Journal of Neuroscience Research 26:488-494 (1990)
Arachidonic Acid Metabolism in Gerbil Cerebra: Effects of Ischemia and Pentobarbital T.F.R. Hamm and R.V. Dorman Department of Biological Sciences, Kent State University, Kent, Ohio
Pentobarbital pretreatment may be used to predict 1978; Steen et al., 1978). These effects appear to be biochemical events involved in ischemic brain dam- related to the observed reduction in tissue necrosis folage following bilateral carotid artery ligations in the lowing acute cerebral ischemia and may depend on the gerbil, since it reduces the subsequent edema and inhibition of synaptic activities (Astrup, 1982). Pentomortality. The effects of this anesthetic on the isch- barbital also reduces the edema (Yoshida et al., 1983; emia-induced modifications of cerebral arachidonic Edgehouse and Dorman, 1987) and mortality (McGraw, acid metabolism were investigated, in order to corre- 1977; Jarrott and Domer, 1980; Yoshida et al., 1983; late observed alterations with tissue damage. Cere- Edgehouse and Dorman, 1987) subsequent to bilateral bral lipids were radiolabeled in vivo with [3H]arach- carotid artcry ligations in the gerbil. These effects may idonic acid prior to 10 rnin of cerebral ischemia and depend on its ability to protect membrane structure and 0-120 min of perfusion. Ischemia stimulated a 97.3% function, since barbiturate anesthesia inhibits ischemiaincrease in unesterified [3H]arachidonate, which was induced free fatty acid accumulation (Flamm et al., due to the loss of label from choline, inositol, and 1977; Tang and Sun, 1982; Shiu et al., 1983; Yoshida et ethanolamine glycerophospholipids. Tissue reperfu- al., 1983). The concentrations of free fatty acids have sion stimulated further reductions in [’H]choline and been shown to correlate with cerebral tissuc damage (Ra[3H]inositolglycerophospholipids, while ethanolamine zan, 1970; Kuwashima et a].. 1976; Yoshida et al., 1980; glycerophospholipid and triglyceride labeling in- Abe et al., 1987). In particular, the accumulation of creased. Inositol glycerophospholipid, but not choline unesterified arachidonic acid appears to be an accurate glycerophospholipid, labeling returned to control indicator of the degree of ischemia-induced brain damlevel by 60 min of reperfusion. Pentobarbital pre- age (Nemoto et al., 1981). Arachidonic acid and its metabolites are involved treatment reduced the accumulation of [‘Hlarachidonate by 56.2% during ischemia. It increased the in a variety of nervous tissue functions. However, uncsrecovery of [3H]ethanolamine glycerophospholipids terified arachidonate has been shown to mimic the damduring the ischemic period and [‘HH]choline glycero- aging effects of cerebral ischemia. It induces cytotoxic phospholipids during the first 5 min of reperfusion. and vasogenic edema (Chan and Fishman, 1978; Chan ct These effects accounted for the reduction of unester- al., 1983), disrupts electrical activities (Kasckow et al., ified [’Hlarachidonate obberved during ischemia and 1986), and affects the uptake and release of neurotransmitters (Chan et al., 1983; Rhoads et al., 1983; Dorman reperfusion. et al., 1986). It also inhibits cerebral Na+-K+-ATPase Key words: cerebral ischemia, lipid metabolism, an- (Ahmed and Thomas, 1971; Chan et al., 1983), disrupts esthesia, phospholipids mitochondria1 functions (Lazarewicz et al., 1972): and induces Ca2+ mobilization (Chan and Turk, 1987). These metabolic perturbations are also observed in ischemic nervous tissue. Thus, the enhanced phospholipid INTRODUCTION catabolism and free fatty acid accumulation observed in Barbiturate anesthesia attenuates the tissue damage associated with cerebral ischemia and, therefore, may be used to assess the metabolic changes related to the damage processes. Barbiturates inhibit ischemia-induced al- Received August 3 1. 1989; revised January 22, 1990: accepted Janterations of ccrebral energy charge, blood flow, lactate uary 23, 1990. production, water content, oxygen consumption, and Address reprint requests to Robert V. Dorman, Dept. Biological Scielectrical activities (Hossmann et al., 1977; Black ct al., ences, Kent State Ilniversity, Kent, OH 44242. 0 1990 Wiley-Liss, Inc.
489
Cerebral Ischemia and Lipid Metabolism
ischemic brain appear to play significant roles in the course of the related tissue damage. The effects of ischemia and reperfusion on the metabolism of the various lipid classes were assessed following ['Hlarachidonate labeling of cerebral lipids in vivo. Experiments were performed, in the presence and absence of pentobarbital anesthesia, in order to distinguish lipid metabolic alterations that may be involved in the mortality and edema development observed with this ischemia model. Cerebral ischemia stimulated the accumulation of free ['Hlarachidonate, which correlated with the loss of radiolabel from the choline, inositol, and ethanolamine glycerophospholipids. Pretreatment with pentobarbital significantly reduced the ischemia-dependent accumulation of [3H]arachidonate. This effect was related to changes in choline and ethanolamine glycerophospholipid metabolism.
MATERIALS AND METHODS Induction of Cerebral Ischemia The surgical procedures and care of the animals were approved by the University's Animal Care Committce, since they fall within established federal guidelines. Male gerbils (50-90 g) were anesthetized with pentobarbital (Nembutal; Abbott Laboratories; 36 mgi kg, i.p.) and their carotid arteries were cxposed and loosely looped with waxed threads (Edgehouse and Dorman, 1987). A small hole was drilled in each animal's left parietal belie at a distance of 2 mm from the sagital suture and 2 rnm behind the bregma. All wounds were closed with skin clips and the animals were allowed to recover for 24 hr. Each gerbil was then given a 10 pl intracerebral injection of 1-2 FCi of ['Hlarachidonic acid (83.6 Ciimmol; New England Nuclear Corp.) suspended in a degassed buffer containing 0.32 M sucrose, 50 mM Tris, 10 mgiml fatty-acid-free bovine serum albumin, and 55 mM glucose; pH 7.4. The animals were restrained with an elastic band on a surgical board and ischemia was induced by occluding both carotid arteries for 10 min with microvascular clamps following 2 hr of isotope incorporation. Blood flow was restored by removal of the clamps and the animals were sacrificed by decapitation at 0, 2, 5, 10, 20, 30, 60 and 120 min of reperfusion. Pentobarbital-treated gerbils were anesthetized 20 min prior to the induction of ischemia. Pentobarbital treatment improved animal survival from 7 1 to 97% throughout the cxpcrimental period, which was consistent with previous results (Edgehouse and Dorman, 1987). Analytical Procedures Cerebra were isolated from other brain regions by dissection on an ice-cold glass plate. Total lipids were
I
r
1
I
I
0
3C
63
93
1
1
'20
I
150
-------~7
150
J
210
243
270
INCOKPOKArION TIME (MIN)
Fig. 1. Time course of lipid labeling in gerhil cerebra following an intracerebral injection of [ 'Hlarachidonate. Cerebral lipids were radiolabeled, extracted, separated, and counted as described in the Materials and Methods following 30-250 niin of incorporation. The results were obtained from duplicate determination5 from five animals and are presented as dpm x 10~3/cerebrum.(0)= CGP, (V)= TGP, (V)= EGP, (m) = SGP, (A)= TG, ((1) = DG, (e)= FFA.
cxtracted according to Folch et al. (1957) prior to lipid class separation by thin-layer chromatography. Phospholipids wcrc scparated by the method of Emilsson and Scindler (1985), while nonpolar lipids were separated according to Freeman and West (1966). The lipid classes were visualized with iodine vapor, scraped into vials containing Ready Solv EP (Beckman Instruments), plus 10% watcr, and counted for radioactivity. These procedures have previously been used to assess cerebral lipid metabolism in the rat (Damron and Dorman, 19x8). Total lipid radioactivity was determined as the amount of isotope recovered from the thin-layer plates. The results were obtained from duplicate determinations from at least four animals and are expressed as % total lipid radioactivity 5 SEM, except for the results from the incorporation time course. which are given as dpm X I O-'!'cerebium, Statistical significance was determined using ANOVA and Duncan's multiple range test with P < 0.05 considcred to bc significant.
RESULTS Cerebral lipids were radiolabeled in vivo following intracerebral injection of [ 'H]arachidonatc. The time courses of incorporation (0-250 min) into the various lipid classes were similar to those reported for rat brain (Damron and Dorman, 1988) and are shown in Figure 1. The maximum incorporations into cholinc (CCP), inosito1 (IGP), and serine (SCP) glycerophospholipids were
Hamm and Dorman
near contol level by 20 min, and was stable through the remainder of the time course. In contrast, TG labeling was unchangcd during 10 min of ischemia, but increased 33.7% during the first 10 rnin of rcperfusion (Table I). The level of ['HITG remained elevated through 60 min of reperfusion before it declined to near control level by 120 min. DG labeling increased 4.4% during the ischemic episode and another 12.6% through 2 min of reperfusion, followed by a return to control level (Table I). Ischemia and reperfusion also affected the ['HIarachidonate labeling of the phospholipids. CGP labeling decreased 2.4% during 10 min of ischemia and this loss continued during the first 5 min of reperfusion (94.7% of control; Fig. 3). The level of ['HH]CCP showed a brief recovery to control value at 10 min of reperfusion, followed by a gradual decline in labeling, which was sigFig. 2. Effects of cerebral ischemia and reperfusion on the nificant by 60 min of reperfusion. IGP labeling (Fig. 4) accumulation of ['HH]free fatty acids in untreated ( o j and pen- was reduced 3.5% during ischemia and 18.0% by 5 min tobarbital-treated ).( gerbils. The in vivo labeling of cerebral of reperfusion. However, IGP labeling returned to conlipids with [3H]arachidonate, the induction of ischemia and trol value by 60 min of reperfusion. EGP labeling (Fig. reperfusion, and the analyses of radiolabeled lipids are de- 5) decreased 3.4% during ischemia, but the level of isoscribed in Materials and Methods. Gerbils were exposed to 10 tope in this pool was increased 18.4% by 5 min of remin of ischemia (shown as black bar) and 0-120 min reperfuperfusion. EGP labeling fell 10.7% between 5 and 10 sion. Anesthetized animals received pentobarbital 20 min bemin of reperfusion and then showed a gradual increase to fore the onset of the ischemic trauma. The results were derived from duplicate determinations from at least four animals and 119% of control at 120 min of reperfusion. Although are expressed as % total lipid radioactivity t SEM. * = sig- SGP contained much less radioactivity, the pattern of niuicantly different from 0 time, group-matched control; Dun- labeling followed that of EGP (data not shown). Pentobarbital pretreatment altered the metabolic can's multiple range test; P < 0.05. flux of isotope through the various lipid classes. The anesthetic reduced the ischemia-induced accumulation of observed at 60 rnin postinjection, while the isotopic la- [3H]arachidonate by 56.2% (Fig. 2). Again, the level of beling of ethanolamine glycerophospholipids (EGP) con- ['HIFFA remained elevated through 10 min, declined to tinued a slow increase during the 250 min of incorpora- control level by 20 min, and was significantly less than tion. Triglyceride (TG) labeling reached its maximum at the control value by 120 rnin of reperfusion. Pentobar60 min postinjection, while [?H]diglyceride (DG) peaked bital anesthesia allowed for a 24.2% reduction in labelat 30 min. The ['Hlarachidonate content of the free fatty ing of the TG during ischemia (Table I). The labeling of acid (FFA) pool declined throughout the time course. this pool returned to control level by 10 rnin of reperfureaching a baseline level at 120 min postinjection. The sion. DG labeling increased 22.2% during 10 min of labeling of the lipid pools remained stable through 250 ischemia in the presence of pentobarbital, but was remin of incorporation, with the exception of EGP. An in duced to control level at 2 min of reperfusion. This revivo incorporation time of 120 min was used for the sponse was followed by a brief increase in labeling at 5 following experiments, since recovered [3H]arachidonate rnin prior to a gradual reduction in 13H]DG that was had reached a minimum lcvcl and experiments could be significantly less than control by 120 rnin of reperfusion conducted through 120 min of reperfusion without de- (Table I). tectable changes in the labeling of the control groups. The Pentobarbital reduced the loss of ['HICGP by % distribution of isotope at 120 min postinjection was 50.0% during ischemia, when compared to the time48.6 k 0.5, 20.8 k 0.8, 17.2 -+ 0.7, 2.9 2 0.1, 2.6 2 matched value obtained from untreated gerbils (Fig. 3). 0.1, 1.7 k 0.1, and 1.9 k 0.2 for CGP, IGP, EGP, SGP, It also caused a significant increase in CGP labeling to 107.7% of control at 2 min of reperfusion. ['HICGP TG, DG, and FFA, respectively. The effects of ischemia and reperfusion on the rcturned to control level by 10 min of reperfusion, but [3H]arachidonate labeling of FFA are shown in Figure 2. was reduced (96% of control) by 120 min. In the presTen minutes of bilateral carotid artery ligations induccd a ence of pentobarbital, IGP labeling was reduced during 97.3% increase in [jH]FFA. The labeling of this pool ischemia and was 78.6% of control by 5 min of reperwas elevated through 5 min of reperfusion, rcturned to fusion, which was similar to the decline observed in
Cerebral Ischemia and Lipid Metabolism TABLE I. Effects of Cerebral Ischemia and Reperfusion on Diglyceride and Triglyceride Labeling in the Presence and Absence of Pentobarbital Pretreatment? O/u total
+
~
0 n_
[ 'H]triglycerides ~
+
*
1.850.2
2.6-'0.1
3.2e0.2
1 . 8 ~ 0 . 12.320.1" 2.120.1 1.920.2 2.020.I 2.2+0.I 1.8+0.1 2 . 0 2 0 . 1 1.7k0.1 1.9+0.1 I.6+0.2 1.8t0.1 1.620.1 1.6k0.1 1.5+0.l 1.320.1"
2.520.2
2.4-tO.1" 2.920.1 2.620.2 3.3-tO.l 3.120.2 3.1?0.2 2.920.2 2.5?0.1"
1.7+0.1
Control R (inin) 0 2 5 10 20 30 60 120
24(n
lipid radioactivity
[3H]diglycerides Pentobarbital
2.620.1 3.120.1" 3.520.2" 3.020.2 3.0-tO.1 3.220.2* 2.320.2
TGcrbil cercbra wcre radiolabclcd in vivo with 13H]areehidonatcprior lo I0 min of ischemia and 0-120 min of reperfusion (R). Total lipids were extracted. separated, and counted as described. The results were derived from duplicate determinations from at least four cerebra and are expressed as % total lipid redioactivity 2 SEM for untreated (-) and pentobarbital-treated ( + ) gerbils. "Significantly different froin control; Duncan's multiple range test: P < 0.05.
52
491
24-
-4 7 '-
C.'
0
'0
4
20 3c
61
120
REPERFUSION TIME (MIN)
Fig. 4. Effects of ischemia and reperfusion on ['HIarachidonatc labeling of inositol glycerophospholipids. The conditions and comparisons are described in the legend for Figure 2. 0 = untreated group = pentobarbital-treated group. * = significantly different from 0 time controls.
1
21
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.d *
13j
*
25 .1
T
5 CTL
3
0
10
20 30 60 REPERFUSION TIME (MIN)
CTL
120
Fig. 3. Effects of ischemia and reperfusion on the ['HIarachidonate labeling of the choline glycerophospholipids. The conditions and comparisons are described in the legend for Figure 2. 0 = untreated group; = pentobarbital-treated group. * = significantly different from 0 time controls.
unanesthetized animals (Fig. 4). The labeling of this phospholipid showed a gradual increase during the remainder of the time course. Pentobarbital pretreatment not only prevented the ischemia-induced decline in EGP labeling, it caused a 19.5% increase in labeling during the 10 niin of ischemia (Fig. 5 ) . The amount of recovered ['HIEGP then declined to control level at 2 min of reperfusion, followed by a 16.1% increase in labeling
0
10
20
30
60
120
REPERFUSION TIME (MIN)
Fig. 5 . Effects of ischemia and reperfusion on ['HIarachidonate labeling of ethanolamine glycerophospholipids. The conditions and comparisons are described in the legend for Figure 2. 0 = untreated group; = pcnlobarbital-treated group. * = significantly diffcrent from 0 time controls.
between 2 and 5 min of reperfusion. EGP labeling was 141.9%of control by 120 min. Again, SGP labeling was low, but appeared to follow a pattern similar to EGP (data not shown).
DISCUSSION Unesterified arachidonic acid has been implicated in ischemic brain damage and pentobarbital has been shown to inhibit the accumulation of this fatty acid and
492
Hamm and Dorman
attenuate many of the consequences of ischemia. 'The effects of cerebral ischemia and subsequent reperfusion on the metabolic flux of arachidonic acid were investigated in awake and pentobarbital-anesthetizedgerbils, in order to correlate the protective cffects of pentobarbital with membrane lipid metabolism. Ten minutes of bilateral carotid artery ligations in the gerbil stimulated the accumulation of ['Hlfree fatty acids in ['H]arachidonic acid-labeled cerebra. Isotope was released from all the major phospholipid classes with the choline glycerophospholipids providing approximately 50% of the radioactivity in the FFA pool. CGP labeling continued to decline through 120 min of reperfusion, while IGP labeling returned to control level and EGP labeling increased during reperfusion. Pentobarbital pretreatment attenuated the ischemia-induced accumulation of ['Hlfree fatty acids. This effect was correlated with the inhibition of isotope loss from the choline glycerophospholipids. The intracerebral injection of [3H]arachidonate resulted in the tirne-dependent incorporation of isotope into gerbil brain complex lipids. Choline glyccrophospholipids showed the greatest level of incorporation at 2 hr postinjection (48.6% of total lipid radioactivity), followed by inositol (20.8%) and ethanolamine glycerophospholipids (17.2%). This pattern of labeling was similar to that observed following intracerebral injections of ['Hlarachidonate in rat (Daniron and Dorman, 1988). Two hour exposures to isotope were routinely used, since the labeling of the free fatty acid pool reached a minimum level by this time, suggesting clearance of unincorporated ['Hlarachidonate. In addition, the labeling of the lipid pools remained constant through 250 min of incorporation, except for EGP, which showed a slow increase in radioactivity. Thus, the changes in radioactivity that were observed through 120 niin of reperfusion reflect metabolic alterations related to the ischemic episode . Ten minutes of bilateral carotid artery ligations caused a redistribution of ['Hlarachidonatc in the various lipid pools. The labeling of the FFA increased 97.3% during this period. Approximately 50% of ['HIFFA was derived from ['HICCP, while IGP and EGP catabolism each provided about 25% of the isotope to this pool. There was also a small increase in ['HIDG recovered following 10 min of ischemia, which appeared to be derived from phospholipid catabolism, since the labeling of the TG pool was not altered during ischemia. These results are consistent with the reported net degradation of all the major phospholipids upon interruption of cerebral blood flow (Porcellati et al., 1978). Pentobarbital pretreatment altered the course of ischeniia-induced phospholipid degradation. This effect was observed as a 56.2% reduction in the accumulation
of unesterified arachidonate during 10 min of ischernia, which was consistent with previous reports (Yoshida et a]., 1983; Shiu et al., 1983). Again, the labeling of the FFA pool with 13HJarachidonate was reflected by the changes observed in phospholipid labeling. The reduced accumulation of I3H]FFA was due to decreased release of isotope from CGP and increased labeling of EGP during 10 min of ischemia. In contrast, IGP labeling was further reduced during the ischemic episode when pentobarbital was present. This may reflect a slowing of acyl group exchange in the presence of anesthetic and may be related to the inhibition of synaptic activities. The return of blood flow to the cerebra caused complex alterations in the metabolism of the glycerolipids. FFA labeling was reduced 50.7% by 10 min of reperfusion, when compared to the labeling observed at the end of the ischemic period. However, the amounts of recovered [3H]CGP and [3H]IGP continued to decline during the same period. The rapid removal of unesterified arachidonate may be due to enhanced clearance, reacylation, or oxidation of the fatly acid. Eicosanoid production is stimulated under these experimental conditions in this model (Dorman, 1988) and reacylation also appears to be involved, since EGP and TG labeling increased during reperfusion. Thc continued loss of [3H]arachidonate from CGP and IGP may reflect the activation of specific phospholipases, since it has been suggested that lipid peroxides can activatc phospholipase C (Snyder and Lamb, 1987) and oxygen radicals stimulate phospholipase A2 (Schwartz et a]., 1988). Also, ischemia stimulatcs the accumulation of intracellular calcium (Siesjo and Wieloch, 1985), which may activate calcium-dependent phospholipases. Pentobarbital pretreatment altered the course of arachidonate flux during the early reperfusion times. The barbiturate reversed some of the effects of reperfusion on EGP and CGP labeling. It was observed that the amount of recovered ['HICGP increased significantly during the first 2 inin of reperfusion, while EGP labeling declined during the same time period. It appeared that the labelings of CGP and EGP were inversely related during tissue reperfusion in both awake and pentobarbital-treated animals. This effect may be due to acyl group exchange between the two lipid classes. Alternatively, it may be due to base exchange processes or alterations in diglyceride utilization by the respective synthetic enzymes. Pentobarbital pretreatment had no significant effect on the recovery of label in the inositol glycerophospholipids. [3H]IGP returned to control levels during the 2 hr time course in both awake and anesthetized animals. The inability of pentobarbital to significantly alter IGP metabolism may indicate that the catabolism of this phos-
Cerebral Ischemia and Lipid Metabolism
pholipid class is not correlated with the mortality or ccrebral edema observed in this stroke model. However, the labeling procedures employed here did not allow for assessment of the effects of ischemia or pentobarbital on polyphosphoinositide metabolism and it has been suggested that ischemia affects the polyphosphoinositides, but not IGP (Yoshida et al., 1986). The anesthetic also altered the recovery of ["HITG . At all times examined, TG labeling was reduced when compared to values obtained from untreated animals. This effect appeared to be due to the decreased availability of unesterified ['Hlarachidonate in pentobarbitaltreated cerebra. The alterations of DG labeling were similar in awake and anesthetized gerbils, except for a pentobarbital-dependent, transient decline in ['HIDG at 2 niin of reperfusion. This effect corresponded to the marked increase in ['H]CCiP and may be related to enhanced utilization of DG for CGP synthesis. In fact, the labeling of both the phospholipids and nonpolar lipids showed transient fluctuations during early reperfusion, which may be related to changes in cerebral blood flow. Pentobarbital pretreatment should allow for discrimination of biochemical alterations that are involved in ischemia-induced brain damage, since it reduces the edema and mortality associated with bilateral carotid artery ligations in the gerbil. This anesthetic was previously used to show that certain prostanoids cannot be implicated in tissue damage (Doman, 1988). In the present study, it was observed that the ischemia-induced release ol [3H]arachidonate from CGP was significantly affected by pentobarbital. A role for CGP metabolism in CNS tissue dysfunctions has been suggested, because the synthetic enzyme choline phosphotransferase (EC 2.8.7.2) is inhibited in ischemic brain (Goldberg et al., 1983) and the stimulation of CGP synthesis with CDPcholine reduces tissue damage. CDPcholine inhibits edema development in incubated cerebral slices (Dittmann, 1971) and cerebral cortex following cold trauma (Rigoulet et al., 1970; Arrigoni et al., 1987). It also protects cultured neurons from hypocapnia-induced damage (Mykita et al., 1986) and both CDPcholine and barbiturates attenuate hypoxic damage in synaptosornes (Benzi et al., 1981). The protective effects of CDPcholine appear to be related to its involvement in lipid metabolism, because it reduces the free fatty acid accumulation associated with cerebral trauma in vivo (Dorman et al., 1983; Horrocks and Dorman, 1985) and in v i m (Damron and Dorman, 1988). Thus, manipulation of CGP metabolism may providc a means for altering the course of ischemic brain damage. However, further investigations are needed to characterize the complex metabolic relationships between arachidonic acid and all the phospholipid classes,
493
ACKNOWLEDGMENTS The technical and editorial assistance of N.L. Edgehouse and D. S . Damron are gratefully acknowledged. This work was supported by NIH grant NS20454.
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F&h J , Lees M. Sloane Stanley GH (1957): A simplc method for the isolation of total lipides from animal tissues. J Biol Chem 226: 497-509. Freeman CP, West D (1966): Complete separation of lipid classes on a single thin-layer plate. J Lipid Res 7:324-327. Goldbcrg WJ. Donnan RV, Horrocks LA (1983): Effects of ischemia and diglycerides on ethanolamine and choline phosphotrdnsferase activities from rat brain. Neurochem Pathol 1 :225-234. Horrocks LA, Doman RV (1985): I’revention by CDPcholine and CDPethanolamine of lipid changes during brain ischemia. In Zappia V, Kennedy EP, Nilsson €31, Galletti P (cds): “Novel Biochemical, Pharmacological and Clinical Aspects of Cytidinediphosphocholine.” New York: Elsevier, pp 205-215. Hossmann K-A, Takagi S , Sakaki S (1977): Barbituate loading following prolonged ischemia of the cat brain. Acta Neurol S c a d 56:376-377. Jarrott DM, Domer FR (1980): A gerbil model of cerebral ischemia suitable for drug evaluation. Stroke I1:203 -209. Kasckow JW, Abood LG, Hoss W, Herndon RM (1986): Mechanism of phospholipiase A2-induced conduction block in bullfrog sciatic nerve 11. Biochcmistry. Brain Res 373:392-398. Kuwashima J , Fujitani B . Nakamura K, Kadokawa T, Yoshida K . Shimizu M (1 976): Biochemical changes in unilateral brain injury in the rat: A possible role of free fatty acid accumulation. Brain Res 110:547-557. LazarewicL JW, Strosznajder J , Gromek A (1972): Effects of ischemia and exogenous fatty acids on the energy metabolism in brain mitochondria. Bull Acad Sci Polon 20:599-606. McGraw CP (1977): Experimental cerebral infarction. Effects of pentobarbital in Mongolian gerbils. Arch Neuml 34334-336, bfykita S , Golly F. Dreyfus H, Frcysz L, Massarelli R (1986): Effect of CDP-choline on hypcicapnic neurons in culture. J Neurochem 47:223-231. Ncmoto EM, Shiu CK. Bleyaert AL ( 1 981): Efficacy of therapies and attcnuation of brain free fatty acid liberation durirlg global ischemia. Crit Care Med 9:397--398. Porcellati G, De Medio GE, Fini C , Floridi A; Goracci G. Horocks IdA, Lazarewic,: JW. Palmerini CA, Strosznajder J. Trovarelli
G (1978): Phospholipid and its metabolism in ischemia. Proc Eur Soc Neurochern 1 :285-302. Rhoads DE. Osburn LD. Peterson NA, Raghupathy E (1983): Release of neurotransmitter amino acids from aynaptosornes: Enhancetncnt of calcium-independent efflux by oleic and arachidonic acids. J Neurochem 41531-537. Rigoulet M, Guerin B, Cohadon F, Vandendreissche M (1979): Unilateral brain injury in the rabbit: Reversible and irreversible damage to the membranal ATPases. J Ncurochem 32:535-541. Schwartz RD, Skolnick P, Paul SM (1988): Regulation of y-aminobutyric acidibarbiturate receptor-gated chloride ion flux in brain vesiclcs by phospholipase A,: Possible role of oxygen radicals. J Ncurochem S0:565-571. Shiu GK, Nemmer JP, Nemoto EM (1983): Reassessment of brain free fatty acid liberation during global ischemia and its attenuation by barbiturate anesthesia. J Neurochem 40:880--884. Siesjo R K , Wieloch T (1985): Molecular mechanisms of ischemic brain damage: Ca“ -related events. In Plum F (ed): “Cerebrovascular Disease.” New York: Raven Press, pp 187-20(). Snyder JW, Lamb RG ( 1987): The role of lipid pcroxidation in the chemical-dependent activation of hepatic phospholipasc C in vitro. Rcs Commun Uhem Pathol Pharmacol 5S:3-16. Steen P.4, Mildc JH. Michenfelder J D (1978): Cerebral metabolic and vascular effects of barbituate therapy following complete global ischemia. J Neurochem 31:1317-1324. Tang W, Sun GY (1982): Factors affecting the frec fatty acids in rat brain cortex. Neurochem Int 4:269-273. Yoshida S, lnoh S , Asano T, Sano K , Kubota M, Shimazaki H , Ueta N ( 1Y80): Effect of transient ischemia on free fatty acids and phospholipids in the gerbil brain. Lipid peroxidation as a possible cause of postischemic injury. J Neurosurg 53323-331. Yoshida S, lnoh S, Asano T. Sano K. Shimazaki H, Ueta N (1983): Brain free fatty acids, edema, and mortality in gerbils subjected to transient ischemia, and the effect of barbituate anesthesia. J Neurochem 40: 1278-1286. Yoshida S, Ikeda M, Busto R , Sanliso M, Martinez E, Ginsberg MD (1 986): Cerebral phosphoinositide, triacplglycerol, and energy metabolism in reversible ischemia: Origin and fate of free fatty acids. J Neurochem 471744-757.
Arachidonic Acid Metabolism in Gerbil Cerebra: Effects of Ischemia and Pentobarbital T.F.R. Hamm and R.V. Dorman Department of Biological Sciences, Kent State University, Kent, Ohio
Pentobarbital pretreatment may be used to predict 1978; Steen et al., 1978). These effects appear to be biochemical events involved in ischemic brain dam- related to the observed reduction in tissue necrosis folage following bilateral carotid artery ligations in the lowing acute cerebral ischemia and may depend on the gerbil, since it reduces the subsequent edema and inhibition of synaptic activities (Astrup, 1982). Pentomortality. The effects of this anesthetic on the isch- barbital also reduces the edema (Yoshida et al., 1983; emia-induced modifications of cerebral arachidonic Edgehouse and Dorman, 1987) and mortality (McGraw, acid metabolism were investigated, in order to corre- 1977; Jarrott and Domer, 1980; Yoshida et al., 1983; late observed alterations with tissue damage. Cere- Edgehouse and Dorman, 1987) subsequent to bilateral bral lipids were radiolabeled in vivo with [3H]arach- carotid artcry ligations in the gerbil. These effects may idonic acid prior to 10 rnin of cerebral ischemia and depend on its ability to protect membrane structure and 0-120 min of perfusion. Ischemia stimulated a 97.3% function, since barbiturate anesthesia inhibits ischemiaincrease in unesterified [3H]arachidonate, which was induced free fatty acid accumulation (Flamm et al., due to the loss of label from choline, inositol, and 1977; Tang and Sun, 1982; Shiu et al., 1983; Yoshida et ethanolamine glycerophospholipids. Tissue reperfu- al., 1983). The concentrations of free fatty acids have sion stimulated further reductions in [’H]choline and been shown to correlate with cerebral tissuc damage (Ra[3H]inositolglycerophospholipids, while ethanolamine zan, 1970; Kuwashima et a].. 1976; Yoshida et al., 1980; glycerophospholipid and triglyceride labeling in- Abe et al., 1987). In particular, the accumulation of creased. Inositol glycerophospholipid, but not choline unesterified arachidonic acid appears to be an accurate glycerophospholipid, labeling returned to control indicator of the degree of ischemia-induced brain damlevel by 60 min of reperfusion. Pentobarbital pre- age (Nemoto et al., 1981). Arachidonic acid and its metabolites are involved treatment reduced the accumulation of [‘Hlarachidonate by 56.2% during ischemia. It increased the in a variety of nervous tissue functions. However, uncsrecovery of [3H]ethanolamine glycerophospholipids terified arachidonate has been shown to mimic the damduring the ischemic period and [‘HH]choline glycero- aging effects of cerebral ischemia. It induces cytotoxic phospholipids during the first 5 min of reperfusion. and vasogenic edema (Chan and Fishman, 1978; Chan ct These effects accounted for the reduction of unester- al., 1983), disrupts electrical activities (Kasckow et al., ified [’Hlarachidonate obberved during ischemia and 1986), and affects the uptake and release of neurotransmitters (Chan et al., 1983; Rhoads et al., 1983; Dorman reperfusion. et al., 1986). It also inhibits cerebral Na+-K+-ATPase Key words: cerebral ischemia, lipid metabolism, an- (Ahmed and Thomas, 1971; Chan et al., 1983), disrupts esthesia, phospholipids mitochondria1 functions (Lazarewicz et al., 1972): and induces Ca2+ mobilization (Chan and Turk, 1987). These metabolic perturbations are also observed in ischemic nervous tissue. Thus, the enhanced phospholipid INTRODUCTION catabolism and free fatty acid accumulation observed in Barbiturate anesthesia attenuates the tissue damage associated with cerebral ischemia and, therefore, may be used to assess the metabolic changes related to the damage processes. Barbiturates inhibit ischemia-induced al- Received August 3 1. 1989; revised January 22, 1990: accepted Janterations of ccrebral energy charge, blood flow, lactate uary 23, 1990. production, water content, oxygen consumption, and Address reprint requests to Robert V. Dorman, Dept. Biological Scielectrical activities (Hossmann et al., 1977; Black ct al., ences, Kent State Ilniversity, Kent, OH 44242. 0 1990 Wiley-Liss, Inc.
489
Cerebral Ischemia and Lipid Metabolism
ischemic brain appear to play significant roles in the course of the related tissue damage. The effects of ischemia and reperfusion on the metabolism of the various lipid classes were assessed following ['Hlarachidonate labeling of cerebral lipids in vivo. Experiments were performed, in the presence and absence of pentobarbital anesthesia, in order to distinguish lipid metabolic alterations that may be involved in the mortality and edema development observed with this ischemia model. Cerebral ischemia stimulated the accumulation of free ['Hlarachidonate, which correlated with the loss of radiolabel from the choline, inositol, and ethanolamine glycerophospholipids. Pretreatment with pentobarbital significantly reduced the ischemia-dependent accumulation of [3H]arachidonate. This effect was related to changes in choline and ethanolamine glycerophospholipid metabolism.
MATERIALS AND METHODS Induction of Cerebral Ischemia The surgical procedures and care of the animals were approved by the University's Animal Care Committce, since they fall within established federal guidelines. Male gerbils (50-90 g) were anesthetized with pentobarbital (Nembutal; Abbott Laboratories; 36 mgi kg, i.p.) and their carotid arteries were cxposed and loosely looped with waxed threads (Edgehouse and Dorman, 1987). A small hole was drilled in each animal's left parietal belie at a distance of 2 mm from the sagital suture and 2 rnm behind the bregma. All wounds were closed with skin clips and the animals were allowed to recover for 24 hr. Each gerbil was then given a 10 pl intracerebral injection of 1-2 FCi of ['Hlarachidonic acid (83.6 Ciimmol; New England Nuclear Corp.) suspended in a degassed buffer containing 0.32 M sucrose, 50 mM Tris, 10 mgiml fatty-acid-free bovine serum albumin, and 55 mM glucose; pH 7.4. The animals were restrained with an elastic band on a surgical board and ischemia was induced by occluding both carotid arteries for 10 min with microvascular clamps following 2 hr of isotope incorporation. Blood flow was restored by removal of the clamps and the animals were sacrificed by decapitation at 0, 2, 5, 10, 20, 30, 60 and 120 min of reperfusion. Pentobarbital-treated gerbils were anesthetized 20 min prior to the induction of ischemia. Pentobarbital treatment improved animal survival from 7 1 to 97% throughout the cxpcrimental period, which was consistent with previous results (Edgehouse and Dorman, 1987). Analytical Procedures Cerebra were isolated from other brain regions by dissection on an ice-cold glass plate. Total lipids were
I
r
1
I
I
0
3C
63
93
1
1
'20
I
150
-------~7
150
J
210
243
270
INCOKPOKArION TIME (MIN)
Fig. 1. Time course of lipid labeling in gerhil cerebra following an intracerebral injection of [ 'Hlarachidonate. Cerebral lipids were radiolabeled, extracted, separated, and counted as described in the Materials and Methods following 30-250 niin of incorporation. The results were obtained from duplicate determination5 from five animals and are presented as dpm x 10~3/cerebrum.(0)= CGP, (V)= TGP, (V)= EGP, (m) = SGP, (A)= TG, ((1) = DG, (e)= FFA.
cxtracted according to Folch et al. (1957) prior to lipid class separation by thin-layer chromatography. Phospholipids wcrc scparated by the method of Emilsson and Scindler (1985), while nonpolar lipids were separated according to Freeman and West (1966). The lipid classes were visualized with iodine vapor, scraped into vials containing Ready Solv EP (Beckman Instruments), plus 10% watcr, and counted for radioactivity. These procedures have previously been used to assess cerebral lipid metabolism in the rat (Damron and Dorman, 19x8). Total lipid radioactivity was determined as the amount of isotope recovered from the thin-layer plates. The results were obtained from duplicate determinations from at least four animals and are expressed as % total lipid radioactivity 5 SEM, except for the results from the incorporation time course. which are given as dpm X I O-'!'cerebium, Statistical significance was determined using ANOVA and Duncan's multiple range test with P < 0.05 considcred to bc significant.
RESULTS Cerebral lipids were radiolabeled in vivo following intracerebral injection of [ 'H]arachidonatc. The time courses of incorporation (0-250 min) into the various lipid classes were similar to those reported for rat brain (Damron and Dorman, 1988) and are shown in Figure 1. The maximum incorporations into cholinc (CCP), inosito1 (IGP), and serine (SCP) glycerophospholipids were
Hamm and Dorman
near contol level by 20 min, and was stable through the remainder of the time course. In contrast, TG labeling was unchangcd during 10 min of ischemia, but increased 33.7% during the first 10 rnin of rcperfusion (Table I). The level of ['HITG remained elevated through 60 min of reperfusion before it declined to near control level by 120 min. DG labeling increased 4.4% during the ischemic episode and another 12.6% through 2 min of reperfusion, followed by a return to control level (Table I). Ischemia and reperfusion also affected the ['HIarachidonate labeling of the phospholipids. CGP labeling decreased 2.4% during 10 min of ischemia and this loss continued during the first 5 min of reperfusion (94.7% of control; Fig. 3). The level of ['HH]CCP showed a brief recovery to control value at 10 min of reperfusion, followed by a gradual decline in labeling, which was sigFig. 2. Effects of cerebral ischemia and reperfusion on the nificant by 60 min of reperfusion. IGP labeling (Fig. 4) accumulation of ['HH]free fatty acids in untreated ( o j and pen- was reduced 3.5% during ischemia and 18.0% by 5 min tobarbital-treated ).( gerbils. The in vivo labeling of cerebral of reperfusion. However, IGP labeling returned to conlipids with [3H]arachidonate, the induction of ischemia and trol value by 60 min of reperfusion. EGP labeling (Fig. reperfusion, and the analyses of radiolabeled lipids are de- 5) decreased 3.4% during ischemia, but the level of isoscribed in Materials and Methods. Gerbils were exposed to 10 tope in this pool was increased 18.4% by 5 min of remin of ischemia (shown as black bar) and 0-120 min reperfuperfusion. EGP labeling fell 10.7% between 5 and 10 sion. Anesthetized animals received pentobarbital 20 min bemin of reperfusion and then showed a gradual increase to fore the onset of the ischemic trauma. The results were derived from duplicate determinations from at least four animals and 119% of control at 120 min of reperfusion. Although are expressed as % total lipid radioactivity t SEM. * = sig- SGP contained much less radioactivity, the pattern of niuicantly different from 0 time, group-matched control; Dun- labeling followed that of EGP (data not shown). Pentobarbital pretreatment altered the metabolic can's multiple range test; P < 0.05. flux of isotope through the various lipid classes. The anesthetic reduced the ischemia-induced accumulation of observed at 60 rnin postinjection, while the isotopic la- [3H]arachidonate by 56.2% (Fig. 2). Again, the level of beling of ethanolamine glycerophospholipids (EGP) con- ['HIFFA remained elevated through 10 min, declined to tinued a slow increase during the 250 min of incorpora- control level by 20 min, and was significantly less than tion. Triglyceride (TG) labeling reached its maximum at the control value by 120 rnin of reperfusion. Pentobar60 min postinjection, while [?H]diglyceride (DG) peaked bital anesthesia allowed for a 24.2% reduction in labelat 30 min. The ['Hlarachidonate content of the free fatty ing of the TG during ischemia (Table I). The labeling of acid (FFA) pool declined throughout the time course. this pool returned to control level by 10 rnin of reperfureaching a baseline level at 120 min postinjection. The sion. DG labeling increased 22.2% during 10 min of labeling of the lipid pools remained stable through 250 ischemia in the presence of pentobarbital, but was remin of incorporation, with the exception of EGP. An in duced to control level at 2 min of reperfusion. This revivo incorporation time of 120 min was used for the sponse was followed by a brief increase in labeling at 5 following experiments, since recovered [3H]arachidonate rnin prior to a gradual reduction in 13H]DG that was had reached a minimum lcvcl and experiments could be significantly less than control by 120 rnin of reperfusion conducted through 120 min of reperfusion without de- (Table I). tectable changes in the labeling of the control groups. The Pentobarbital reduced the loss of ['HICGP by % distribution of isotope at 120 min postinjection was 50.0% during ischemia, when compared to the time48.6 k 0.5, 20.8 k 0.8, 17.2 -+ 0.7, 2.9 2 0.1, 2.6 2 matched value obtained from untreated gerbils (Fig. 3). 0.1, 1.7 k 0.1, and 1.9 k 0.2 for CGP, IGP, EGP, SGP, It also caused a significant increase in CGP labeling to 107.7% of control at 2 min of reperfusion. ['HICGP TG, DG, and FFA, respectively. The effects of ischemia and reperfusion on the rcturned to control level by 10 min of reperfusion, but [3H]arachidonate labeling of FFA are shown in Figure 2. was reduced (96% of control) by 120 min. In the presTen minutes of bilateral carotid artery ligations induccd a ence of pentobarbital, IGP labeling was reduced during 97.3% increase in [jH]FFA. The labeling of this pool ischemia and was 78.6% of control by 5 min of reperwas elevated through 5 min of reperfusion, rcturned to fusion, which was similar to the decline observed in
Cerebral Ischemia and Lipid Metabolism TABLE I. Effects of Cerebral Ischemia and Reperfusion on Diglyceride and Triglyceride Labeling in the Presence and Absence of Pentobarbital Pretreatment? O/u total
+
~
0 n_
[ 'H]triglycerides ~
+
*
1.850.2
2.6-'0.1
3.2e0.2
1 . 8 ~ 0 . 12.320.1" 2.120.1 1.920.2 2.020.I 2.2+0.I 1.8+0.1 2 . 0 2 0 . 1 1.7k0.1 1.9+0.1 I.6+0.2 1.8t0.1 1.620.1 1.6k0.1 1.5+0.l 1.320.1"
2.520.2
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1.7+0.1
Control R (inin) 0 2 5 10 20 30 60 120
24(n
lipid radioactivity
[3H]diglycerides Pentobarbital
2.620.1 3.120.1" 3.520.2" 3.020.2 3.0-tO.1 3.220.2* 2.320.2
TGcrbil cercbra wcre radiolabclcd in vivo with 13H]areehidonatcprior lo I0 min of ischemia and 0-120 min of reperfusion (R). Total lipids were extracted. separated, and counted as described. The results were derived from duplicate determinations from at least four cerebra and are expressed as % total lipid redioactivity 2 SEM for untreated (-) and pentobarbital-treated ( + ) gerbils. "Significantly different froin control; Duncan's multiple range test: P < 0.05.
52
491
24-
-4 7 '-
C.'
0
'0
4
20 3c
61
120
REPERFUSION TIME (MIN)
Fig. 4. Effects of ischemia and reperfusion on ['HIarachidonatc labeling of inositol glycerophospholipids. The conditions and comparisons are described in the legend for Figure 2. 0 = untreated group = pentobarbital-treated group. * = significantly different from 0 time controls.
1
21
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.d *
13j
*
25 .1
T
5 CTL
3
0
10
20 30 60 REPERFUSION TIME (MIN)
CTL
120
Fig. 3. Effects of ischemia and reperfusion on the ['HIarachidonate labeling of the choline glycerophospholipids. The conditions and comparisons are described in the legend for Figure 2. 0 = untreated group; = pentobarbital-treated group. * = significantly different from 0 time controls.
unanesthetized animals (Fig. 4). The labeling of this phospholipid showed a gradual increase during the remainder of the time course. Pentobarbital pretreatment not only prevented the ischemia-induced decline in EGP labeling, it caused a 19.5% increase in labeling during the 10 niin of ischemia (Fig. 5 ) . The amount of recovered ['HIEGP then declined to control level at 2 min of reperfusion, followed by a 16.1% increase in labeling
0
10
20
30
60
120
REPERFUSION TIME (MIN)
Fig. 5 . Effects of ischemia and reperfusion on ['HIarachidonate labeling of ethanolamine glycerophospholipids. The conditions and comparisons are described in the legend for Figure 2. 0 = untreated group; = pcnlobarbital-treated group. * = significantly diffcrent from 0 time controls.
between 2 and 5 min of reperfusion. EGP labeling was 141.9%of control by 120 min. Again, SGP labeling was low, but appeared to follow a pattern similar to EGP (data not shown).
DISCUSSION Unesterified arachidonic acid has been implicated in ischemic brain damage and pentobarbital has been shown to inhibit the accumulation of this fatty acid and
492
Hamm and Dorman
attenuate many of the consequences of ischemia. 'The effects of cerebral ischemia and subsequent reperfusion on the metabolic flux of arachidonic acid were investigated in awake and pentobarbital-anesthetizedgerbils, in order to correlate the protective cffects of pentobarbital with membrane lipid metabolism. Ten minutes of bilateral carotid artery ligations in the gerbil stimulated the accumulation of ['Hlfree fatty acids in ['H]arachidonic acid-labeled cerebra. Isotope was released from all the major phospholipid classes with the choline glycerophospholipids providing approximately 50% of the radioactivity in the FFA pool. CGP labeling continued to decline through 120 min of reperfusion, while IGP labeling returned to control level and EGP labeling increased during reperfusion. Pentobarbital pretreatment attenuated the ischemia-induced accumulation of ['Hlfree fatty acids. This effect was correlated with the inhibition of isotope loss from the choline glycerophospholipids. The intracerebral injection of [3H]arachidonate resulted in the tirne-dependent incorporation of isotope into gerbil brain complex lipids. Choline glyccrophospholipids showed the greatest level of incorporation at 2 hr postinjection (48.6% of total lipid radioactivity), followed by inositol (20.8%) and ethanolamine glycerophospholipids (17.2%). This pattern of labeling was similar to that observed following intracerebral injections of ['Hlarachidonate in rat (Daniron and Dorman, 1988). Two hour exposures to isotope were routinely used, since the labeling of the free fatty acid pool reached a minimum level by this time, suggesting clearance of unincorporated ['Hlarachidonate. In addition, the labeling of the lipid pools remained constant through 250 min of incorporation, except for EGP, which showed a slow increase in radioactivity. Thus, the changes in radioactivity that were observed through 120 niin of reperfusion reflect metabolic alterations related to the ischemic episode . Ten minutes of bilateral carotid artery ligations caused a redistribution of ['Hlarachidonatc in the various lipid pools. The labeling of the FFA increased 97.3% during this period. Approximately 50% of ['HIFFA was derived from ['HICCP, while IGP and EGP catabolism each provided about 25% of the isotope to this pool. There was also a small increase in ['HIDG recovered following 10 min of ischemia, which appeared to be derived from phospholipid catabolism, since the labeling of the TG pool was not altered during ischemia. These results are consistent with the reported net degradation of all the major phospholipids upon interruption of cerebral blood flow (Porcellati et al., 1978). Pentobarbital pretreatment altered the course of ischeniia-induced phospholipid degradation. This effect was observed as a 56.2% reduction in the accumulation
of unesterified arachidonate during 10 min of ischernia, which was consistent with previous reports (Yoshida et a]., 1983; Shiu et al., 1983). Again, the labeling of the FFA pool with 13HJarachidonate was reflected by the changes observed in phospholipid labeling. The reduced accumulation of I3H]FFA was due to decreased release of isotope from CGP and increased labeling of EGP during 10 min of ischemia. In contrast, IGP labeling was further reduced during the ischemic episode when pentobarbital was present. This may reflect a slowing of acyl group exchange in the presence of anesthetic and may be related to the inhibition of synaptic activities. The return of blood flow to the cerebra caused complex alterations in the metabolism of the glycerolipids. FFA labeling was reduced 50.7% by 10 min of reperfusion, when compared to the labeling observed at the end of the ischemic period. However, the amounts of recovered [3H]CGP and [3H]IGP continued to decline during the same period. The rapid removal of unesterified arachidonate may be due to enhanced clearance, reacylation, or oxidation of the fatly acid. Eicosanoid production is stimulated under these experimental conditions in this model (Dorman, 1988) and reacylation also appears to be involved, since EGP and TG labeling increased during reperfusion. Thc continued loss of [3H]arachidonate from CGP and IGP may reflect the activation of specific phospholipases, since it has been suggested that lipid peroxides can activatc phospholipase C (Snyder and Lamb, 1987) and oxygen radicals stimulate phospholipase A2 (Schwartz et a]., 1988). Also, ischemia stimulatcs the accumulation of intracellular calcium (Siesjo and Wieloch, 1985), which may activate calcium-dependent phospholipases. Pentobarbital pretreatment altered the course of arachidonate flux during the early reperfusion times. The barbiturate reversed some of the effects of reperfusion on EGP and CGP labeling. It was observed that the amount of recovered ['HICGP increased significantly during the first 2 inin of reperfusion, while EGP labeling declined during the same time period. It appeared that the labelings of CGP and EGP were inversely related during tissue reperfusion in both awake and pentobarbital-treated animals. This effect may be due to acyl group exchange between the two lipid classes. Alternatively, it may be due to base exchange processes or alterations in diglyceride utilization by the respective synthetic enzymes. Pentobarbital pretreatment had no significant effect on the recovery of label in the inositol glycerophospholipids. [3H]IGP returned to control levels during the 2 hr time course in both awake and anesthetized animals. The inability of pentobarbital to significantly alter IGP metabolism may indicate that the catabolism of this phos-
Cerebral Ischemia and Lipid Metabolism
pholipid class is not correlated with the mortality or ccrebral edema observed in this stroke model. However, the labeling procedures employed here did not allow for assessment of the effects of ischemia or pentobarbital on polyphosphoinositide metabolism and it has been suggested that ischemia affects the polyphosphoinositides, but not IGP (Yoshida et al., 1986). The anesthetic also altered the recovery of ["HITG . At all times examined, TG labeling was reduced when compared to values obtained from untreated animals. This effect appeared to be due to the decreased availability of unesterified ['Hlarachidonate in pentobarbitaltreated cerebra. The alterations of DG labeling were similar in awake and anesthetized gerbils, except for a pentobarbital-dependent, transient decline in ['HIDG at 2 niin of reperfusion. This effect corresponded to the marked increase in ['H]CCiP and may be related to enhanced utilization of DG for CGP synthesis. In fact, the labeling of both the phospholipids and nonpolar lipids showed transient fluctuations during early reperfusion, which may be related to changes in cerebral blood flow. Pentobarbital pretreatment should allow for discrimination of biochemical alterations that are involved in ischemia-induced brain damage, since it reduces the edema and mortality associated with bilateral carotid artery ligations in the gerbil. This anesthetic was previously used to show that certain prostanoids cannot be implicated in tissue damage (Doman, 1988). In the present study, it was observed that the ischemia-induced release ol [3H]arachidonate from CGP was significantly affected by pentobarbital. A role for CGP metabolism in CNS tissue dysfunctions has been suggested, because the synthetic enzyme choline phosphotransferase (EC 2.8.7.2) is inhibited in ischemic brain (Goldberg et al., 1983) and the stimulation of CGP synthesis with CDPcholine reduces tissue damage. CDPcholine inhibits edema development in incubated cerebral slices (Dittmann, 1971) and cerebral cortex following cold trauma (Rigoulet et al., 1970; Arrigoni et al., 1987). It also protects cultured neurons from hypocapnia-induced damage (Mykita et al., 1986) and both CDPcholine and barbiturates attenuate hypoxic damage in synaptosornes (Benzi et al., 1981). The protective effects of CDPcholine appear to be related to its involvement in lipid metabolism, because it reduces the free fatty acid accumulation associated with cerebral trauma in vivo (Dorman et al., 1983; Horrocks and Dorman, 1985) and in v i m (Damron and Dorman, 1988). Thus, manipulation of CGP metabolism may providc a means for altering the course of ischemic brain damage. However, further investigations are needed to characterize the complex metabolic relationships between arachidonic acid and all the phospholipid classes,
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ACKNOWLEDGMENTS The technical and editorial assistance of N.L. Edgehouse and D. S . Damron are gratefully acknowledged. This work was supported by NIH grant NS20454.
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