newborn pigs (1) and, following cerebral ischemia, such "prostanoid dependent" dilation does not occur (2-4). Nonetheless, the brain maintains the ability to metabolize exogenous arachidonic acid through the prostaglandin endoperoxide synthase pathway following ischemia. Therefore, it appears that the mechanism responsible for the loss of "prostanoid dependent" cerebral dilator responses is not an inhibition of prostaglandin endoperoxide synthase. ACKNOWLEDGEMENTS We thank J. Quetel, M. Jackson, O. Burks, and M. Craig for excellent technical assistance. These studies were supported by the NIH. Dr. Mirro is supported by a Clinical Investigatorship from the NIH.
I~ffier, C.W. and D.W. Busija. Arachidonic acid metabolites and perinatal cerebral hemodynamics. Sere. Perinatol. 11:31-42, 1987. .
Leffier, C.W., D.G. Beasley, and D.W. Busija. Cerebral ischemia alters cerebral microvascular reactivity in newborn pigs. Am. J. Physiol. 257:H266-H271, 1989.
.
Leffler, C.W., D.W. Busija, W.M. Armstead, R. Mirro, and D.G. Beasley. Ischemia alters cerebral vascular responses to hypcreapnia and acetylcholine in piglets. Pediatr. Res. 25:180-183, 1989.
4.
Leffler, C.W., D.W. Busija, D.G. Beasley, W.M. Armstead, and R. Mirro. Postischemic cerebral microvascular responses to norepinepbrine and hypotension in newborn pigs. Stroke 20:541-546, 1989.
5.
Leffler, C.W., D.W. Busija, R. Mirro, W.M. Armstead, and D.G. Beasley. Effects of ischemia on brain blood flow and oxygen consumption of newborn pigs. Am. J. Physiol. 257:H (in press), 1989.
6.
Leffler, C.W. and D.W. Busija. Arachidonate metabolism on the cerebral surface of newborn pigs. Prostaglandins 30:811-818, 1985.
.
Abe, K., K. Kogure, H. Yamamoto, M, Imazawa, and K. Miyamoto. Mechanism of arachidonic acid liberation during ischemia in gerbil cerebral cortex. J. Neurochem. 48:503-509, 1987.
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PROSTAGLANDINS
.
Wieloch, T. and B.K. Siesjo. Ischemic brain injury: the importance of calcium, lipolytic activities, and free fatty acids. Pathol. Biol. (Paris) 30:269-277, 1982.
9.
Gandet, R.L, I. Alam, and L. Levine. Accumulation of cyclooxygenase products of arachidonic acid metabolism in gerbil brain during reperfusion after bilateral common carotid occlusion. J. Neurochem. 35:653-658, 1980.
10.
Needleman, P., J. Turk, B.A. Jakscluk, A.R. Morison, and J.B. Lefkowith. Arachidonic acid metabolism. Annu. Rev. Biochem. 55:69-102, 1986.
I1.
Armstead, W.M., R. Mirro, D.W. Busija, and C.W. Leffler. Post-ischemic generation of superoxide anion by newborn pig brain. Am. J. Physiol. 255:H401H408, 1988.
12.
Setty, B.N.Y. and MJ. Smart. 15-hydroxy 5, 8, 11, 13 eicosatetraenoic acid inhibits human vascular cyclooxygenase. J. Clin. Invest. 77:202-211, 1986.
13.
Lovelady, G.K., R. Mirro, W.M. Armstead, D.W. Busija and C.W. Leffler. Effect of 15-HETE or cerebral arterioles of newborn pigs. Prostaglandins 36:507513, 1988.
14.
Isakson, P.C., A. Raz, W. Hsueh, and P. Needleman. Lipases and prostaglandin biosynthesis. Adv. Prostagl. Thrombox. Res. 3:113-120, 1978.
15.
Bentham, J.M., AJ. Higgins, and B. WoodwarcL The effects of ischemia, lysophosphatidylcholine and palmitoylcarnitine on rat heart phospholipase/%2 activity. Basic Res. Cardio. 82, Suppl. !:127-136, 1987.
16.
Shaikh, N.A., and E. Downer. Time course of changes in porcine myocardial phospholipid levels during ischemia. Circ. Res. 49:316-325, 1981.
17.
de Groot, H. and A. Littaner. Hypoxia, reactive oxygen, and cell injury. Free Radical Biol. and Medicine 6:541-551, 1989.
18.
Leffler, C.W., D.W. Busija, W.M. Armstead, and R. Mirro. H202 effects on cerebral prostanoids and pial artedolar diameter in piglets. Am. J. Physiol. 2~:(in press), 1990.
19.
Loftier, C.W., D.W. Busija, W.M. Armstead, R. Mirro, and O. Theiin. Activated oxygen alters cerebral microvascular responses in newborn pigs. FASEB J. 4:A692, 1990.
20.
Wci, E.P., C.W. Christman, H.A. Kontos, and J.T. Povlishock. Effects of oxygen radicals on cerebral arterioles. Am. J. Physiol. 248:HI57-H162, 1985.
21.
Rosenblum, W.I. Effects of free radical generation on mouse pial arterioles: possible role of hydroxyl radicals. Am. J. Physiol. 24~:H139-H142, 1983.
22.
Armstead, W.M., R. Mirro, D.W. Busija, and C.W. Leffler. Permissive role of prostanoids in acctylcholine-induced cerebral vasoconstriction. J. Pharmacol. Expd. Thcrap. 25h1012-1019, 1989. Editor: F. Coceani
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Received: 11-13-89
AoeeIM:ed: 6-15-90
SEPTEMBER 1990 VOL. 40 NO. 3
PROSTANOID SYNTHESIS AND VASCULAR RESPONSES TO EXOGENOUS ARACRIDONIC ACID FOLLOWING CEBEBRU ISCHEMIA IN PIGLETS C.W. Leffler, R. Mirro, W.M. Armstead, D.W. Busija, and 0. Thelin Laboratory Departments of
for Research in Neonatal Physiology c Biophysics, Obstetrics P Gynecology, University of Tennessee, Meraphis, Tennessee 38163
Physiology, Pediatrics,
and
ABSTRACT In newborn pigs, cerebral ischemiaabolishes both increasedcerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuationof prostaglandinendoperoxidesynthaseactivitycould accountfor thefailureto increase prostanoid synthesis and loss of responses to these stimuli. To test this
possibility, arachidonic acid (3,6, or 30 &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 dosedependent increases of PGEz in cortical periarachnoid cerebral spinal fluid. 6keto-PGFI, and TXB2 only increased at the highest concentration of arachidonic acid (30 &ml). Cerebral ischemia did not decrease the conversion of any concentration of arachidonic acid to PGE2, 6-keto_PGFt,, 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 ti-ee arachidonic acid. INTRODUCTION Cerebral hemodynamic responses of newborn pigs to hypercapnia and hypotension are dependent upon products of prostaglandin endoperoxide synthase (“prostanoid dependent”) (1). Increases in cortical periarachnoid pmstanoids in response to hypercapnia and hypotension occur concomitantly with increases in pial arteriolar diameter. In addition, treatment with indomethacin abolishes increases in cerebral prostanoid concentrations and markedly attenuates or abolishes pial arteriolar dilation in response to arterial hypercapnia and hypotension. Following total cerebral ischemia, these “prostanoid dependent” responses are lost, while prostanoid independent constrictor (norepinephrine) and dilator (isoproterenol) responses are not altered (2-4). Further, exogenous PGE;! dilates pial arterioles similarly both before and after ischemia, indicating that failure of the “prostanoid dependent” responses is not due to an inability of the vessels to respond to prostanoids (2). The
SEPTEMBER 1990 VOL. 40 NO. 3
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PROSTAGLANDINS responses to other products of prostaglandin endoperoxide synthase have not been examined following ischemia. The altered responses appear to result from failure of the stimuli to increase arachidonic acid metabolism via the prostaglandin endoperoxide synthase pathway following ischemia (2-4). The mechanisms responsible for these changes are unclear. Loss of arachidonic acid metabolism via the prostaglandin endoperoxide synthase pathway could result from failure to generate free arachidonic acid or from inactivation of prostaglandin endoperoxide synthase. Therefore, the present study tests the hypothesis that cerebral ischemia reduces prostaglandin endoperoxide synthase activity in the brain. We examine effects of cerebral ischemia on cerebral conversion of exogenous arachidonic acid to prostanoids and on pial arteriolar responses to topical arachidonic acid.
METHODS The animal protocols used were reviewed and approved by the Animal Care and Use Committee of the University of Tennessee, Memphis. Cranial Window Imnlantation. Newborn pigs (I-3 days old) were anesthetized with ketamine hydrochlol~de (33 mg/kg i.m.) and acepromazine (3.3 mg/kg i.m.) and maintained on ct-chloralose (50 mg/kg i.v. initially, plus 5 mg-kg-l,h-1). The animals were intubated and ventilated with air. Catheters were inserted in the femoral vein for maintenance of anesthesia and blood withdrawal and in the femoral artery to record blood pressure and draw samples for blood gas and pH analysis. Body temperature was maintained between 37-38°C. The scalp was retracted and a hole 2 cm in diameter was made in the skull over the parietal cortex. The dura was cut without touching the brain and all cut edges were retracted over the bone so that the periarachnoid space was not exposed to damaged bone or damaged membranes. A stainless steel and glass cranial window was placed in the hole and cemented into place with dental acrylic. The space under the window was filled with artificial CSF (150 mEq Na+/1, 3 mEq K+/1, 2.5 mEq Ca++/1, 1.2 rnEq Mg++/1,132 mEq CI-/1, 3.7 mM glucose, 6 mM urea, 25 mEq HC03-/1, pH, 7.33; PC02, 46 mm Hg; P02, 43 mm Hg) through needles incorporated into the sides of the window. The volume of fluid directly under the window was 500 pl and was contiguous with the periarachnoid space. At the same time that the windows were implanted, at a site remote from the window, a hollow stainless steel bolt was implanted in the skull of each piglet without damaging the dura. The bolt also was secured with dental acrylic. After implantation of the window and the hollow bolt, 20 min. were allowed before experimentation was begun. Pial arterioles were observed with a stereomicroscope. Pial arteriolar diameter was measured with a television camera mounted on the microscope, a video monitor, and a video mieroscaler. Cerebral surface CSF (300 i11)was collected by placing a syringe on an injection port of the cranial window. Fresh artificial CSF was placed under the cranial window and collection made 10 rain. later. CSF was collected by slowly infusing artificial CSF into one side of the window and allowing the CSF under the window to drip freely into a collection tube on the opposite side. To determine the loss of exogenously placed prostanoids, we measured the recovery of PGE2 placed under the cranial window for 10
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PROSTAGLANDINS rain in indomethacin pretreated piglets. The recovery was 63 + 4% (n = 5) indicating that some relatively consistent dispersion, uptake, brain-to-blood transport, or dilution occurs. Exoerimental Desien. Piglets were divided into 3 no ischemia groups and 3 ischemia groups, given different concentrations of arachidonic acid. In the no ischemia groups, the hollow bolt was implanted, but intracranial pressure was not altered. In the ischemia groups, 20 rain of total brain ischemia was produced by increasing the intracranial pressure. Artificial CSF (37°C) was infused into the hollow bolt in the skull in order to maintain intracranial pressure (measured at a cranial window needle port) 15 rnm Hg above mean arterial pressure. Blood was withdrawn as necessary to maintain the mean arterial pressure no higher than I00 mm Hg. We found that this procedure results in reduction of blood flow throughout the brain and spinal cord to a level that is not detectable using radioactively labeled microspheres (5). At the end of the 20-rain ischemia period, the intracranial pressure was returned to atmospheric pressure, the infusion tube removed from the hollow bolt, and the bolt sealed with bone wax. After 1 hr of reperfusion, measurements of pial arteriolar diameter and arterial pressure were made before and during application of arachidonic acid. Two collections of cortical periarachnoid CSF were made in each piglet: 60 rain following ischemia (or similar timing in no ischemia group) and during the next 10 min with arachidonic acid (3, 6, or 30 ~tg/rnl in artificial CSF) under the cranial window. P r 0 ~ a n o i d Analvsis. Prostanoids (6-keto-prostaglandin F l a [6-keto-PGFlct], thromboxane B2 [;rXB2], and prostaglandin E 2 [PGE2]), in cortical periarachnoid CSF were analyzed by radioimmunoassay against an artificial CSF matrix as described previously (6). All unknowns were processed at 3 dilutions, with parallelism between the unknown dilution curve and the standard curve required before the result was used. Sample dilutions used in the present study allowed analysis of prostanoid concentrations between 100-100,000 pg/ml. Previously, we demonstrated large proportional increases in prostanoids examined after topical application of arachidonic acid and greater than 90% decreases in concentrations of all prostanoids examined in the cortical periarachnoid fluid following treatment with indomethacin (10 mg/kg i.v.) under basal conditions and when stimulated with exogenous arachidonic acid (6). Our antibodies cross-react minimally (less than 1%) with other prostanoids studied. Further, target ligands are not displaced from the antibodies by arachidonic acid (30 l~g/ml); 5-HETE, 12-HETE, or 15-HETE (l~tg/ml); LTB4, LTC4, LTD4, or LTE4 (5 lag/ml); or lipoxin A4, or lipoxin B4 (10 ~tg/ml). Statistical Analvses. All values are presented as means + SEM. Comparisons between two populations were made using t-tests for planned comparisons (paired or unpaired, as appropriate). P < 0.05 was required for inference that populations were different. RESULTS The blood pressures of the piglets in the different groups are shown in Table 1. Ischemia did not alter the mean arterial pressure. The blood pressure in the ischen~ia group given 30 ~tg/ml of arachidonic acid was lower than those in the other groups before any procedure. However, we see no way that this difference would affect the interpretation of the data. Ischemia had no effect on pial arteriolar diameter at 60 minutes of reperfusion. Further, the diameters of pial arterioles before application of arachidonic acid in the different groups were not different: no ischemia, 3 ~tg arachidonic acid/ml, 129 + 15 tzm; no ischemia, 6 ~tg ar~/chidonic acid/ml, 105 + 11 ~tm; no ischemia, 30 ~tg arachidonic acid/ml, 130 + 16 I~m; ischemia, 3 ~tg arachidonic acid/ml, 132 + 17 ~tm; ischemia, 6 I~g arachidonic acid/rnl, 125 + 19 lwn; ischemia, 30 ~tg arachidonic acid/ml, 143 + 9 Wn.
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PROSTAGLANDINS T A B L E 1.
Mean arterial pressures (ram Hg) of newborn pigs. Preischemia
2ug2mLml
Postischemia
Arachidonic Acid (Min.~* 1 5 9
NO ISCHEMIA (n = 5) ISCHEMIA (n = 5)
72+4
NA
72+4
71+5
72+4
68 5:8
57 + 4
57 + 4
57 + 4
57 + 4
NO ISCHEMIA (n = 5) ISCHEMIA (n = 5)
68 + 5
NA
73 + 9
68 + 5
69 + 5
74 + 5
63 + 1
63 + 2
61 -t- 1
62 + 1
68 + 6
NA
74 + 6
66 + 7
65 + 7
50 + 1
50 + 2
49 + 2
49 + 2
49 + 2
NO ISCHEMIA (n = 6) ISCHEMIA (n = 5)
*Time after arachidonic acid at 3, 6, or 30 gg/ml was placed under the cranial window. NA = not applicable Mean + SEM Topical application of arachidonic acid caused dilation of pial arterioles (Fig. 1). No dose-response relationships could be detected in the range of arachidonic acid concentrations examined (3-30 ~tghnl). The time to the maximum increase in diameter was similar among groups (no significant differences): no ischemia, 4 + I rain, 5 + 2 rain, 6 + 1 rain, with 3, 6, or 30 ~tg arachidonic acid/ml respectively; postischemia, 4 + 2 rain, 6 + 2 min, and 4 + 1 rain with 3, 6, 30 ~tg arachidonic acid/ml, respectively. Topical arachidonic acid caused dose dependem increases in cortical periarachnoid PGE2 concentrations in both no ischemia and ischemia piglets (Table 2). These increases were similar in no ischemia and ischemia piglets. Apparent dose-dependent increases were seen in 6-keto-PGFlct as well, but these reached significance only at 30 gg arachidonic acid/ml. The changes were the same in no ischemia and ischemia piglets. The increases in TXB2 in no ischemia and ischemia piglets did not show consistent dose-response relationships to arachidonic acid.
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PROSTAGLANDINS 20
e~ llu i11 IE
e~ < ..1
IO ISCHEMIA 1 ISCHEMIA ~
A
10
J~ o v
m
a.
3
6
30
ARACHIDONATE CONC. ( ~. glm I) Figure 1. Effect of topical arachidonic acid on pial arteriolar diameters of piglets not exposed to cerebral ischemia (no ischemia) and 1 hr following cerebral ischemia (ischemia). Values are the maximum increase in diameter during the 10 rain. period of exposure to arachidonic acid. *P < 0.05 compared to zero. N = 5 for all groups except no ischemia, 30 ~tg arachidonic acid/ml, where n = 6. T A B L E 2.
Effect of topical arachidonate (AA) on cortical CSF prostanoids CSF Prostanoid Concentration (pg/ml)
NO ISCHEMIA
6-keto-PGFlct
N o A A (n = 5) 3 ~tg AA/ml
847 ± 141
955 4- 301
TXB2
PGE2
258 + 155 376 + 228
1403 ± 205 4106± 1475"
141 + 58 375 4- 105"
4822± 1996 85664- 2754*
No A A (n = 5) 6 rtg AA/ml
1323 ± 336
No A A (n = 6) 30 mg AMml
1545 ± 234 5356 4- 1860"
354 4- 114 1030 + 244*
29594- 352 401664- 13407*
No A A (n = 5) 3 ~tg AA/rnl
2487 4- 1214 29454-1605
726 + 362 1217+889
2908 4- 920 46954-801"
No A A (n = 5) 6 txg AMml
2331 + 930
5012 + 2226
284 + 165 1049 -I- 465
3060 + 834 17850 + 5635*
No A A (n = 5) 30 Ilg AA/ml
2072 + 601 6231 4- 616"
129 + 49 1383 + 418"
2327 + 744 50575 + 12627*
834 5 : 1 2 4
I~O-mMIA
*P < 0.05 compared to no AA. Mean + SEM
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245
PROSTAGLANDINS
The present studies demonstrate that ischemia and subsequent reperfusion do not inhibit prostaglandin endoperoxide synthase in the newborn pig brain, suggesting that the mechanism for the decreased stimulation-induced prostanoid synthesis following ischemia, indicating decreased arachidonic acid metabolism via prostaglandin endoperoxide synthase, involves inhibition of release of free arachidonic acid. These results are surprising as various known mechanisms could inhibit prostaglandin endoperoxide synthase following ischemia. For example, ischemia causes an increase in free arachidonic acid (7, 8) with rapid conversion to prostanoids upon reperfusion (9). Such an increase in prostanoid endoperoxide synthase activity could result in a decrease in prostaglandin endoperoxide synthase activity because this enzyme is autodestructive (10). Also, during postischemie reperfusion, superoxide anion is generated in large quantities on the piglet brain surface as a result of arachidonic acid metabolism by prostaglandin endoperoxide synthase (I I). Free radicals, in particular hydroxyl radical, could inhibit prostaglandin endoperoxide synthase. Another potential inhibitor is 15HETE, which may be synthesized in large quantities during postischemic reperfusion and is an endogenous inhibitor of prostaglandin endoperoxide synthase (12). However, exogenous 15-HETE does not appear to inhibit prostanoid synthesis by the newborn pig cerebral cortical surface (13). None of these mechanisms appear to significantly inhibit prostaglandin endoperoxide synthase following cerebral ischemia in newborn pigs. Therefore, the mechanism preventing the increase in prostanoid synthesis in response to appropriate stimuli following ischemia appears to be prevention of release of free arachidonic acid. However, it remains possible that the increase in prostanoid synthesis triggered by hypercapnia and hypotension is isolated to a specific cell type, for example, endothelium, and that increased arachidonic acid metabolism only in this cell type is necessary for dilation in response to hypercapnia and hypotension. If ischemia/reperfusion inhibited prostaglandin endoperoxide synthase in this cell type only, conversion of topical arachidonic acid to prostanoids could appear normal. This possibility cannot be excluded. Arachidonic acid release from membrane phospholipids occurs via specific phospholipases (14). Therefore, phospholipase inhibition by events occurring during or following ischemia could account for a reduction in prostanoid synthesis in response to stimuli following ischemia. For example, lysophosphatidylcholine and palmitoylcamitine, which increase during ischemia (16), inhibit phospholipase A2 (15). After 20 min of ischemia, phospholipase A2 activity is decreased in homogenates and mitochondria fractions of ischemic rat myocardium (15). Oxygen centered free radical damage to phospholipases is another possibility. Oxygen centered free radicals generated in the immediate postischemia period cause lipid peroxidation in membranes (17). It is possible that peroxidation of membrane lipids, including arachidonic acid, prevents release or metabolism to prostanoids. Finally, the mechanism(s) by which hypercapnia and hypotension stimulate prostanoid synthesis by the newborn pig brain are unknown. The ability of such stimuli to activate phospholipase(s) may be lost following ischemia. The vasodilator products of prostaglandin endoperoxide synthase involved in arachidonic acid induced dilation were not determined in the present experiments. Prostaglandin endoperoxide synthase catalyzes conversion of arachidonic acid to a variety of both dilators and constrictors. The predominant stable prostanoids produced dilate pial arterioles (I). However, the clear dose-response relationship between prostanoid production and araehidonie acid concentrations coupled with the absence of a dose-response relationship between pial arteriolar dilation and arachidonic acid concentxation suggests that other products of arachidonic acid must be involved in the effects on pial arteriolar tone.
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PROSTAGLANDINS One group of arachidonic acid metabolic products are the activated oxygen species. These compounds also predominantly dilate pial arterioles of newborn pigs (18, 19) and of adults of other species (20, 21). By contrast, the cyclic endoperoxide intermediates in prostanoid synthesis cause constriction of newborn pig pial arterioles via thromboxane receptor activation (22). PGG2/PGH2 probably are the main pial arteriolar constrictor metabolites of topical arachidonic acid. Increasing concentrations of cyclic endoperoxides during very rapid metabolism of arachidonic acid at the higher concentrations could partially counteract the effects of higher prostanoid concentrations. Such a circumstance could account for the poor dose-response relationship between pial arteriolar dilation and arachidonic acid concentration even though concentrations of stable prostanoids increase progressively with increasing arachidonic acid concentration. Dilator products of prostaglandin endopemxide synthase are important mediators of cerebral vasodilation in response to hypercapnia and hypotension in newborn pigs (1) and, following cerebral ischemia, such "prostanoid dependent" dilation does not occur (2-4). Nonetheless, the brain maintains the ability to metabolize exogenous arachidonic acid through the prostaglandin endoperoxide synthase pathway following ischemia. Therefore, it appears that the mechanism responsible for the loss of "prostanoid dependent" cerebral dilator responses is not an inhibition of prostaglandin endoperoxide synthase. ACKNOWLEDGEMENTS We thank J. Quetel, M. Jackson, O. Burks, and M. Craig for excellent technical assistance. These studies were supported by the NIH. Dr. Mirro is supported by a Clinical Investigatorship from the NIH.
I~ffier, C.W. and D.W. Busija. Arachidonic acid metabolites and perinatal cerebral hemodynamics. Sere. Perinatol. 11:31-42, 1987. .
Leffier, C.W., D.G. Beasley, and D.W. Busija. Cerebral ischemia alters cerebral microvascular reactivity in newborn pigs. Am. J. Physiol. 257:H266-H271, 1989.
.
Leffler, C.W., D.W. Busija, W.M. Armstead, R. Mirro, and D.G. Beasley. Ischemia alters cerebral vascular responses to hypcreapnia and acetylcholine in piglets. Pediatr. Res. 25:180-183, 1989.
4.
Leffler, C.W., D.W. Busija, D.G. Beasley, W.M. Armstead, and R. Mirro. Postischemic cerebral microvascular responses to norepinepbrine and hypotension in newborn pigs. Stroke 20:541-546, 1989.
5.
Leffler, C.W., D.W. Busija, R. Mirro, W.M. Armstead, and D.G. Beasley. Effects of ischemia on brain blood flow and oxygen consumption of newborn pigs. Am. J. Physiol. 257:H (in press), 1989.
6.
Leffler, C.W. and D.W. Busija. Arachidonate metabolism on the cerebral surface of newborn pigs. Prostaglandins 30:811-818, 1985.
.
Abe, K., K. Kogure, H. Yamamoto, M, Imazawa, and K. Miyamoto. Mechanism of arachidonic acid liberation during ischemia in gerbil cerebral cortex. J. Neurochem. 48:503-509, 1987.
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PROSTAGLANDINS
.
Wieloch, T. and B.K. Siesjo. Ischemic brain injury: the importance of calcium, lipolytic activities, and free fatty acids. Pathol. Biol. (Paris) 30:269-277, 1982.
9.
Gandet, R.L, I. Alam, and L. Levine. Accumulation of cyclooxygenase products of arachidonic acid metabolism in gerbil brain during reperfusion after bilateral common carotid occlusion. J. Neurochem. 35:653-658, 1980.
10.
Needleman, P., J. Turk, B.A. Jakscluk, A.R. Morison, and J.B. Lefkowith. Arachidonic acid metabolism. Annu. Rev. Biochem. 55:69-102, 1986.
I1.
Armstead, W.M., R. Mirro, D.W. Busija, and C.W. Leffler. Post-ischemic generation of superoxide anion by newborn pig brain. Am. J. Physiol. 255:H401H408, 1988.
12.
Setty, B.N.Y. and MJ. Smart. 15-hydroxy 5, 8, 11, 13 eicosatetraenoic acid inhibits human vascular cyclooxygenase. J. Clin. Invest. 77:202-211, 1986.
13.
Lovelady, G.K., R. Mirro, W.M. Armstead, D.W. Busija and C.W. Leffler. Effect of 15-HETE or cerebral arterioles of newborn pigs. Prostaglandins 36:507513, 1988.
14.
Isakson, P.C., A. Raz, W. Hsueh, and P. Needleman. Lipases and prostaglandin biosynthesis. Adv. Prostagl. Thrombox. Res. 3:113-120, 1978.
15.
Bentham, J.M., AJ. Higgins, and B. WoodwarcL The effects of ischemia, lysophosphatidylcholine and palmitoylcarnitine on rat heart phospholipase/%2 activity. Basic Res. Cardio. 82, Suppl. !:127-136, 1987.
16.
Shaikh, N.A., and E. Downer. Time course of changes in porcine myocardial phospholipid levels during ischemia. Circ. Res. 49:316-325, 1981.
17.
de Groot, H. and A. Littaner. Hypoxia, reactive oxygen, and cell injury. Free Radical Biol. and Medicine 6:541-551, 1989.
18.
Leffler, C.W., D.W. Busija, W.M. Armstead, and R. Mirro. H202 effects on cerebral prostanoids and pial artedolar diameter in piglets. Am. J. Physiol. 2~:(in press), 1990.
19.
Loftier, C.W., D.W. Busija, W.M. Armstead, R. Mirro, and O. Theiin. Activated oxygen alters cerebral microvascular responses in newborn pigs. FASEB J. 4:A692, 1990.
20.
Wci, E.P., C.W. Christman, H.A. Kontos, and J.T. Povlishock. Effects of oxygen radicals on cerebral arterioles. Am. J. Physiol. 248:HI57-H162, 1985.
21.
Rosenblum, W.I. Effects of free radical generation on mouse pial arterioles: possible role of hydroxyl radicals. Am. J. Physiol. 24~:H139-H142, 1983.
22.
Armstead, W.M., R. Mirro, D.W. Busija, and C.W. Leffler. Permissive role of prostanoids in acctylcholine-induced cerebral vasoconstriction. J. Pharmacol. Expd. Thcrap. 25h1012-1019, 1989. Editor: F. Coceani
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Received: 11-13-89
AoeeIM:ed: 6-15-90
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