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Leukotriene C4 and Prostaglandin E2 Activities in the Serum and Cerebrospinal Fluid During Acute Cerebral Ischemia S. Aktan, C. Aykut and S. Ercan* Department of Neurology, Marmara University School of Medicine, Marmara University Hospital, 81190, Istanbul, Turkey, * Department of Pharmacology, Gazi University School of Medicine, Ankara, Turkey (Reprint requests to SA) ABSTRACT. Lipoxygenase pathway products of arachidonic acid (AA) metabolism (known as leukotrienes, LTs) are produced in the brain durhtg,pathologic conditions such as ischemia, hemorrhage, trauma, and seizure in which the release of AA is sustained by the activation of local phospholipases. The most common type of LT in the central nervous system is an LTCd which is a highly potent vasoconstrictor leading to increase in vascular permeability. In this study, we compared the serum (S) and cerebrospioal fluid (CSF) prostaglandm EZ (PC&) and LTCl levels in 13 consecutively admitted patients with acute cerebral ischemia aged 55-80 years with 10 age-matched controls. Patients with previous glucocorticosteroid and antiiiammatory drug usage were not included in the study. S and CSF samples were drawn during the first 72 h of the attack, and samples were evaluated by bioassay. There was no significant difference in S PGEz and LTC, values, whereas a significant difference was observed between CSF PGEz and LTC4 values as compared with the control group. The high levels ,of CSF PGEz and LTCJike activity in acute cerebral ischemia may indicate that these mediators have a role to play in cerebral edema. The CSF PGE&TCd ratio was also found to be reduced in the ischemic group implying higher LTC,, synthesis than PGEZ synthesis. In the light of these findings, we suggest that use of a selective antagonist of LTs may be helpful in reducing the ischemic penumbra during acute cerebral ischemia by controlling the vasogenic edema.

INTRODUCTION

MATERIALS AND METHODS

It is known that prostaglandins (PGs), especially prostaglandin Ez (PGE,) which are the cyclooxygenase metabolites of arachidonic acid (AA) may decrease cerebral blood flow and induce cerebral edema during cerebral ischemia of affected brain region (l-6). On the other hand, Indomethacin, one of the cyclooxygenase inhibitors cannot control vasogenic edema after stroke (l-7). Recently, attention has been focused on the biological effects of other AA metabolites, particularly of those produced by lipoxygenase pathways such as leukotrienes (LTs), especially in pathologic conditions, like cerebral ischemia, subarachnoid hemorrhage, head trauma, and seizure (6, 8, 9, 10-12). LTC4 and LTD4 are highly potent vasoconstrictors of cerebral arteries leading to increase vascular permeability (13, 14).

In this study, we included patients aged 55-80 years whose main diagnosis was acute cerebral ischemia. The sample consisted of 13 patients who were consecutively admitted to the Department of Neurology, Marmara University, School of Medicine between January and April 1989. We excluded patients with previous glucocorticoid and antiinflammatory drug usage. As a control group, we included 10 age-matched abdominal surgery patients undergoing spinal anesthesia who had no previous history of cerebral ischemia. Clinically 5 of 13 patients were classified as ‘Reversible Ischemic Neurologic Deficit’ (RIND) while the others were classified as ‘Acute Completed Stroke’. Blood serum and cerebrospinal fluid (CSF) samples were drawn separately for each method and were stored at -24°C. All samples were studied simultaneously. For the determination of PGEz and LTG levels, a bioassay method was carried out as outlined below.

Date received 29 October 1990 Date accepted 25 February i991

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Prostanlandins Leukotrienes

and Essential Fattv Acids

The determination of serum LTCclike activity After separation of blood serum, 1 mg/ml acetylsalicylic acid and after 2 min 1 N HCl (1 ml) were added. Then the sample was centrifuged at 3000 g for 15 min. Supernatant was removed and 2 ml ethylacetate was added. After stirring, the sample was centrifuged again and the ethlylacetate phase was removed and vaporized. The remainder was frozen at -24°C. The determination of CSF LTCJike activity After adding 1 mg/ml acetylsalicylic acid into the CSF samples, they were frozen at -24°C. LTC6like activity was evaluated in a guinea-pig ileum bioassay method by the Anderson technique (15, 16). The determination of serum PGEz-like activity After separation of blood serum (1 ml), 1 N HCl (1 ml) was added and centrifuged at 3000 g for 15 min. Then the supematant was removed and 2 ml ethylacetate was added; after stirring, the sample was centrifuged again. The ethylacetate phase was removed and vaporized and then frozen at -24°C.

There was no statistically significant difference between two groups (Table 1). The mean value of serum LTCb-like activity was found to be 0.5 0.1 ng/ml in the ischemic group. The range was between 0.1-1.2 ng/ml. The mean value of serum LTCd-like activity was found to be 0.2 0.1 &ml in the control group. There was no significant difference for the serum LTC4 values between the cerebral ischemia and the control group (Table 1). The mean value of CSF PG&-like activity was found to be 2.6 0.4 rig/ml in the ischemic group. The range was between 0.4-4.2 ng/ml. The mean value of CSF PGE*-like activity was 0.1 0.1 ng/ml in the control group. There was a statistically significant difference for the CSF PGE2 values in patients with cerebral ischemia compared to the controls (p ~0.01) (Table 2). The mean value of CSF LTC4-like activity was found to be 0.6 0.1 ng/ml in the ischemic group. The range was between 0.1-1.2 ng/ml. The mean value of CSF LTC4-like activity was found to be 0.2 0.04 ng/ml in the control group. There was again a significant difference for the CSF LTCd-like activity between the two groups (p ~0.02) (Table 2).

Table 2 The values of CSF PGEz and LTC,

The determination of CSF PGE&ke activity A sufficient amount of the CSF sample was kept at -24°C. PGEZ-like activity was evaluated on rat stomach fundus muscle by bioassay (17, 18).

Groups

PGE$-like activity @g/ml)

LTC,-like activity @g/ml)

Cerebral ischemia Control

2.59 + 0.37** 1.14 + 0.08’

0.55 + 0.10** 0.20 + 0.04*

*Results are expressed as Means _+ SEM **There was statistically significant difference between two groups

Statistical methods Results are expressed as means SEM (Standard Error of the Mean). Statistically evaluation of the data was performed by Student’s t-test.

DISCUSSION RESULTS The mean value of serum PGErlike activity was found to be 3.2 0.4 ng/ml in the ischemic group. The range of values was between 0.4-5.4 ngml. The mean value of serum PGErlike activity was found to be 2.6 0.3 ng/ml in the control group.

Table 1 The values of serum PGE, and LTC, Groups

PGQ-like activity @s/ml)

LTCJike @g/ml)

Cerebral &hernia Control

3.21 It 0.14** 2.60 -+ 0.32”

0.46 t o.ot3** 0.24 + 0.06*

activity

* Results are expressed as Means + SEM ** There was no statistically significant difference between two groups

Leukotrienes (LTs) and prostaglandins (PGs) are products derived from AA by the activation of local phospholipases (19-21). Many experimental studies have shown that PG levels are increased in the brain during pathologic conditions such as cerebral ischemia, subarachnoid hemorrhage brain tumor, head trauma and seizure (22-24). Recently, it has been shown LTs are also produced in the brain during these pathologic conditions (1, 25). It has been suggested that these AA metabolites are responsible for the formation of cerebral edema in the ischemic brain tissue (1, 26, 27). However, the development of brain edema could not be prevented with indomethacin which inhibits cyclooxygenase and reduces PGs (2, 14). Moscowitz stated that LTs with the biologic activity of enhancing vascular permeability might be involved in the pathogenesis of brain edema (1). Indeed, Black reported that direct

LTC, and PGE, Activities During Acute Cerebral Ischemia

application of LTs to brain parenchyma enhanced Evans blue extravasation in rats (28). In this study, the significantly high levels of CSF LTC, and PGE2 may indicate that they are involved in the development of vasogenic edema in acute ischemia. The CSF PGE&TC., ratio was found to be reduced in the patient group, implying higher synthesis of LTCJ than PGE2 which is in the line with similar findings reported in the recent literature (6, 8-12). Several more aspects of LT formation remain to be investigated further. We still do not know whether LTs are produced by neuronal cells, glial cells, or infiltrating leukocytes. Recent studies in experimental animals have shown that leukocytes accumulate in the postischemic hemisphere within hours of stroke (29, 30). In this study, despite the high levels of PGEz and LT&like activities in the CSF of the patient group, we did not observe a statistically significant difference in serum AA metabolites between the groups. As a result, we concluded that these mediators are locally generated in the brain and do not reach the brain from plasma. In conclusion, it is possible that LTC4, a highly potent vasoconstrictor of human cerebral arteries, increases vascular permeability leading to vasogenic edema which produces an ischemic penumbra, and enlarges the peri-infarct area. In the light of these findings, the use of a selective antagonist of LTs or may be helpful in protecting the ischemic penumbra during acute cerebral ischemia. References 1. Moskowitz M A. Synthesis of compounds with properties leukotrienes C4 and D4 in gerbil brains after ischemia and reperfusion. Science 224: 886, 1984. 2. Ment L R, Steward W B, Duncan C C. Beagle pup model of brain injury: Regional cerebral blood flow and cerebral prostaglandins. J. Neurosurg. 67: 278, 1987. 3. Goddard F T, Michael L H. Effects of prostaglandin E, on cerebral blood flow in the newborn beagle. Ann. Neurol. 12: 222, 1982 (Abstract). 4. Weinderfeld T, Lysy T, Shohami E. Effect of dexamethasone on prostaglandin synthesis in various areas of the rat brain. J. Neurochem. 48: 1351, 1987. 5. Ment L R, Steward W B, Duncan C C. Beagle puppy model of perinatal cerebral infarction. J. Neurosurg. 65: 851, 1986. 6 Holst H V, Granstrom E, Hammarstrom S. Effect of leukotrienes C4, D4, prostacyclin and tromboxane A2 on isolated human cerebral arteries. Acta Neurochirurgica 62: 177, 1982. Filep T, Filep E, Frolich J C. Vascular responses to leukotriene B4, C4, and D4 following indomethacin. Br. J. Pharmac. 90: 431, 1987. Gazzaniga P P, Ferroni P, Lenti D. Identification of blood leukotrienes in classical migraine. Headache 27: 211, 1987. Miyamoto T, Lindgren T A, Hokfelt T. Regional distribution of leukotriene and

mono-hydroxyeicosatetraenoic acid production in the rat brain. Febs Letters 216: 123, 1987. 10. Lysz T W, Centra M, Markey K. Evidence for increased activity of mouse brain fatty acid lipoxygenase following drug-induced convulsions. Brain Research 408: 6, 1987. 11. Shimizu T, Takusagawa Y, Izumi T. Enzymic synthesis of leukotriene B4 in guine pig brain. J. Neurosurg. 48: 1541, 1987. 12. Eimerl T, Siren A L, Feuerstein G. Systemic and regional hemodynamic effects of leukotrienes D4 and E4 in conscious rat. Prostaglandins Leukotrienes Med. 17: 229, 1985. 13. Gulati 0 P, Malmsten C, Ponard G. The local edemogenic effects of Ieukotriene C4 and prostaglandin E2 in rats. Prostaglandins Leukotrienes Med. 10: 11, 1983. 14. Ban M, Tonai T, Kalino T, Matsumoto K. A flavonoid inhibitor of lipoxygenase inhibits leukotriene production following ischemia in gerbil brain. Stroke 20: 248, 1989. 15. Anderson W H, O’Donnel M, Simko B A, Welton A F. An in vivo model for measuring antigen-induced SRS-A mediated bronchoconstriction and plasma SRS-A levels in the guine pig. Br. J. Pharmacol. 78: 67, 1983. 16. Samhoon M N, Piper P J. A combined use of isolated strips of guine pig lung parenchyma and ileum as a sensitive bioassay for leukotriene C4. Prostaglandins 27: 711, 1984. 17. Gilmore M, Vane J R, Wyllie S H: Prostaglandin release by the spleen. Nature 213: 1135, 1968. 18. Vane J R. A sensitive method for the assay of 5-hydroxytryptamine. Br. J. Pharmacol. 12: 344, 1957. 19. Brus R, Krzeminski T, Kurcok A. Central effects of leukotriene C4 and D4 in rats and mice. Biomed. Biochem. Acta 45, 9: 1153, 1986. 20. Simmet T, Luck W, Delank W K. Formation of cisteinyl-leukotriens by human brain tissue. Brain Research 456: 344, 1988. 21. Hartung H P, Toyka K V. Leukotriene production by cultured astroglial cells. Brain Research 435: 367. 1987. 22. Gaudet R J. Levine L. Transient cerebral ischemia and brain prostaglandins. Biochem. Biophys. Res. Commun. 86: 893. 1979. 23. Gaudet R J, Levine L, Alam I. Accumulation of cyclooxygenase products of arachidonic acid metabolism in gerbil brain during reperfusion after bilateral common carotid artery occlusion. J. Neurochem. 35: 653, 1980. 24. Crockard H A, Bhakoo K K, Lascelles P T. Regional prostaglandin levels in cerebral ischemia. J. Neurochem. 38: 1311. 1982. 25. Kiwak K J, Moskowitz M A, Levine L. Leukotriene production in gerbil brain after ischemic insult, subarachnoid hemorrhage, and concussive injury. J. Neurosurg. 62: 865, 1985. 26. Bhakoo K K. Crockard H A. Lascelles P C. Prostaglandin synthesis and dedema formation during reperfusion following experimental brain ischemia in the gerbil. Stroke 15: 865, 1984. 27. Black K L, Hoff J T, Radin N S. Eicosapentaenoic acid: Effect on brain prostaglandins, cerebral blood flow and edema in ischemic gerbils. Stroke 15: 65, 1984. 28. Black K L. Hoff J T. Leukotrienes increases blood-brain barrier permeability following intraparenchymal injections in rats. Ann. Neurol. 18: 349, 1985. 29. Saito K, Levine L, Michael A, Moskowitz M A. Blood components contribute to rise in gerbil brain levels of leukotriene-like immunoreactivity after ischemia and reperfusion. Stroke 19: 1395, 1988. 30. Busija D W, Leffler C W, Beasley D G. Effects of leukotrienes C4, D4 and E4 on cerebral arteries of newborn pigs. Pediatrics Research 20: 973, 1986.

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Leukotriene C4 and prostaglandin E2 activities in the serum and cerebrospinal fluid during acute cerebral ischemia.

Lipoxygenase pathway products of arachidonic acid (AA) metabolism (known as leukotrienes, LTs) are produced in the brain during pathologic conditions ...
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