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reoxygenation and glutamate-induced injury
Neuroscience Letters, 160 (1993)106,108 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
106
NSL 09819
Prostacyclin (PGI2) protects rat cortical neurons in culture against hypoxia/reoxygenation and glutamate-induced injury Chantal Cazevieille, Agnrs Muller and Claude Bonne Laboratoire de Physiologic Cellulaire, Facult~ de Pharmacie, Universitk Montpellier I, Montpellier (France) (Received 5 March 1993; Revised version 18 June 1993; Accepted 18 June 1993)
Key words. Prostacyclin; PGI2; Ischemia; Glutamate; Excitotoxicity; Neuron culture; Hypoxia/reoxygenation Arachidonic acid and its metabolites are released in brain extracellular fluids as a result of ischemia and may participate in either damaging or protecting neural tissues. This study investigates the neuroprotective effect of prostacyclin (PGI2) on hypoxia (5 h)lreoxygenation (3 h) and on the excitotoxic neurotransmitter, glutamate (10/.tM), in rat cortical neuron cultures. At/~M concentrations, PGI2 inhibits lactate dehydrogenase release, a cell-injury marker. These results, showing a direct cytoprotective effect of PGI2 on brain cells, reinforce its beneficial properties on vessels and circulating cells in cerebral ischemia.
Arachidonic acid has been shown to be released by cerebral tissues as a result of ischemia [6, 8]. It has been reported that this fatty acid itself modulates synaptic transmission as a retrograde messenger [14]. In particular, arachidonic acid could participate in ischemia-induced neuronal damage through glutamate-uptake inhibition [3] which leads to toxic concentrations of this neurotransmitter. Otherwise, arachidonic acid metabolites, especially cyclooxygenase products, have been detected in brain extracellular fluid during postischemic reperfusion period in vivo [7]. From a functional point of view, some prostanoids, such as thromboxane A2, could have deleterious effects on neuroglial tissues related to their vasoconstrictive activity and aggregating action on platelets. By contrast, other prostaglandins, such as prostacyclin (PGI2), could afford protection by opposite activities [10]. Moreover, it has also been shown that prostaglandins could have direct cytoprotective effects in isolated tissues against different stresses, including hypoxia [4, 12]. The aim of the present study was to investigate the neuroprotective effect of PGI 2 on pure neuron cultures exposed to hypoxia/reoxygenation. Furthermore, cytoprotective activity has also been studied in glutamateinjured neurons, since this excitatory neurotransmitter Correspondence." C. Bonne, Laboratoire de Physiologic Cellulaire, Universit6 Montpellier I, Facult6 de Pharmacie, 15 avenue Charles Flahault, 34060 Montpellier CI~DEX, France.
plays a major role in hypoxia/reoxygenation-induced neurotoxicity [2, 13]. Pregnant rats were sacrificed with diethyl-ether vapor, the 16-18-day-old fetal rats were killed by rapid decapitation and their forebrain was removed. Dissociated cortical cells (1.5-2 x 106) were plated in 35-mm dishes precoated with 0.1 mg/ml poly-D-lysine and then with a serum-containing medium (Dulbecco's modified Eagle's medium; DMEM) supplemented with 4 mM L-gtutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 5% fetal calf serum and 5% horse serum. Primary cultures were grown in a 5% CO2/humidified atmosphere at 37°C, in DMEM supplemented with L-glutamine (4 mM), glucose (6 g/l), penicillin (100 U/ml), streptomycin (100 pg/ml) and 10% hormonal medium containing transferrin (1 mg/ml), insulin (250 /lg/ml), putrescine (6.10 -4 M ) , Na selenite (3.10 -7 M ) , progesterone (2.10 -7 M) and fl-estradiol (10-11 M). Neuronal cell purity (95 %) was assessed by immunocytochemical characterization of glial fibriilary acid protein-containing glial cells. 10 days after plating, cortical neurons were incubated in DMEM 1 g/1 glucose with L-glutamine and penicillin/ streptomycin without hormonal medium (simplified medium) in an anoxic atmosphere (95% NJ5% CO2) at 37°C for 5 h. Reoxygenation was further obtained by replacing cells in normoxic conditions (95% air/5% CO2) for 3 h. Control cultures were kept in normoxic conditions. Prostaglandins (Cascade; Biochem, UK), were added to cell cultures before hypoxia. The stock solution of PGI2 was prepared in a Tris-HCl buffer (pH = 9) and
107 20"
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0.1~M
I~M
PGI2 -
10~M
-
0.1~M
I~M
10~M
6-keto-PGF1 (x - -
Fig. 1. Effect of prostacyclin (PGIz) and 6-keto-PGFlc~ on hypoxia/ reoxygenation-induced LDH release from cultured neurons. Results are expressed as increase in LDH release from controls kept in normoxic conditions which are 25.9 _+1.9 mU/ml per mg protein. Results are mean _+S.E.M. from at least fivedistinct experimentsmade in triplicate. *P < 0.05; **P < 0.01 one-way ANOVA followed by Dunnett's test.
The present study demonstrates for the first time that PGI2 at ~tM concentrations directly protects isolated neurons from hypoxia/reoxygenation and glutamate-induced damage. Renkawek et al. [12] reported that PGIz exerted cytoprotective effect after anoxia on nervous tissues, predominantly glial cells. The PGI2 mechanism of action in this effect is unknown and, at present, can only be subject of hypotheses. The fact that a cell-permeable c A M P analogue did not afford any protection (not shown) and the fact that 6 - k e t o - P G F l ~ presented some protective activity do not support a receptor-mediated process. However, the accuracy of the structure-activity relationship requires further experiments with other prostanoids tested in a larger range of concentrations. It is rather surprising that PGI2, which has a half-life o f - 3 min in the medium, can be active for several hours in our experiments. These data imply that P G I 2 triggers a long-lasting event which persists after the prostaglandin degradation. In D y m o n d and Kalmus [4], P G E 2 has been shown to be neuroprotective against actinomycin C toxicity through a cAMP-independent mechanism. These authors have proposed that P G E 2 may alter the plasma membrane by stabilizing it, an effect previously suggested by others from experiments carried out with P G I 2 15
not used for up to 10 days. F o r glutamate-treatment experiments, cortical cultures were incubated under normoxic conditions with 10 ~tM L-glutamate in simplified medium for 3 h at 37°C. Prostaglandins were added to cultures just before glutamate exposure. Cellular injury was assessed by lactate dehydrogenase ( L D H ) release into the cell-culture supernatant after hypoxia/reoxygenation or glutamate exposure as previously described [2]. When cortical neurons were exposed for 5 h to an anoxic atmosphere, followed by 3 h reoxygenation, they released the cytoplasmic enzyme, L D H , a cytotoxicity marker. Treatment with PGI2 ( 1 - 1 0 / I M ) inhibited this release but 6-keto-PGFlct, the stable degradation product of PGI2, only at the highest concentration significantly protected the neuron from the oxidative stress (Fig. 1). In addition, when cultured cortical neurons were incubated for 3 h with glutamate (10/.tM), the same sign of cell injury was observed, i.e., increase in L D H release into the culture medium. Addition of PGI2 (1-10/.tM) to the cultures significantly decreased this enzyme leakage. The effect of the same concentrations of 6 - k e t o - P G F l ~ was not significant (Fig. 2).
_z ¢L
lO g X
5
_c
10~M Glu
I~M
10~M + PGI2
I~M
10~M
÷6-keto-PGF1
(x--
Fig. 2. Effect of prostacyclin (PGI2) and 6-keto-PGFlct on glutamateinduced neuron LDH release from cultured neurons. Results are expressed as increase in LDH release from controls which are 23.4 _+2.5 mU/ml per mg protein. Results are mean _+S.E.M. from at least five distinct experiments made in triplicate. **P < 0.01 one-way ANOVA followed by Dunnett's test.
108 on isolated liver l y s o s o m e s [9]. Besides, in an earlier s t u d y [I], showing t h a t PGI2 can p r o t e c t h e p a t o c y t e s a g a i n s t CC14 toxicity, it has been p r o p o s e d that this p r o s t a g l a n d i n c o u l d act by inhibiting l i p o p e r o x i d a t i o n , a possible m e c h a n i s m which is u n c l e a r a n d even c o n t r o versial [12]. O u r results, d e m o n s t r a t i n g a n e u r o p r o t e c t i v e effect a g a i n s t g l u t a m a t e toxicity, c o u l d also be linked to an a n t i o x i d a t i v e m e c h a n i s m since g l u t a m a t e toxicity is related, at least in p a r t , to O2-derived free radical generation [2]. P r o s t a g l a n d i n s , a n d P G I 2 in particular, c o u l d be n a t u rally occurring c y t o p r o t e c t i v e agents a g a i n s t a variety o f stresses. The p a t h o p h y s i o l o g i c a l significance o f this rem a i n s to be elucidated. In particular, it is n o t k n o w n if PGI2, which is p r o d u c e d in b r a i n u n d e r ischemia [7], can effectively reach a sufficient c o n c e n t r a t i o n , at n e u r o n a l level, to exert such a protective effect. F u r t h e r m o r e , p h a r m a c o l o g i c a l i m p l i c a t i o n s o f this activity is questionable. Indeed, PGI2 clinical trials in b r a i n ischemic stroke, b a s e d on v a s o d i l a t o r a n d a n t i t h r o m b o t i c actions o f this p r o s t a g l a n d i n , have given rise to d i s a p p o i n t i n g results
4
5
6
7
8
9
10
[51. These conclusions reinforce the interest in further investigating the direct n e u r o p r o t e c t i v e m e c h a n i s m o f action o f PGI2 a n d o t h e r eicosanoids.
11
12 l Bursch, W., Taper, H.S., Somer, M.R, Meyer, S., Putz, B. and Schulte-Hermann, R., Histochemical and biochemical studies on the effect of the prostacyclin derivative Iloprost on CC14-induced lipid peroxidation in rat liver and its significance for hepatoprotection, Hepatology, 9 (1989) 830 838. 2 Cazevieille, C., Muller, A., Meynier F. and Bonne, C., Superoxide and nitric oxide cooperation in hypoxia/reoxygenation-induced neuron injury, Free Radic. Biol. Med., 14 (1993) 389-395. 3 Chan, RH., Kerlan, R. and Fishman, R.A., Reductions of y-aminobutyric acid and glutamate uptake and (Na++K+)-ATPase activ-
13
14
ity in brain slices and synaptosomes by arachidonic acid, J. Neurochem., 40 (1983) 309 316. Dymond, J.B. and Kalmus, G.W., The cytoprotective properties of" prostaglandin E2 against the toxic effects of actinomycin C on embryonic neural retina cells, Prostaglandins, 44 (1992) 129 134. Hsu, C.Y., Faught, Jr., R.E., Furlan, A.J., Coull, B.M.. Huang, D.C., Hogan, E.L., Linet, O.I. and Yatsu, F.M., Intravenous prostacyclin in acute nonhemorrhagic stroke: a placebo-controlled double-blind trial, Stroke, 18 (1987) 352-358. Hsu, C.Y., Liu, T.H., Xu, J., Hogan, E.L., Chao, J., Sun, G., Tai, H.H., Beckman, J.S. and Freeman, B.A., Arachidonic acid and its metabolites in cerebral ischemia. In A,I. Barkai and N.G. Bazan (Eds.), Arachidonic Acid Metabolism in The Nervous System, Ann. N.Y. Acad. Sci., 559 (1989) 282 295. Huttemeier, RC., Kamiyama, Y., Su, M., Watkins, W.D. and Benveniste, H., Microdialysis measurements of PGD2, TXB2, and 6keto-PGFla in rat CA1 hippocampus during transient cerebral ischemia, Prostaglandins, 45 (1993) 177 187. Kunievsky, B., Bazan, N.G. and Yavin, E., Generation of arachidonic acid and diacylglycerol second messengers from polyphosphoinositides in ischemic fetal brain, J. Neurochem., 59 (1992) 1812-1819. Lefer, A.M. and Smith, IlI, E.F., Protective action of prostacyclin in myocardial ischemia and trauma. In J.R. Vane and S. Bergstr6m (Eds.), Prostacyclin, Raven, New York, 1979, pp. 339-348. Miller, V.T., Coull, B.M., Yatsu, F.R, Shah, A.B. and Beamer, N.B., Prostacyclin infusion in acute cerebral infarction, Neurology, 34 (1984) 1431-1435. Paller, M.S. and Manivel, J.C., Prostaglandins protect kidneys against isehemic and toxic injury by a cellular effect, Kidney Int., 42 (1992) 1345 1354. Renkawek, K., Herbaeyznska-Cedro, K. and Mossakowski, M.J., The effect of prostacyclin on the morphological and enzymatic properties of CNS cultures exposed to anoxia, Acta Neurol. Scan&, 73(1986) 111 118. Simon, R.R, Swan, J.H., Griffith, T. and Meldrum, B.S., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. Williams, J.H., Errington, M.L., Lynch, M.A. and Bliss, T.V.R, Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus, Nature (London), 341 (1989) 739-742.
106
NSL 09819
Prostacyclin (PGI2) protects rat cortical neurons in culture against hypoxia/reoxygenation and glutamate-induced injury Chantal Cazevieille, Agnrs Muller and Claude Bonne Laboratoire de Physiologic Cellulaire, Facult~ de Pharmacie, Universitk Montpellier I, Montpellier (France) (Received 5 March 1993; Revised version 18 June 1993; Accepted 18 June 1993)
Key words. Prostacyclin; PGI2; Ischemia; Glutamate; Excitotoxicity; Neuron culture; Hypoxia/reoxygenation Arachidonic acid and its metabolites are released in brain extracellular fluids as a result of ischemia and may participate in either damaging or protecting neural tissues. This study investigates the neuroprotective effect of prostacyclin (PGI2) on hypoxia (5 h)lreoxygenation (3 h) and on the excitotoxic neurotransmitter, glutamate (10/.tM), in rat cortical neuron cultures. At/~M concentrations, PGI2 inhibits lactate dehydrogenase release, a cell-injury marker. These results, showing a direct cytoprotective effect of PGI2 on brain cells, reinforce its beneficial properties on vessels and circulating cells in cerebral ischemia.
Arachidonic acid has been shown to be released by cerebral tissues as a result of ischemia [6, 8]. It has been reported that this fatty acid itself modulates synaptic transmission as a retrograde messenger [14]. In particular, arachidonic acid could participate in ischemia-induced neuronal damage through glutamate-uptake inhibition [3] which leads to toxic concentrations of this neurotransmitter. Otherwise, arachidonic acid metabolites, especially cyclooxygenase products, have been detected in brain extracellular fluid during postischemic reperfusion period in vivo [7]. From a functional point of view, some prostanoids, such as thromboxane A2, could have deleterious effects on neuroglial tissues related to their vasoconstrictive activity and aggregating action on platelets. By contrast, other prostaglandins, such as prostacyclin (PGI2), could afford protection by opposite activities [10]. Moreover, it has also been shown that prostaglandins could have direct cytoprotective effects in isolated tissues against different stresses, including hypoxia [4, 12]. The aim of the present study was to investigate the neuroprotective effect of PGI 2 on pure neuron cultures exposed to hypoxia/reoxygenation. Furthermore, cytoprotective activity has also been studied in glutamateinjured neurons, since this excitatory neurotransmitter Correspondence." C. Bonne, Laboratoire de Physiologic Cellulaire, Universit6 Montpellier I, Facult6 de Pharmacie, 15 avenue Charles Flahault, 34060 Montpellier CI~DEX, France.
plays a major role in hypoxia/reoxygenation-induced neurotoxicity [2, 13]. Pregnant rats were sacrificed with diethyl-ether vapor, the 16-18-day-old fetal rats were killed by rapid decapitation and their forebrain was removed. Dissociated cortical cells (1.5-2 x 106) were plated in 35-mm dishes precoated with 0.1 mg/ml poly-D-lysine and then with a serum-containing medium (Dulbecco's modified Eagle's medium; DMEM) supplemented with 4 mM L-gtutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 5% fetal calf serum and 5% horse serum. Primary cultures were grown in a 5% CO2/humidified atmosphere at 37°C, in DMEM supplemented with L-glutamine (4 mM), glucose (6 g/l), penicillin (100 U/ml), streptomycin (100 pg/ml) and 10% hormonal medium containing transferrin (1 mg/ml), insulin (250 /lg/ml), putrescine (6.10 -4 M ) , Na selenite (3.10 -7 M ) , progesterone (2.10 -7 M) and fl-estradiol (10-11 M). Neuronal cell purity (95 %) was assessed by immunocytochemical characterization of glial fibriilary acid protein-containing glial cells. 10 days after plating, cortical neurons were incubated in DMEM 1 g/1 glucose with L-glutamine and penicillin/ streptomycin without hormonal medium (simplified medium) in an anoxic atmosphere (95% NJ5% CO2) at 37°C for 5 h. Reoxygenation was further obtained by replacing cells in normoxic conditions (95% air/5% CO2) for 3 h. Control cultures were kept in normoxic conditions. Prostaglandins (Cascade; Biochem, UK), were added to cell cultures before hypoxia. The stock solution of PGI2 was prepared in a Tris-HCl buffer (pH = 9) and
107 20"
A
o
15"
m
.t,',,,,',',"
:,;+;,',;,
g e~ .J
10"
iiiiiti!iii! . .....
:';';';'-';!
"13
":';':':'9 :'."""4 :"''";t
m ~p
c 5'
1
TT
:,;.;,~,~+
:!:!;t:!:!;t
;,~,~,~,;,: ,R4,R
:-'-;,,;,-;,,;~ 0
0.1~M
I~M
PGI2 -
10~M
-
0.1~M
I~M
10~M
6-keto-PGF1 (x - -
Fig. 1. Effect of prostacyclin (PGIz) and 6-keto-PGFlc~ on hypoxia/ reoxygenation-induced LDH release from cultured neurons. Results are expressed as increase in LDH release from controls kept in normoxic conditions which are 25.9 _+1.9 mU/ml per mg protein. Results are mean _+S.E.M. from at least fivedistinct experimentsmade in triplicate. *P < 0.05; **P < 0.01 one-way ANOVA followed by Dunnett's test.
The present study demonstrates for the first time that PGI2 at ~tM concentrations directly protects isolated neurons from hypoxia/reoxygenation and glutamate-induced damage. Renkawek et al. [12] reported that PGIz exerted cytoprotective effect after anoxia on nervous tissues, predominantly glial cells. The PGI2 mechanism of action in this effect is unknown and, at present, can only be subject of hypotheses. The fact that a cell-permeable c A M P analogue did not afford any protection (not shown) and the fact that 6 - k e t o - P G F l ~ presented some protective activity do not support a receptor-mediated process. However, the accuracy of the structure-activity relationship requires further experiments with other prostanoids tested in a larger range of concentrations. It is rather surprising that PGI2, which has a half-life o f - 3 min in the medium, can be active for several hours in our experiments. These data imply that P G I 2 triggers a long-lasting event which persists after the prostaglandin degradation. In D y m o n d and Kalmus [4], P G E 2 has been shown to be neuroprotective against actinomycin C toxicity through a cAMP-independent mechanism. These authors have proposed that P G E 2 may alter the plasma membrane by stabilizing it, an effect previously suggested by others from experiments carried out with P G I 2 15
not used for up to 10 days. F o r glutamate-treatment experiments, cortical cultures were incubated under normoxic conditions with 10 ~tM L-glutamate in simplified medium for 3 h at 37°C. Prostaglandins were added to cultures just before glutamate exposure. Cellular injury was assessed by lactate dehydrogenase ( L D H ) release into the cell-culture supernatant after hypoxia/reoxygenation or glutamate exposure as previously described [2]. When cortical neurons were exposed for 5 h to an anoxic atmosphere, followed by 3 h reoxygenation, they released the cytoplasmic enzyme, L D H , a cytotoxicity marker. Treatment with PGI2 ( 1 - 1 0 / I M ) inhibited this release but 6-keto-PGFlct, the stable degradation product of PGI2, only at the highest concentration significantly protected the neuron from the oxidative stress (Fig. 1). In addition, when cultured cortical neurons were incubated for 3 h with glutamate (10/.tM), the same sign of cell injury was observed, i.e., increase in L D H release into the culture medium. Addition of PGI2 (1-10/.tM) to the cultures significantly decreased this enzyme leakage. The effect of the same concentrations of 6 - k e t o - P G F l ~ was not significant (Fig. 2).
_z ¢L
lO g X
5
_c
10~M Glu
I~M
10~M + PGI2
I~M
10~M
÷6-keto-PGF1
(x--
Fig. 2. Effect of prostacyclin (PGI2) and 6-keto-PGFlct on glutamateinduced neuron LDH release from cultured neurons. Results are expressed as increase in LDH release from controls which are 23.4 _+2.5 mU/ml per mg protein. Results are mean _+S.E.M. from at least five distinct experiments made in triplicate. **P < 0.01 one-way ANOVA followed by Dunnett's test.
108 on isolated liver l y s o s o m e s [9]. Besides, in an earlier s t u d y [I], showing t h a t PGI2 can p r o t e c t h e p a t o c y t e s a g a i n s t CC14 toxicity, it has been p r o p o s e d that this p r o s t a g l a n d i n c o u l d act by inhibiting l i p o p e r o x i d a t i o n , a possible m e c h a n i s m which is u n c l e a r a n d even c o n t r o versial [12]. O u r results, d e m o n s t r a t i n g a n e u r o p r o t e c t i v e effect a g a i n s t g l u t a m a t e toxicity, c o u l d also be linked to an a n t i o x i d a t i v e m e c h a n i s m since g l u t a m a t e toxicity is related, at least in p a r t , to O2-derived free radical generation [2]. P r o s t a g l a n d i n s , a n d P G I 2 in particular, c o u l d be n a t u rally occurring c y t o p r o t e c t i v e agents a g a i n s t a variety o f stresses. The p a t h o p h y s i o l o g i c a l significance o f this rem a i n s to be elucidated. In particular, it is n o t k n o w n if PGI2, which is p r o d u c e d in b r a i n u n d e r ischemia [7], can effectively reach a sufficient c o n c e n t r a t i o n , at n e u r o n a l level, to exert such a protective effect. F u r t h e r m o r e , p h a r m a c o l o g i c a l i m p l i c a t i o n s o f this activity is questionable. Indeed, PGI2 clinical trials in b r a i n ischemic stroke, b a s e d on v a s o d i l a t o r a n d a n t i t h r o m b o t i c actions o f this p r o s t a g l a n d i n , have given rise to d i s a p p o i n t i n g results
4
5
6
7
8
9
10
[51. These conclusions reinforce the interest in further investigating the direct n e u r o p r o t e c t i v e m e c h a n i s m o f action o f PGI2 a n d o t h e r eicosanoids.
11
12 l Bursch, W., Taper, H.S., Somer, M.R, Meyer, S., Putz, B. and Schulte-Hermann, R., Histochemical and biochemical studies on the effect of the prostacyclin derivative Iloprost on CC14-induced lipid peroxidation in rat liver and its significance for hepatoprotection, Hepatology, 9 (1989) 830 838. 2 Cazevieille, C., Muller, A., Meynier F. and Bonne, C., Superoxide and nitric oxide cooperation in hypoxia/reoxygenation-induced neuron injury, Free Radic. Biol. Med., 14 (1993) 389-395. 3 Chan, RH., Kerlan, R. and Fishman, R.A., Reductions of y-aminobutyric acid and glutamate uptake and (Na++K+)-ATPase activ-
13
14
ity in brain slices and synaptosomes by arachidonic acid, J. Neurochem., 40 (1983) 309 316. Dymond, J.B. and Kalmus, G.W., The cytoprotective properties of" prostaglandin E2 against the toxic effects of actinomycin C on embryonic neural retina cells, Prostaglandins, 44 (1992) 129 134. Hsu, C.Y., Faught, Jr., R.E., Furlan, A.J., Coull, B.M.. Huang, D.C., Hogan, E.L., Linet, O.I. and Yatsu, F.M., Intravenous prostacyclin in acute nonhemorrhagic stroke: a placebo-controlled double-blind trial, Stroke, 18 (1987) 352-358. Hsu, C.Y., Liu, T.H., Xu, J., Hogan, E.L., Chao, J., Sun, G., Tai, H.H., Beckman, J.S. and Freeman, B.A., Arachidonic acid and its metabolites in cerebral ischemia. In A,I. Barkai and N.G. Bazan (Eds.), Arachidonic Acid Metabolism in The Nervous System, Ann. N.Y. Acad. Sci., 559 (1989) 282 295. Huttemeier, RC., Kamiyama, Y., Su, M., Watkins, W.D. and Benveniste, H., Microdialysis measurements of PGD2, TXB2, and 6keto-PGFla in rat CA1 hippocampus during transient cerebral ischemia, Prostaglandins, 45 (1993) 177 187. Kunievsky, B., Bazan, N.G. and Yavin, E., Generation of arachidonic acid and diacylglycerol second messengers from polyphosphoinositides in ischemic fetal brain, J. Neurochem., 59 (1992) 1812-1819. Lefer, A.M. and Smith, IlI, E.F., Protective action of prostacyclin in myocardial ischemia and trauma. In J.R. Vane and S. Bergstr6m (Eds.), Prostacyclin, Raven, New York, 1979, pp. 339-348. Miller, V.T., Coull, B.M., Yatsu, F.R, Shah, A.B. and Beamer, N.B., Prostacyclin infusion in acute cerebral infarction, Neurology, 34 (1984) 1431-1435. Paller, M.S. and Manivel, J.C., Prostaglandins protect kidneys against isehemic and toxic injury by a cellular effect, Kidney Int., 42 (1992) 1345 1354. Renkawek, K., Herbaeyznska-Cedro, K. and Mossakowski, M.J., The effect of prostacyclin on the morphological and enzymatic properties of CNS cultures exposed to anoxia, Acta Neurol. Scan&, 73(1986) 111 118. Simon, R.R, Swan, J.H., Griffith, T. and Meldrum, B.S., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. Williams, J.H., Errington, M.L., Lynch, M.A. and Bliss, T.V.R, Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus, Nature (London), 341 (1989) 739-742.