PROSTAGLANDINS EFFECTS OF ISCHEMIA/REPERFUSION ON BRAIN TISSUE PROSTANOIDS AND LEUKOTRIENES IN NEWBORN PIGS P. Hsu,
S. luckerman, R. Mirro, and C.W. Leffer
W.M. Armstead,
Laboratory for Research in Neonatal Physiology, Departments of Physiology/Biophysics, Pediatrics, and Obstetrics and Gynecology, University of Tennessee, and St. Judes Research Hospital, Memphis, Tennessee 38163
ABSTRACT We i n v e s t i g a t e d t h e h y p o t h e s i s t h a t c e r e b r a l p r o s t a n o i d a n d peptidoleukotriene (LTs) (LTC4/D4/E4/F4) s y n t h e s i s are increased during postischemic reperfusion of newborn pig brains. Prostanoids and LTs extracted from brain tissue were determined by RIA in shamcontrol piglets a n d at lh, 3h, or 12h after a 20-min period of total cerebral ischemia. During reperfusion following ischemia, all regional brain tissue (cerebrum, brain stem and cerebellum) prostanoids (6keto-PGF1~, TXB2, PGE2 and PGF2a) w e r e increased at l h compared with those in sham-control piglets. Only cerebral and brain stem 6k e t o - P G F l a and cerebral TXB 2 remained elevated at 3h postischemia and all prostanoids r e t u r n e d to control levels by 12h postischemia. Brain tissue LTs were lower t h a n prostanoids and were not altered 1, 3, or 12h following ischemia. These data indicate t h a t 1) newborn pig brain tissue prostanoids are increased initially, and t h e n r e t u m e d to control levels at later stages of reperfusion following ischemia; 2) LTs are p r e s e n t in newborn pig brain tissue, b u t are not increased by ischemia/reperfusion injury and therefore probably do not play a signii~cant role in cerebral ischemia-reperfusion injury. INTRODUCTION A variety of studies in adult animals have shown t h a t ischemia and the following reperfusion increase prostanoid (1-7) a n d peptidoleukotriene (LT) (8,9) production in cerebral tissue. These eicosanoids have been proposed to be involved in the cerebral blood flow (CBF) changes (1013), the loss of cerebrovascular responsiveness (12-15), or cerebral edema (1, 2, 12, 13, 16) after discrete periods of cerebral ischemia. Previously. it h a s been observed t h a t prostanoids are i m p o r t a n t in m o d u l a t i n g perinatal cerebral h e m o d y n a m i c s (17). We also reported t h a t reperfusion following a 20-min period of total cerebral ischemia abolished prostanoid-mediated microvascular responses (18-20) and altered blood-brain barrier (BBB) permeability (21). However, whether b r a i n t i s s u e p r o s t a n o i d s or LTs are altered d u r i n g reperfusion of ischemic newborn pig brain is unknown. To our knowledge, there are no published d a t a of m e a s u r e m e n t s of eicosanoids in newborn animal brain tissue after ischemia-reperfusion injury.
DECEMBER 1991 VOL. 42 NO. 6
557
PROSTAGLANDINS T h e p r e s e n t s t u d y w a s d e s i g n e d to t e s t t h e h y p o t h e s i s t h a t r e p e r f u s i o n following c e r e b r a l i s c h e m i a i n c r e a s e s b r a i n p r o s t a n o i d a n d / o r LT b i o s y n t h e s i s w h i c h c a n b e d e t e c t e d a s a n i n c r e a s e d c o n c e n t r a t i o n in the b r a i n t i s s u e of n e w b o r n pigs. M E T H O D S AND MATERIALS All s u r g i c a l a n d e x p e r i m e n t a l p r o c e d u r e s involving a n i m a l s u s e d w e r e reviewed a n d a p p r o v e d b y t h e A n i m a l C a r e a n d U s e C o m m i t t e e a t t h e U n i v e r s i t y of T e n n e s s e e , M e m p h i s . N e w b o r n pigs (1-6 d a y s old) of e i t h e r s e x w e r e v e n t i l a t e d w i t h a positive p r e s s u r e i n f a n t r e s p i r a t o r a f t e r i n t u b a t i o n w i t h a n e n d o t r a c h e a l t u b e . B o d y t e m p e r a t u r e w a s m a i n t a i n e d b e t w e e n 37 ° a n d 3 8 ° C. U n d e r h a l o t h a n e a n d n i t r o u s oxide a n e s t h e s i a , c a t h e t e r s w e r e i n s e r t e d into a f e m o r a l v e i n for b l o o d w i t h d r a w a l a n d v e n o u s a c c e s s , a n d a f e m o r a l a r t e r y to r e c o r d b l o o d p r e s s u r e a n d d r a w s a m p l e s for b l o o d g a s a n d p H a n a l y s i s . T h e s c a l p w a s r e t r a c t e d a n d a 3 m m hole w a s m a d e in t h e s k u l l over the r i g h t p a r i e t a l c o r t e x w i t h o u t d a m a g i n g t h e d u r a u s i n g a n electric drill w i t h a hollow t o o t h l e s s bit. A hollow s t a i n l e s s s t e e l b o l t w a s s c r e w e d into t h e hole to a d e p t h f l u s h w i t h t h e i n n e r s k u l l s u r f a c e a n d t h e b o l t s e a l e d in p l a c e w i t h d e n t a l acrylic. T h e h o l l o w b o l t s a l l o w e d i n f u s i o n of artificial c e r e b r o s p i n a l fluid (ACSF) (150 N a ÷ m e q / l , 3 K + m e q / l , 2.5 C a 2÷ m e q / l , 1.2 Mg 2÷ m e q / l , 132 CI- m e q / l , 3.7 m M glucose, 6 m M u r e a , 25 H C O 3 m e q / l ; p H 7.33) into t h e c r a n i u m to i n c r e a s e i n t r a c r a n i a l p r e s s u r e , p r o d u c i n g i s c h e m i a .
Experimental design.
In t h e i s c h e m i a g r o u p , 20 rain of t o t a l b r a i n ischemia was produced by increasing the intracranial pressure as d e s c r i b e d p r e v i o u s l y (18-21). Artificial C S F (37°C) w a s i n f u s e d into t h e h o l l o w b o l t in t h e s k u l l to m a i n t a i n i n t r a c r a n i a l p r e s s u r e 15 m m H g above m e a n arterial pressure. Venous blood was withdrawn as n e c e s s a r y to m a i n t a i n t h e m e a n a r t e r i a l p r e s s u r e n o h i g h e r t h a n 100 m m H g . T h i s p r o c e d u r e r e s u l t s in r e d u c t i o n of b l o o d flow t h r o u g h o u t t h e b r a i n a n d s p i n a l c o r d to a level t h a t is n o t d e t e c t a b l e u s i n g r a d i o a c t i v e l y l a b e l e d m i c r o s p h e r e s . At t h e e n d of t h e 2 0 - m i n i s c h e m i a p e r i o d , t h e i n t r a c r a n i a l p r e s s u r e w a s r e t u r n e d to a t m o s p h e r i c , a n d t h e b o l t s e a l e d w i t h b o n e wax. In t h e s h a m - c o n t r o l g r o u p , t h e hollow b o l t w a s i m p l a n t e d , b u t i n t r a c r a n i a l p r e s s u r e w a s n o t altered. In t h e NDGA (Nordihydroguaiaretic acid, a preferential 5-1ipoxygenase inhibitor, Sigma) t r e a t e d group, NDGA w a s dissolved in s a l i n e w i t h 2 0 m e q N a H C O 3 / I a t a p p r o x i m a t e l y 50°C w i t h c o n s t a n t s t i r r i n g (1-2 hours). T h e NDGA s o l u t i o n w a s i n f u s e d i n t r a v e n o u s l y 5 m g / k g initially a n d followed b y i n f u s i o n a t a r a t e of 2 m g / k g . h r .
558
DECEMBER 1991 VOL. 42 NO. 6
PROSTAGLANDINS At the end of reperfusion (lh, 3h, 12h), the anesthetized piglet was sacrificed with s a t u r a t e d KCI (i.v.), the head elevated 10 cm above the h e a r t a n d the b r a i n t i s s u e was removed rapidly (within 1 min), immediately rinsed with ice-cold normal saline, brain tissue regions separated and frozen in liquid nitrogen for storage at -60 °C until extraction. E x t r a c t i o n o f LTs a n d P r o s t a n o i d s . Dissected brain t i s s u e s were weighed a n d homogenized in 0°C m e t h a n o l , t h e n centrifuged at 20,000g for 20 min at 4°C. The s u p e m a t a n t was passed t h r o u g h a #3 filter paper, acidified to pH 3 with formic acid and diluted to 15% aqueous methanol with water, for application to a C 18 Sep-Pak column (Waters) primed with 1 ml m e t h a n o l a n d 15 ml 15% a q u e o u s methanol containing 0.5% EDTA (pH 3 with formic acid). The column was washed with 5 ml 15% aqueous methanol, then 5 ml 30°/o aqueous methanol. LTs and prostanoids were eluted with 100% methanol, and evaporated to dryness. LT and prostanoids were analyzed by RIA. E x t r a c t i o n recovery for LT was approximately 39% a n d 60% for prostanoids. All u n k n o w n s were assayed at two dilutions. P r o s t a n o i d a n a l y s i s . Prostanoids [6-keto-prostaglandin F l a (6-ketoP G F I ~ , PGE2, thromboxane B2 (TXB2), and PGF2a] were analyzed by r a d i o i m m u n o a s s a y (RIA). Our antibodies cross-react minimally (
S. luckerman, R. Mirro, and C.W. Leffer
W.M. Armstead,
Laboratory for Research in Neonatal Physiology, Departments of Physiology/Biophysics, Pediatrics, and Obstetrics and Gynecology, University of Tennessee, and St. Judes Research Hospital, Memphis, Tennessee 38163
ABSTRACT We i n v e s t i g a t e d t h e h y p o t h e s i s t h a t c e r e b r a l p r o s t a n o i d a n d peptidoleukotriene (LTs) (LTC4/D4/E4/F4) s y n t h e s i s are increased during postischemic reperfusion of newborn pig brains. Prostanoids and LTs extracted from brain tissue were determined by RIA in shamcontrol piglets a n d at lh, 3h, or 12h after a 20-min period of total cerebral ischemia. During reperfusion following ischemia, all regional brain tissue (cerebrum, brain stem and cerebellum) prostanoids (6keto-PGF1~, TXB2, PGE2 and PGF2a) w e r e increased at l h compared with those in sham-control piglets. Only cerebral and brain stem 6k e t o - P G F l a and cerebral TXB 2 remained elevated at 3h postischemia and all prostanoids r e t u r n e d to control levels by 12h postischemia. Brain tissue LTs were lower t h a n prostanoids and were not altered 1, 3, or 12h following ischemia. These data indicate t h a t 1) newborn pig brain tissue prostanoids are increased initially, and t h e n r e t u m e d to control levels at later stages of reperfusion following ischemia; 2) LTs are p r e s e n t in newborn pig brain tissue, b u t are not increased by ischemia/reperfusion injury and therefore probably do not play a signii~cant role in cerebral ischemia-reperfusion injury. INTRODUCTION A variety of studies in adult animals have shown t h a t ischemia and the following reperfusion increase prostanoid (1-7) a n d peptidoleukotriene (LT) (8,9) production in cerebral tissue. These eicosanoids have been proposed to be involved in the cerebral blood flow (CBF) changes (1013), the loss of cerebrovascular responsiveness (12-15), or cerebral edema (1, 2, 12, 13, 16) after discrete periods of cerebral ischemia. Previously. it h a s been observed t h a t prostanoids are i m p o r t a n t in m o d u l a t i n g perinatal cerebral h e m o d y n a m i c s (17). We also reported t h a t reperfusion following a 20-min period of total cerebral ischemia abolished prostanoid-mediated microvascular responses (18-20) and altered blood-brain barrier (BBB) permeability (21). However, whether b r a i n t i s s u e p r o s t a n o i d s or LTs are altered d u r i n g reperfusion of ischemic newborn pig brain is unknown. To our knowledge, there are no published d a t a of m e a s u r e m e n t s of eicosanoids in newborn animal brain tissue after ischemia-reperfusion injury.
DECEMBER 1991 VOL. 42 NO. 6
557
PROSTAGLANDINS T h e p r e s e n t s t u d y w a s d e s i g n e d to t e s t t h e h y p o t h e s i s t h a t r e p e r f u s i o n following c e r e b r a l i s c h e m i a i n c r e a s e s b r a i n p r o s t a n o i d a n d / o r LT b i o s y n t h e s i s w h i c h c a n b e d e t e c t e d a s a n i n c r e a s e d c o n c e n t r a t i o n in the b r a i n t i s s u e of n e w b o r n pigs. M E T H O D S AND MATERIALS All s u r g i c a l a n d e x p e r i m e n t a l p r o c e d u r e s involving a n i m a l s u s e d w e r e reviewed a n d a p p r o v e d b y t h e A n i m a l C a r e a n d U s e C o m m i t t e e a t t h e U n i v e r s i t y of T e n n e s s e e , M e m p h i s . N e w b o r n pigs (1-6 d a y s old) of e i t h e r s e x w e r e v e n t i l a t e d w i t h a positive p r e s s u r e i n f a n t r e s p i r a t o r a f t e r i n t u b a t i o n w i t h a n e n d o t r a c h e a l t u b e . B o d y t e m p e r a t u r e w a s m a i n t a i n e d b e t w e e n 37 ° a n d 3 8 ° C. U n d e r h a l o t h a n e a n d n i t r o u s oxide a n e s t h e s i a , c a t h e t e r s w e r e i n s e r t e d into a f e m o r a l v e i n for b l o o d w i t h d r a w a l a n d v e n o u s a c c e s s , a n d a f e m o r a l a r t e r y to r e c o r d b l o o d p r e s s u r e a n d d r a w s a m p l e s for b l o o d g a s a n d p H a n a l y s i s . T h e s c a l p w a s r e t r a c t e d a n d a 3 m m hole w a s m a d e in t h e s k u l l over the r i g h t p a r i e t a l c o r t e x w i t h o u t d a m a g i n g t h e d u r a u s i n g a n electric drill w i t h a hollow t o o t h l e s s bit. A hollow s t a i n l e s s s t e e l b o l t w a s s c r e w e d into t h e hole to a d e p t h f l u s h w i t h t h e i n n e r s k u l l s u r f a c e a n d t h e b o l t s e a l e d in p l a c e w i t h d e n t a l acrylic. T h e h o l l o w b o l t s a l l o w e d i n f u s i o n of artificial c e r e b r o s p i n a l fluid (ACSF) (150 N a ÷ m e q / l , 3 K + m e q / l , 2.5 C a 2÷ m e q / l , 1.2 Mg 2÷ m e q / l , 132 CI- m e q / l , 3.7 m M glucose, 6 m M u r e a , 25 H C O 3 m e q / l ; p H 7.33) into t h e c r a n i u m to i n c r e a s e i n t r a c r a n i a l p r e s s u r e , p r o d u c i n g i s c h e m i a .
Experimental design.
In t h e i s c h e m i a g r o u p , 20 rain of t o t a l b r a i n ischemia was produced by increasing the intracranial pressure as d e s c r i b e d p r e v i o u s l y (18-21). Artificial C S F (37°C) w a s i n f u s e d into t h e h o l l o w b o l t in t h e s k u l l to m a i n t a i n i n t r a c r a n i a l p r e s s u r e 15 m m H g above m e a n arterial pressure. Venous blood was withdrawn as n e c e s s a r y to m a i n t a i n t h e m e a n a r t e r i a l p r e s s u r e n o h i g h e r t h a n 100 m m H g . T h i s p r o c e d u r e r e s u l t s in r e d u c t i o n of b l o o d flow t h r o u g h o u t t h e b r a i n a n d s p i n a l c o r d to a level t h a t is n o t d e t e c t a b l e u s i n g r a d i o a c t i v e l y l a b e l e d m i c r o s p h e r e s . At t h e e n d of t h e 2 0 - m i n i s c h e m i a p e r i o d , t h e i n t r a c r a n i a l p r e s s u r e w a s r e t u r n e d to a t m o s p h e r i c , a n d t h e b o l t s e a l e d w i t h b o n e wax. In t h e s h a m - c o n t r o l g r o u p , t h e hollow b o l t w a s i m p l a n t e d , b u t i n t r a c r a n i a l p r e s s u r e w a s n o t altered. In t h e NDGA (Nordihydroguaiaretic acid, a preferential 5-1ipoxygenase inhibitor, Sigma) t r e a t e d group, NDGA w a s dissolved in s a l i n e w i t h 2 0 m e q N a H C O 3 / I a t a p p r o x i m a t e l y 50°C w i t h c o n s t a n t s t i r r i n g (1-2 hours). T h e NDGA s o l u t i o n w a s i n f u s e d i n t r a v e n o u s l y 5 m g / k g initially a n d followed b y i n f u s i o n a t a r a t e of 2 m g / k g . h r .
558
DECEMBER 1991 VOL. 42 NO. 6
PROSTAGLANDINS At the end of reperfusion (lh, 3h, 12h), the anesthetized piglet was sacrificed with s a t u r a t e d KCI (i.v.), the head elevated 10 cm above the h e a r t a n d the b r a i n t i s s u e was removed rapidly (within 1 min), immediately rinsed with ice-cold normal saline, brain tissue regions separated and frozen in liquid nitrogen for storage at -60 °C until extraction. E x t r a c t i o n o f LTs a n d P r o s t a n o i d s . Dissected brain t i s s u e s were weighed a n d homogenized in 0°C m e t h a n o l , t h e n centrifuged at 20,000g for 20 min at 4°C. The s u p e m a t a n t was passed t h r o u g h a #3 filter paper, acidified to pH 3 with formic acid and diluted to 15% aqueous methanol with water, for application to a C 18 Sep-Pak column (Waters) primed with 1 ml m e t h a n o l a n d 15 ml 15% a q u e o u s methanol containing 0.5% EDTA (pH 3 with formic acid). The column was washed with 5 ml 15% aqueous methanol, then 5 ml 30°/o aqueous methanol. LTs and prostanoids were eluted with 100% methanol, and evaporated to dryness. LT and prostanoids were analyzed by RIA. E x t r a c t i o n recovery for LT was approximately 39% a n d 60% for prostanoids. All u n k n o w n s were assayed at two dilutions. P r o s t a n o i d a n a l y s i s . Prostanoids [6-keto-prostaglandin F l a (6-ketoP G F I ~ , PGE2, thromboxane B2 (TXB2), and PGF2a] were analyzed by r a d i o i m m u n o a s s a y (RIA). Our antibodies cross-react minimally (
