Activated oxygen and arachidonate on newborn cerebral arterioles
effects
CHARLES W. LEFFLER, DAVID W. BUSIJA, WILLIAM M. ARMSTEAD, DOUGLAS R. SHANKLIN, R. MIRRO, AND 0. THELIN Laboratory for Research in Neonatal Physiology, Departments of Physiology and Biophysics, Pediatrics, Obstetrics and Gynecology, and Pathology, University of Tennessee, Memphis, Tennessee 38163
LEFFLER, CHARLES UT., DAVID W. BUSIJA, WILLIAM M. ARMSTEAD, DOUGLAS R. SHANKLIN, R. MIRRO, AND 0. THELIN. Activated oxygen and arachidonate effects on newborn
cerebral arterioles. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): Hl230-Hl238, 1990.-We have observed that pial arteriolar dilation in response to hypercapnia and hypotension is abolished after cerebral ischemia in newborn pigs. We determined whether direct generation of activated oxygen on the brain surface (OX: xanthine oxidase, hypoxanthine, FeC13, and FeS04) or topical arachidonate altered pial arteriolar responsiveness in a manner similarly to cerebral ischemia. OX, which generated more brain surface superoxide than reperfusion after ischemia, dilated pial arterioles. This dilation was reversed within 10 min of the end of exposure. OX produced ultrastructural changes in pial vessel endothelium and appeared to cause intravascular aggregation of granulocytes. After OX, prostanoid-dependent pial arteriolar dilations in response to hypercapnia and hypotension were attenuated, whereas constrictor responses to norepinephrine and acetylcholine and dilator responses to prostaglandin E2 and isoproterenol were not affected. After OX, hypercapnia increased cortical periarachnoid cerebrospinal fluid prostanoids modestly, whereas acetylcholine produced the normal strong stimulation of prostanoid synthesis. Arachidonate (low4 M and 7 x 10s4 M) also caused reversible pial arteriolar dilation but did not alter subsequent pial arteriolar responses. Therefore, although arachidonate did not mimic the effects of ischemia-reperfusion on pial arteriolar reactivity, OX produced alterations that are qualitatively similar, although quantitatively less, than those produced by ischemia. cerebral
circulation;
activated
vascular responses to various stimuli after ischemia also have been observed in adult rats (6), cats (18, 22), monkeys (21), and humans (19). The precise mechanism by which ischemia and reperfusion prevent the hypercapnia- and hypotension-induced cerebral prostanoid synthesis and pial arteriolar dilation is not known. Reperfusion after cerebral ischemia increases superoxide anion generation by the newborn pig brain (2). This increase in superoxide anion production is blocked by pretreatment with indomethacin, suggesting endoperoxide synthase metabolism of arachidonic acid is the source. In cats, topical application of a high dose of arachidonic acid causespial arteriolar dilation that is not completely reversible and vascular injury via a free radical mechanism (7). Ischemia results in massive increases in free arachidonic acid in the brain (1, 27) that can be rapidly metabolized to prostanoids and free radicals on reperfusion. Therefore, the present experiments in newborn pigs were designed to examine the possibility that generation of activated oxygen on the brain surface or topical application of arachidonic acid would produce similar changes to those caused by ischemia and reperfusion; specifically, inhibition of hypercapnia- and hypotension-induced cerebral prostanoid synthesis and inhibition of pial arteriolar dilation in response to these two prostanoid-dependent dilator mechanisms, without changing prostanoidindependent responses.
oxygen species; prostanoids METHODS
followed by reperfusion decreases prostanoid-dependent pial arteriolar dilation in newborn pigs. Specifically, in piglets, pial arteriolar dilation in response to systemic hypotension (13, 16) and hypercapnia (11, 25) is dependent on synthesis of dilator prostanoids by the brain. After ischemia, both the increase in cortical periarachnoid cerebrospinal fluid prostanoid concentration and the pial arteriolar dilation normally produced by either hypercapnia (14) or systemic hypotension (15) are absent. In contrast, the constrictor response to norepinephrine (9,15) and the dilator response to isoproterenol (9), both of which are prostanoid independent (12), are not altered by prior cerebral ischemia. Pial arteriolar dilation in response to exogenous prostaglandin E, also is normal (9). Alterations in cerebral CEREBRAL
H1230
ISCHEMIA
0363-6135/90
$1.50
Copyright
Cranial window. Newborn pigs (l-3 days old) were anesthetized with ketamine hydrochloride (33 mg/kg im) and acepromazine (3.3 mg/kg im) and maintained on (xchloralose (50 mg/kg iv initially, plus 7 mg. kg-l. h-l). 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 and 38OC. 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
0 1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
PIGLET
CEREBRAL
cemented into place with dental acrylic. The space under the window was filled with artificial cerebrospinal fluid (aCSF; 150 Na+ meq/l, 3 K+ meq/l, 2.5 Ca2+ meq/l, 1.2 Mg2+ meq/l, 132 Cl- meq/l, 3.7 mM glucose, 6 mM urea, 25 HCO: meq/l; pH, 7.33; 46 mmHg Pco~; 43 mmHg Po2) through needles incorporated into the sides of the window. The volume of fluid directly under the window was 500 ~1 and was contiguous with the periarachnoid space. Pial arterioles were observed with a trinocular stereomicroscope. Pial arteriolar diameter was measured with a video micrometer coupled to a television camera mounted on the microscope and a video monitor. Using a stage micrometer, we determined that the scale is linear over the range of O-1,000 pm. Cerebral surface CSF (300 ~1) was collected by placing a l-ml syringe on an injection port of the cranial window. CSF was collected by slowly infusing aCSF into one side of the window and allowing the CSF under the window to drip freely into a collection tube on the opposite side. Experimental design. Separate groups of piglets were examined that were defined by treatments. The treatments were an activated oxygen-generating system, the inactivated system, 10B4 M arachidonic acid, or 7 X low4 arachidonic acid. The activated oxygen-generating (OX) system consisted of xanthine oxidase (1 U/ml), hypoxanthine (0.2 mM), FeS04 (0.02 mM), and FeC& (0.02 mM), which were placed under the cranial window for 15 min. The xanthine oxidase was added to the other components within l-2 s of placement on the brain. In vitro reduction of nitroblue tetrazolium demonstrates that exogenous substrate is depleted between 50 and 60 s after mixing with xanthine oxidase. Continuous reaction with any endogenous substrate would occur. This treatment results in a large amount of superoxide anion generated on the brain surface (22 t 3 pmol rnrnB2. 15 min-‘; n = 3), as measured by previously described methods (2). In three other piglets, the generation of activated oxygen and application procedure was modified to produce continued generation of superoxide over 20 min. Repeated applications at 5-min intervals of 0.2 U/ml of xanthine oxidase, 0.6 mM hypoxanthine, and 0.02 mM FeC13EDTA were used. With the reduced concentration of enzyme the increased substrate is sufficient for continued generation over each 5-min period. In these three piglets, hypercapnia was administered both before and after treatment with the activated oxygen system for comparison. Piglets treated with the inactivated OX system (control) were initially treated with oxypurinol(50 mg/kg, 30 min before experimentation) to inhibit endogenous xanthine oxidase. They were treated as above, but the xanthine oxidase in the system was replaced with xanthine oxidase that had been boiled for 30 min to inactivate the enzyme. Because the piglets treated with the inactivated OX system (heat-inactivated xanthine oxidase and oxypurinol) showed posttreatment responses similar to those of untreated controls, this group was used as control for all experiments to minimize the number of piglets needed.
VASCULAR
ACTIVITY
H1231
Arachidonic acid treatments consisted of placing 10m4 M or 7 x 10m4 M arachidonic acid under the cranial window for two successive lo-min periods. Experiments measuring pial arteriolar reactivity were begun 20 min after each of the above treatments. All the reactivity experiments described below were not used in each piglet (see figure legends for n). Treatments were performed in the order described below, because previous experience has shown that the effects of topical norepinephrine and hypercapnia are completely reversible and do not compromise subsequent responses, whereas topical acetylcholine may alter responses to subsequent stimuli, and hypotension may cause deterioration of the preparation. The experimental protocol for investigating microvascular reactivity and for collecting CSF for prostanoid analysis involved topical application of norepinephrine, hypercapnia, hypotension, topical application of prostaglandin ( PG)E2, topical application of isoproterenol, and topical application of acetylcholine. Twenty minutes after the treatment (activated oxygen-generating systems, inactivated control, or arachidonic acid), the window was flushed with aCSF, and control measurements of pial arteriolar diameter, arterial blood pressure, blood gases, and pH were taken. Norepinephrine dissolved in aCSF (low4 M) was injected under the cranial window while pial arteriolar diameter and arterial blood pressure were measured. Ten minutes later, the cranial window was flushed with aCSF. The window was flushed with aCSF two more times at 5-min intervals, followed by a repeat lo-min control period, during which pial arteriolar diameter and arterial blood pressure were measured. Then the CSF from under the cranial window was collected for prostanoid analysis. Hypercapnia was produced by ventilating the piglet with a 10% CO2, 21% 02, 69% N2 mixture. Pial arteriolar diameter and arterial pressure were measured. After 10 min of hypercapnia, the CSF from under the cranial window was collected for prostanoid analysis, and an arterial blood sample for blood gas and pH analysis was drawn. The ventilation mixture was returned to air, and the window was flushed with aCSF four times at 5-min intervals. Hypotension was induced by withdrawing venous blood into acidcitrate-dextrose to decrease mean arterial blood pressure to -50% of the before-phlebotomy pressure. After 10 min of hypotension, the shed blood was reinfused. The CSF under the window was replaced four times at 5-min intervals. The ability of the pial arterioles to dilate in response to topical application of PGE2 was examined by measuring responses of pial arterioles to 3 X 10m7 M PGE2. Isoproterenol (lo-” M) was then placed under the cranial window, and the maximal response within 10 min was recorded. When the arteriole returned to its control value, the response to topical acetylcholine (10e3 M) was determined. The CSF under the window was collected for prostanoid analyses after 1 0 min of exposure. Vascular ultrastructure. To examine pial vascul .ar ultrastructure, bilateral cortical windows were made. One was filled with OX, whereas the other served as control. both cortical surfaces were After 15-min exposure, washed briefly with aCSF and then covered with 2%
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
H1232
PIGLET
CEREBRAL
VASCULAR
ACTIVITY
1. Arterial blood pressure, gases, and pH
TABLE
Group
Beginning
Control (n = 8) Mean arterial pressure, Arterial blood PO,, mmHg Pco~, mmHg
62zk7
mmHg
(n = 13) Mean arterial pressure, Arterial blood PO,, mmHg Pco~, mmHg PH
Arachidonic acid (lo-* M, n = 8) Mean arterial pressure, mmHg Arterial blood Po2, mmHg Pco~, mmHg
PH
are means t with beginning.
86t5 32kl 7.51kO.02
9Ok3 74&2* 7.13t0.03*
SE; n = no. of pigs.
OX,
E ru 5 E
40
g
30
9$ 6= E "
20
Wg#
5 z! E
10
0 1
3
6
10
12
15
POST
TIME (min)
FIG. 1. Effects of active xanthine oxidase (hatched bars, n = 6) and oxypurinol pretreatment (50 mg/kg) and inactive xanthine oxidase (solid bars, n = 6) with hypoxanthine and FeS04/FeClx on pial arteriolar diameter of newborn pigs. Pretreatment diameters were 165 t 21 pm in active group and 107 t 21 pm in inactive control group. Post, 10 min after exposure. Values are means k SE. * P < 0.05 compared with 0 change.
aqueous glutaraldehyde. Ten minutes later, the pia, arachnoid, and surface vessels were peeled away and diced in 2% aqueous glutaraldehyde. Routine thick cuts, 1.0-1.4 pm, were stained by toluidine blue for survey before thin cuts, 0.4 pm, and lead citrate staining for electron microscopy using a Zeiss 1oc. Prostanoid analysis. Prostanoids [6-keto-prostaglandin-F1, (6-keto-PGF1,), thromboxane BP (TxB& and PGE,] in cortical periarachnoid CSF were analyzed by radioimmunoassay against an aCSF matrix as described previously (10). All unknowns were processed at three dilutions, with parallelism between the unknown dilution curve and the standard curve required before the result was used. Sample dilutions allowed analysis of prosta-
78k4 3822 7.41kO.03 28t2"
43t4 81t4 39t3 7.45t0.02
31t3"
61tlO 80-1-4 36k4 7.41t0.07
69kll
83*4 38t2 7.5320.04
50
58k9
82-+-7 68&l* 7.27t0.01*
66k6
oxygen-generating
34&2*
57*5
83k4 30t2 7.57kO.02
PH
Values compared
431k2
53-+6
Arachidonic acid (7 X lo-* M, n = 5) Mean arterial pressure, mmHg Arterial blood PO,, mmHg Pco~, mmHg
End
8827 68t2* 7.20&0.01*
51t3
mmHg
Hypotension
48k3
87t4 33tl 7.5OkO.02
PH
ox
Hypercapnia
38t4
87t16 63t7* 7.25-r-0.02* system:
xanthine
oxidase,
80tO 36t4 7.3620.09 hypoxanthine,
FeC13,
and
FeS04.
*
P < 0.05
noid concentrations between 100 and 50,000 pg/ml. Previously, using this assay, we demonstrated large proportional increases in prostanoids after topical application of arachidonic acid and >9O% decreases in concentrations of all prostanoids examined in the cortical periarachnoid fluid after treatment with indomethacin (10 mg/ kg iv) in basal conditions and when stimulated with exogenous arachidonic acid (10). Our antibodies crossreact minimally (
effects
CHARLES W. LEFFLER, DAVID W. BUSIJA, WILLIAM M. ARMSTEAD, DOUGLAS R. SHANKLIN, R. MIRRO, AND 0. THELIN Laboratory for Research in Neonatal Physiology, Departments of Physiology and Biophysics, Pediatrics, Obstetrics and Gynecology, and Pathology, University of Tennessee, Memphis, Tennessee 38163
LEFFLER, CHARLES UT., DAVID W. BUSIJA, WILLIAM M. ARMSTEAD, DOUGLAS R. SHANKLIN, R. MIRRO, AND 0. THELIN. Activated oxygen and arachidonate effects on newborn
cerebral arterioles. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): Hl230-Hl238, 1990.-We have observed that pial arteriolar dilation in response to hypercapnia and hypotension is abolished after cerebral ischemia in newborn pigs. We determined whether direct generation of activated oxygen on the brain surface (OX: xanthine oxidase, hypoxanthine, FeC13, and FeS04) or topical arachidonate altered pial arteriolar responsiveness in a manner similarly to cerebral ischemia. OX, which generated more brain surface superoxide than reperfusion after ischemia, dilated pial arterioles. This dilation was reversed within 10 min of the end of exposure. OX produced ultrastructural changes in pial vessel endothelium and appeared to cause intravascular aggregation of granulocytes. After OX, prostanoid-dependent pial arteriolar dilations in response to hypercapnia and hypotension were attenuated, whereas constrictor responses to norepinephrine and acetylcholine and dilator responses to prostaglandin E2 and isoproterenol were not affected. After OX, hypercapnia increased cortical periarachnoid cerebrospinal fluid prostanoids modestly, whereas acetylcholine produced the normal strong stimulation of prostanoid synthesis. Arachidonate (low4 M and 7 x 10s4 M) also caused reversible pial arteriolar dilation but did not alter subsequent pial arteriolar responses. Therefore, although arachidonate did not mimic the effects of ischemia-reperfusion on pial arteriolar reactivity, OX produced alterations that are qualitatively similar, although quantitatively less, than those produced by ischemia. cerebral
circulation;
activated
vascular responses to various stimuli after ischemia also have been observed in adult rats (6), cats (18, 22), monkeys (21), and humans (19). The precise mechanism by which ischemia and reperfusion prevent the hypercapnia- and hypotension-induced cerebral prostanoid synthesis and pial arteriolar dilation is not known. Reperfusion after cerebral ischemia increases superoxide anion generation by the newborn pig brain (2). This increase in superoxide anion production is blocked by pretreatment with indomethacin, suggesting endoperoxide synthase metabolism of arachidonic acid is the source. In cats, topical application of a high dose of arachidonic acid causespial arteriolar dilation that is not completely reversible and vascular injury via a free radical mechanism (7). Ischemia results in massive increases in free arachidonic acid in the brain (1, 27) that can be rapidly metabolized to prostanoids and free radicals on reperfusion. Therefore, the present experiments in newborn pigs were designed to examine the possibility that generation of activated oxygen on the brain surface or topical application of arachidonic acid would produce similar changes to those caused by ischemia and reperfusion; specifically, inhibition of hypercapnia- and hypotension-induced cerebral prostanoid synthesis and inhibition of pial arteriolar dilation in response to these two prostanoid-dependent dilator mechanisms, without changing prostanoidindependent responses.
oxygen species; prostanoids METHODS
followed by reperfusion decreases prostanoid-dependent pial arteriolar dilation in newborn pigs. Specifically, in piglets, pial arteriolar dilation in response to systemic hypotension (13, 16) and hypercapnia (11, 25) is dependent on synthesis of dilator prostanoids by the brain. After ischemia, both the increase in cortical periarachnoid cerebrospinal fluid prostanoid concentration and the pial arteriolar dilation normally produced by either hypercapnia (14) or systemic hypotension (15) are absent. In contrast, the constrictor response to norepinephrine (9,15) and the dilator response to isoproterenol (9), both of which are prostanoid independent (12), are not altered by prior cerebral ischemia. Pial arteriolar dilation in response to exogenous prostaglandin E, also is normal (9). Alterations in cerebral CEREBRAL
H1230
ISCHEMIA
0363-6135/90
$1.50
Copyright
Cranial window. Newborn pigs (l-3 days old) were anesthetized with ketamine hydrochloride (33 mg/kg im) and acepromazine (3.3 mg/kg im) and maintained on (xchloralose (50 mg/kg iv initially, plus 7 mg. kg-l. h-l). 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 and 38OC. 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
0 1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
PIGLET
CEREBRAL
cemented into place with dental acrylic. The space under the window was filled with artificial cerebrospinal fluid (aCSF; 150 Na+ meq/l, 3 K+ meq/l, 2.5 Ca2+ meq/l, 1.2 Mg2+ meq/l, 132 Cl- meq/l, 3.7 mM glucose, 6 mM urea, 25 HCO: meq/l; pH, 7.33; 46 mmHg Pco~; 43 mmHg Po2) through needles incorporated into the sides of the window. The volume of fluid directly under the window was 500 ~1 and was contiguous with the periarachnoid space. Pial arterioles were observed with a trinocular stereomicroscope. Pial arteriolar diameter was measured with a video micrometer coupled to a television camera mounted on the microscope and a video monitor. Using a stage micrometer, we determined that the scale is linear over the range of O-1,000 pm. Cerebral surface CSF (300 ~1) was collected by placing a l-ml syringe on an injection port of the cranial window. CSF was collected by slowly infusing aCSF into one side of the window and allowing the CSF under the window to drip freely into a collection tube on the opposite side. Experimental design. Separate groups of piglets were examined that were defined by treatments. The treatments were an activated oxygen-generating system, the inactivated system, 10B4 M arachidonic acid, or 7 X low4 arachidonic acid. The activated oxygen-generating (OX) system consisted of xanthine oxidase (1 U/ml), hypoxanthine (0.2 mM), FeS04 (0.02 mM), and FeC& (0.02 mM), which were placed under the cranial window for 15 min. The xanthine oxidase was added to the other components within l-2 s of placement on the brain. In vitro reduction of nitroblue tetrazolium demonstrates that exogenous substrate is depleted between 50 and 60 s after mixing with xanthine oxidase. Continuous reaction with any endogenous substrate would occur. This treatment results in a large amount of superoxide anion generated on the brain surface (22 t 3 pmol rnrnB2. 15 min-‘; n = 3), as measured by previously described methods (2). In three other piglets, the generation of activated oxygen and application procedure was modified to produce continued generation of superoxide over 20 min. Repeated applications at 5-min intervals of 0.2 U/ml of xanthine oxidase, 0.6 mM hypoxanthine, and 0.02 mM FeC13EDTA were used. With the reduced concentration of enzyme the increased substrate is sufficient for continued generation over each 5-min period. In these three piglets, hypercapnia was administered both before and after treatment with the activated oxygen system for comparison. Piglets treated with the inactivated OX system (control) were initially treated with oxypurinol(50 mg/kg, 30 min before experimentation) to inhibit endogenous xanthine oxidase. They were treated as above, but the xanthine oxidase in the system was replaced with xanthine oxidase that had been boiled for 30 min to inactivate the enzyme. Because the piglets treated with the inactivated OX system (heat-inactivated xanthine oxidase and oxypurinol) showed posttreatment responses similar to those of untreated controls, this group was used as control for all experiments to minimize the number of piglets needed.
VASCULAR
ACTIVITY
H1231
Arachidonic acid treatments consisted of placing 10m4 M or 7 x 10m4 M arachidonic acid under the cranial window for two successive lo-min periods. Experiments measuring pial arteriolar reactivity were begun 20 min after each of the above treatments. All the reactivity experiments described below were not used in each piglet (see figure legends for n). Treatments were performed in the order described below, because previous experience has shown that the effects of topical norepinephrine and hypercapnia are completely reversible and do not compromise subsequent responses, whereas topical acetylcholine may alter responses to subsequent stimuli, and hypotension may cause deterioration of the preparation. The experimental protocol for investigating microvascular reactivity and for collecting CSF for prostanoid analysis involved topical application of norepinephrine, hypercapnia, hypotension, topical application of prostaglandin ( PG)E2, topical application of isoproterenol, and topical application of acetylcholine. Twenty minutes after the treatment (activated oxygen-generating systems, inactivated control, or arachidonic acid), the window was flushed with aCSF, and control measurements of pial arteriolar diameter, arterial blood pressure, blood gases, and pH were taken. Norepinephrine dissolved in aCSF (low4 M) was injected under the cranial window while pial arteriolar diameter and arterial blood pressure were measured. Ten minutes later, the cranial window was flushed with aCSF. The window was flushed with aCSF two more times at 5-min intervals, followed by a repeat lo-min control period, during which pial arteriolar diameter and arterial blood pressure were measured. Then the CSF from under the cranial window was collected for prostanoid analysis. Hypercapnia was produced by ventilating the piglet with a 10% CO2, 21% 02, 69% N2 mixture. Pial arteriolar diameter and arterial pressure were measured. After 10 min of hypercapnia, the CSF from under the cranial window was collected for prostanoid analysis, and an arterial blood sample for blood gas and pH analysis was drawn. The ventilation mixture was returned to air, and the window was flushed with aCSF four times at 5-min intervals. Hypotension was induced by withdrawing venous blood into acidcitrate-dextrose to decrease mean arterial blood pressure to -50% of the before-phlebotomy pressure. After 10 min of hypotension, the shed blood was reinfused. The CSF under the window was replaced four times at 5-min intervals. The ability of the pial arterioles to dilate in response to topical application of PGE2 was examined by measuring responses of pial arterioles to 3 X 10m7 M PGE2. Isoproterenol (lo-” M) was then placed under the cranial window, and the maximal response within 10 min was recorded. When the arteriole returned to its control value, the response to topical acetylcholine (10e3 M) was determined. The CSF under the window was collected for prostanoid analyses after 1 0 min of exposure. Vascular ultrastructure. To examine pial vascul .ar ultrastructure, bilateral cortical windows were made. One was filled with OX, whereas the other served as control. both cortical surfaces were After 15-min exposure, washed briefly with aCSF and then covered with 2%
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
H1232
PIGLET
CEREBRAL
VASCULAR
ACTIVITY
1. Arterial blood pressure, gases, and pH
TABLE
Group
Beginning
Control (n = 8) Mean arterial pressure, Arterial blood PO,, mmHg Pco~, mmHg
62zk7
mmHg
(n = 13) Mean arterial pressure, Arterial blood PO,, mmHg Pco~, mmHg PH
Arachidonic acid (lo-* M, n = 8) Mean arterial pressure, mmHg Arterial blood Po2, mmHg Pco~, mmHg
PH
are means t with beginning.
86t5 32kl 7.51kO.02
9Ok3 74&2* 7.13t0.03*
SE; n = no. of pigs.
OX,
E ru 5 E
40
g
30
9$ 6= E "
20
Wg#
5 z! E
10
0 1
3
6
10
12
15
POST
TIME (min)
FIG. 1. Effects of active xanthine oxidase (hatched bars, n = 6) and oxypurinol pretreatment (50 mg/kg) and inactive xanthine oxidase (solid bars, n = 6) with hypoxanthine and FeS04/FeClx on pial arteriolar diameter of newborn pigs. Pretreatment diameters were 165 t 21 pm in active group and 107 t 21 pm in inactive control group. Post, 10 min after exposure. Values are means k SE. * P < 0.05 compared with 0 change.
aqueous glutaraldehyde. Ten minutes later, the pia, arachnoid, and surface vessels were peeled away and diced in 2% aqueous glutaraldehyde. Routine thick cuts, 1.0-1.4 pm, were stained by toluidine blue for survey before thin cuts, 0.4 pm, and lead citrate staining for electron microscopy using a Zeiss 1oc. Prostanoid analysis. Prostanoids [6-keto-prostaglandin-F1, (6-keto-PGF1,), thromboxane BP (TxB& and PGE,] in cortical periarachnoid CSF were analyzed by radioimmunoassay against an aCSF matrix as described previously (10). All unknowns were processed at three dilutions, with parallelism between the unknown dilution curve and the standard curve required before the result was used. Sample dilutions allowed analysis of prosta-
78k4 3822 7.41kO.03 28t2"
43t4 81t4 39t3 7.45t0.02
31t3"
61tlO 80-1-4 36k4 7.41t0.07
69kll
83*4 38t2 7.5320.04
50
58k9
82-+-7 68&l* 7.27t0.01*
66k6
oxygen-generating
34&2*
57*5
83k4 30t2 7.57kO.02
PH
Values compared
431k2
53-+6
Arachidonic acid (7 X lo-* M, n = 5) Mean arterial pressure, mmHg Arterial blood PO,, mmHg Pco~, mmHg
End
8827 68t2* 7.20&0.01*
51t3
mmHg
Hypotension
48k3
87t4 33tl 7.5OkO.02
PH
ox
Hypercapnia
38t4
87t16 63t7* 7.25-r-0.02* system:
xanthine
oxidase,
80tO 36t4 7.3620.09 hypoxanthine,
FeC13,
and
FeS04.
*
P < 0.05
noid concentrations between 100 and 50,000 pg/ml. Previously, using this assay, we demonstrated large proportional increases in prostanoids after topical application of arachidonic acid and >9O% decreases in concentrations of all prostanoids examined in the cortical periarachnoid fluid after treatment with indomethacin (10 mg/ kg iv) in basal conditions and when stimulated with exogenous arachidonic acid (10). Our antibodies crossreact minimally (
