- Email: [email protected]
Brain tissue injury and blood-brain barrier opening induced by injection of LGE2 or PGE2
PROSTAGLANDINSLEUBO~S ANDEMNTIALFATTYACIDS Prostaglandins Leukotneneb and Essential ,Q Longman Group UK Ltd 1992
Fatty Acids
t1992)
41. 105-I IO
Brain Tissue Injury and Blood-Brain Barrier Opening Induced by Injection of LGE2 or PGE2 J. W. Schmidley, J. Dadson, R. S. Iyer* and R. G. Salomon” Deparrments of Neurology and *Chemistry, Case Western Reserve University, Cleveland, Ohio 441062699, USA (Reprint requests to JWS)
Room S404.3395 Scranton Road
ABSTRACT.
The hypothesis that the accumulation of prostaglandin (PG)E, during reperfusion of severely ischemic tissue contributes to a breakdown in the blood-brain barrier (BBB) was expanded to include a parallel role for levuglandins(LGs), y-ketoaldehydes produced by rearrangement of PGH2. LGEz was shown to be more potent than PGEz in causing breakdown of the BBB when injected intrahemispherically. Brain tissue necrosis was clearly evident with total doses of levuglandin as low as 100 nmole.
cerebral ischemia offer a potential environment for generation of LGs. In severe cerebral ischemia, free fatty acids (FFAs), in particular arachidonic acid (C2&, accumulate to levels many times normal. This increase is largest, in ,both absolute and relative terms for CZo:.. Thus, 0.4 mM concentrations of unesterified CZoZ4 are generated in the brain after 30 min of cerebral ischemia (17-20). During recirculation, levels of FFAs decline, but in both absolute and relative terms, the decline is greatest for C2a+ This fall in [email protected] is associated with a burst of eicosanoid synthesis (21-23). It has been proposed that superoxide radical produced during PG synthesis (24, 25) the PGs themselves (26, 27) other cyclooxygenase products (21, 22, 28, 29), or leukotrienes (30) may be responsible for reperfusion injury following cerebral ischemia (31-33). C20:4and other polyunsaturated fatty acids (PUFAs) have also been implicated directly (34). Furthermore, since synthesis of known eicosanoids accounts for only a fraction of FFAs released during ischemia, it is possible that LGs may also be generated, and participate in this process. Since there is as yet no reliable way of detecting either the LGs, of their protein adducts in normal of diseased tissues, we took an alternative, albeit indirect, approach, and examined the effects of these newly discovered molecules on brain parenchyma and the BBB following intracerebral injection.
INTRODUCTION In 1977, model studies (1) suggested that the endoperoxide intermediated of the cyclooxygenase cascade, prostaglandin (PG) H,, should yield products in addition to the PC&, prostacyclin (PGJ,) thromboxanes (TXs), malondialdehyde (MDA), and hydroxyheptadecatrienoic acid (HHT). In 1982, the production of novel y-ketoaldehydes was detected in the physiological aqueous environment of PGH;? biosynthesis (2, 3). These vinylogous P-hydroxy carbonyl compounds were designated the levuglandins (LGs) (Fig. 1). LGEz (a 9, lo-secoprostaglandin) and LGDp (a 10, I I-secoprostaglandin) both readily eliminate HZ0 to form anhydro derivatives, anLGEz and anLGD, (4). Subsequent studies showed that anLGDz selectively and reversibly binds to PGD2 and PGF2 receptors in the rat uterus (5) while a&GE2 binds to PGE, and weakly to PGD, receptors in this tissue (6). This discovery of biologically significant activity suggests that LGs may act to modulate or modify prostaglandin activities at the local level. The recent observation that the LGs avidly and covalently bind proteins (7) crosslinking and polymerizing them (8), suggest that they are the ‘unidentified metabolites’ of PGH, which covalently bind to proteins (916). This may explain the difficulty encountered in detecting the LGs in biological systems, and also offers a potential mechanism whereby they might cause tissue damage in ischemia or other disease processes. The conditions which exist during reperfusion following severe
MATERIALS AND METHODS Adult rats of either sex were anesthetized with pentobarbital, 50 mg per kg, given intraperitoneally. The scalp
Date received 4 November 199 1 Date accepted 3 1 March 1992 105
106
Prostaglandins Leukotrienes and Essential Fatty Acids
PGD 2
PGE 2
0
TXA2 +
MDA +
HHT
LGE 2 AnLGE2 OHC\,~\*~~CHWOH
)/+,%%I LG&
was infiltrated with 2% lidocaine, and the cranium exposed. A burr hole was drilled, and experimental or control solution was then infused slowly into the brain through a stereotaxically placed (5 mm below the dura) 26 gauge needle, over 10 min. Both the total dose of eicosanoid and volume of solution injected were chosen to be less than those used in a previous study (34) which injected solutions containing 2.5 pm01 of CZo14 in 500 ~1 of Krebs Ringer buffer. Since the bioconversion of Czo:. is not a practical source of supply for LGs, LGE2 was prepared by an asymmetric total synthesis as reported previously (35). Solutions (50 ~1) injected in the present study contained either 0.1 M pH 7.4 phosphate-buffered saline (PBS-control), or LGE2 (0.5 of 1.25 pmol) in (0.1 M PBS, of PGE2 (1.25 ymol) in 0.1 M PBS. Following infusion, the needle was withdrawn, the burr hole occluded with bone wax, and the scalp sutured. The animals were returned to their cages, and allowed to recover. One h before decapitation, they were reanesthetized, and 1 ml of 2% Evans Blue was injected via internal jugular vein. 4 or 24 h after intracerebral injection, the animals were killed by decapitation. The brains were removed and sectioned in a coronal plane at the level of the needle entry site, which was easily identified on the surface of the hemisphere. The slice (3 mm thick) anterior to the plane of section was used for de-
-H20- ‘j&+./W,, AnLGD2
termination of water content; the slice (5 mm in thickness) immediately posterior to the plane of section was weighed, rinsed thoroughly in normal saline, and placed in 5 ml of 100% formamide at 60 “C for determination of Evans Blue concentration. Water content was determined by first weighing the fresh brain slices, then drying them to constant weight at 100 “C. Water content was calculated using the formula (wet weight - dry weight)/wet weight x 100. After extraction in formamide for 24 h, the Evans Blue content of the tissue was determined calorimetrically using a Varian Cary 2300 UV-Visible NIR spectrophotometer by monitoring the absorption at 620 nm. For histological examination, other animals were anesthetized and injected in a similar manner, and after 3 days, the brains fixed by vascular perfusion with, or immersion in, 4% buffered parafotmaldehyde, embedded in paraffin, sectioned and stained with methylene blue for microscopic examination. For the histological studies, animals were injected with 50~1 of experimental solution containing 0.1, 0.5, of 2.5 pm01 of LGE2 in 0.1 M PBS, or with the PBS control solution, T-tests were used for statistical analysis, which was accomplished using the ABSTAT program (Anderson Bell, Parker, CO). P values co.05 were considered significant; except as noted, the p values reported were for the two-tailed t-test.
Brain Tissue Injury and Blood-Brain
Barrier Opening Induced by Injection of LGE, or F’GE,
107
Fig. 2 Light micrograph of lesion induced by intraparenchymal injection of 100 nmol of LGE;. Normal neuropil at lower left, pale. edematous area of lesion at upper right; with cellular reaction running diagonally from upper Ieft to lower right along border of lesion. Final magnification 175x.
RESULTS All the animals recovered consciousness at approximately the same time after surgery. Those injected with higher doses of LGE2 (0.5 or 2.5 pmol) were less active than those injected with lower doses of LGE2, PGE2 or controls.
Histopathological observations The histopathological
findings were similar in the animals injected with 0.1, 0.5, or 2.5 pm01 of LGE*. The only difference was in the size of the lesion; animals injected with the higher dose of LGE;? had very large lesions, occupying approximately one-third to one-half of the cross-sectional area of the hemisphere, and easily visible to the unaided eye. The lesions in the other animals were smaller, but otherwise similar. Even the lowest dose (100 nmol) caused lesions which were readily apparent at 175x magnification (Fig. 2). They consisted of a central area of pallor and loss of normal cellular constituents, with hypercellular margins. The cellular infiltrate consisted largely of lipid-laden macrophages. The lesions were sharply demarcated from the surrounding brain; the only detectable abnormality in neurons of adjoining brain was a change in the nucleolar morphology. In neurons immediately adjacent to the area of necrosis, the nucleoli were rod-like, rather than round, as in normal neurons. There was no obvious vascular damage of intravascular thrombosis. In two animals the LG solution entered the ventricle, and caused extensive denudation of the ependyma and choroid plexus epithelium (Fig. 3). The latter structure and the subependyma1 regions were also infiltrated with cells, leading to obliteration of normal architecture. Hemispheres injected with control solution showed no abnormality
beyond the area immediately needle penetration.
adjacent
to the site of
Evans Blue Intrahemispheric injection of either 0.5 of 1.25 pm01 of LGE,, or 1.25 pm01 of PGE,, caused marked extravasation of Evans Blue compared with hemispheres injected with the control solution. This was most marked for the higher dose of LGE,, and was significantly greater than that produced by the same amount of PGEz or the lower dose of LGE2 which were equivalent. Evans Blue was also increased in the contralateral hemisphere, although to a lesser extent, probably because of spread of the fluid across the corpus callosum (see Discussion). The amount of Evans Blue extravasation 4 h after intrahemispheric injection of LGEz was significantly less than that found 24 h after injection. Injection of the control solution alone caused a significant extravasation of Evans Blue in comparison to the noninjected hemisphere, probably as a result of needle trauma. This was somewhat less marked at 24 h compared with 4 h and, although greater than the noninjected hemisphere at 24 h, was significantly less than the values obtained following injection of LGE, of PGE, (Table 1).
Water content The water content of hemispheres injected with control solution, or with LGE, of PGE,, was significantly greater than that in the contralateral hemisphere, both at 4 h and at 24 h. The water content of hemispheres injected with LGE, of PGE, was not significantly different from that of hemispheres injected with control solution (Table 2).
108
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Fig. 3 Light micrograph of changes induced in ependyma, choroid plexus and periventricular neuropil by presence of LG& in CSF. Animal had been injected with 0.5 pm01 of LGE*, some of which presumably entered the ventricle. Left panel shows denudation of ependyma (E) with cellular infiltration of subjacent neuropil and of choroid plexus (C); right panel shows normal structures for comparison.
DISCUSSION The experiments described demonstrate that LGE2 causes tissue necrosis, accompanied by breakdown of the blood-brain barrier (BBB), when injected into the substance of the cerebral hemisphere. This effect was dose-dependent, as demonstrated by both the histological and the biochemical studies. The finding of increased Table 1
Extravasation
of Evans Blue, /@lo0
mg wet weight brain
N’
Injected Mean (& SD)
Non-injected Mean (*SD)
24 hour Control
14
LGE, 0.5 pm01
17
0.291 (H.245) 0.864” (ti.427)
LGE, 1.25 ,umol
11
0.507d (fl.266) I .2.57 a.d (&0.520) 2.409”. b. c, d. e (kl.057) 1.5O84d (ti.411)
(M.888) 1.219” (kO.321)
0.670 (~.lll) 1.255d* (f0.684)
0.534 (ti.053) 1.055” (ti.420)
PGE, 1.25 pm01
11
4 hour Control
4
LGE, 1.25 ~01
11
, ,862”
b, c. e
Evans Blue content of injected regions, and the same areas of noninjected contralateral hemispheres at 4 and 24 h following injection of control solution, LGE, or PGl$ ‘Number of animals. “indicates value for region was significantly > that for same region in brain injected with control solution; b indicates that value for the region was significantly > than same region in brain injected with LGE, 10 mM; c indicates that the value for the region was significantly > that for same region of brain injected with PGE, 25 mM; d indicates that the value for the injected hemisphere was significantly > that for noninjected hemisphere; c indicates that the value at 24 h is significantly > the value at 4 h for same region injected with the same solution. *indicates p value of 0.04 for one-tailed t-test, p of 0.08 for two-tailed t-test; all other p values were co.05 for two-tailed t-test.
Evans Blue in the contralateral hemisphere is probably explained by the tendency of the extravasated proteinrich fluid to follow white matter tracts such as the corpus callosum (36). We were unable to demonstrate an increase in brain water, despite the obvious tissue damage. It is possible that brain water had already increased and then was decreasing by 4 h, when the first determinations of brain water were made, whereas the Evans Blue extravasation was slower to develop, and to resolve. During reperfusion of severely ischemic gerbil brain, edema, as judged by specific gravity, increases to a maximum after about 1 h of reperfusion and then decreases, whereas extravasation of Evans Blue increases steadily in parallel with the concentration of PGE, generated during reperfusion over 3 h (27). Alternatively, it is possible that this measure of brain edema is merely less sensitive than histologic evaluation of Evans Blue extravasation. Table 2
Percent Hz0 N+
Injected Mean (+ SD)
Non-injected Mean (&SD)
24 hour Control
14
LGEz 0.5 ~01
21
LGE, 1.25 ~01
11
PG& 1.25 pm01
11
79.886d (? 1.567) 79.616d (f 1.847) 80.023d (fl.199) 79.307d (_+I,905)
79.121 (* 1.548) 78.7381 (_+1.664) 78.669 (kO.995) 78.878 (_+1.805)
4 hour Control
4
LG& 1.25 ~01
11
79.890” (fi.422) 79.573d (kO.859)
79.180 (+o. 105) 79.056 (f0.761)
Percent water determinations for injected regions, and the same areas of noninjected contralateral hemisphere at 4 and 24h. Superscripts as in legend for Table 1. The only statistical significance was as noted.
Brain Tissue Injury and Blood-Brain
The histologic changes observed were nonspecific but, in general, quite similar to those caused by injection of CZo14and other PUFAs in somewhat larger doses (2.5 pmol) and solution volumes (500 pL) into cerebral hemisphere (34) or ventricle (37). The increase in BBB permeability evoked by the higher dose of LGE? was proportionally greater than that evoked by C2,,+ The effects of Cz0:4 injection on BBB permeability were assayed using the radioactive serum albumin technique, rather than the Evans Blue technique, nonetheless, the amount of Evans Blue extravasation caused by intrahemispheric injection of 1.25 pm01 of LGE-,, was nearly 5 times that induced by intrahemispheric injection of control solution. This is comparatively greater than the 3- to 3.5fold increase in radioactive serum albumin space, from roughly 2% in animals injected with buffer control, to approximately 6.7% in animals injected with 2.5 pm01 of CZti4 (34). Given the negative results obtained by Chan and Fishman (38) for PGE2 with the in vitro brain slice model, it is surprising that PGE, had an effect following intrahemispheric injection. It is possible that higher local concentrations of PGE2 were responsible; in their in vitro incubation system, Chan and Fishman used 0.5 mM concentrations of PGs and PUFAs. BBB permeability. of course, could not be assessed in vitro. To summarize, the experiments reported establish that LGE,, injected in amounts ranging from 100 nmol to 2.5 pm01 total dose, is capable of causing brain tissue necrosis and, in doses greater than 500 nmol, breakdown of the BBB as manifested by extravasation of Evans Blue. It is well established that in cerebral ischemia free fatty acids, in particular the PUFAs, accumulate rapidly. The most striking increases occur for C& (17-20). With recirculation and resupply of oxygen, there is prompt synthesis of both cyclooxygenase and lipoxygenase products, and a sharp decline in free CZ0:4concentration. Yet eicosanoid synthesis can account for only a small percentage of the total CR4 released during ischemia. Studies of cyclooxygenase and lipoxygenase products in ischemic/reperfused brain generally find concentrations in the ng/gm range (21-23, 30, 40), whereas the PUFAs are released in amounts up to hundreds of ,ug/gm of wet weight tissue ( 17-20). Undoubtedly, reacylation, and possibly peroxidative damage, also account for some of the decrease in FFAs which occurs upon reperfusion (33). It is plausible, although as yet unproven, that a tiny fraction of the massive quantities of C,,:, released during severe ischemia is converted to LGs during the burst of cyclooxygenase-promoted oxidative metabolism that occurs in the reperfused brain, and that this contributes to tissue necrosis, edema, and increased vascular permeability. Several factors may suppress enzymatic conversions of PGH? and consequently favor rearrangement to LGs. For example, it is possible the superoxide radical, which may be a byproduct of cyclooxygenase activity, acts to inhibit PG12 synthetase. Similarly, high concentrations of Co+ which are typical during &hernia,
Barrier Opening Induced by Injection of LGE, or PGEz
109
inhibit PGE isomerase (42) and therefore make it more likely that PGHZ will yield other end products including the LGs. Since the extravasation of Evans Blue, indicative of the breakdown of the BBB, coincides with PGE, accumulation during reperfusion through severely ischemic tissue, it was postulated that PGE, may cause egress of Evans Blue by increasing vascular permeability (27). Our demonstration that LGE, is more potent than PGE, in causing breakdown of the BBB when injected intrahemispherically provides presumptive evidence that LGs may also contribute to BBB damage associated with repefusion through severely ischemic tissue. Since the LGs must be produced from PGH2, complete inhibition of cyclooxygenase should prevent their production, as well as that of the other PGs and TXs. Observations on the effects of cyclooxygenase inhibitors such as indomethacin and aspirin on cerebral ischemia have been contradictory, with some studies showing protection (22, 40) but others failing to do so (41). However, it appears that the effects of these inhibitors depend on the severity of the ischemia. Thus, inhibition of the cyclooxygenase pathway by indomethacin does not affect edema formation upon reperfusion after moderate ischemia but does after severe ischemia (43). Further definition of the role, if any, of LGs in ischemic injury to the central nervous system will depend on detecting them in ischemic or ischemic/reperfused tissues; this, in turn, will depend on development of a relatively sensitive assay. The development of an immunoassay for this purpose has not, thus far, been possible, probably because of the tendency of the LGs to covalently bind proteins. The exact mechanisms whereby the LGs contribute to tissue damage will, of course, need extensive further investigation. One potential mechanism, which also explains the difficulty with detecting them in vitro by usual chemical methods, is their remarkable tendency to covalently bind to, and crosslink proteins. The potential role of protein cross-linking agents in the damage associated with cerebral ischemia has already been extensively discussed in the context of free radical mechanism (41), but may apply equally to the damage caused by the LGs. We believe that the results reported in this paper represent an encouraging first step in the investigation of the properties of these newly discovered eicosanoid products.
Acknowledgement This research was supported and NS22524 (to JWS).
by NIH grants GM21249-12
(to RGS)
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22. Gaudet, R. J., L. Levine. Transient cerebral ischemia and brain prostaglandins. Biochem. Biophys. Res. Commun. 86: 893, 1979. 23. Horrocks, L. A., M. VanRollins, A. J. Yates. Lipid changes in the ageing brain, pp. 601630 in The Molecular Basis of Neuropathology (A. N Davison and R. H. S. Thompson, Eds) Edward Arnold (Publishers), London, 1981. 24. Demopoulos. H., E. Flamm, M. Seligmann, R. Power, D. Pietronigro, I. Ransohoff. Molecular pathology of lipids in CNS membranes pp. 491-508 in Oxygen and Physiological Function (F. F. Jobsis, Ed), Professional Information Library. Dallas, 1977. 25. Nordstrom, C. H.. Rehncrona, D., Siesjo. B. K. Effects of phenobarbital in cerebral ischemia. Stroke. 9: 335, 1978. 26. Shohami. E., J. Rosenthal, Lavy, S. The effect of incomplete cerebral ischemia on prostaglandin levels in rat brain. Stroke 13: 494, 1982. 27. Crockard, H. A.. K. K. Bhakoo, P. T. Lascelles, S. F. Avery. Prostaglandins and free fatty acids in experimental cerebral ischaemia. Clin. Res. Rev. 4: 89,1984. thromboxanes. 28. Wolfe, L. S. Eicosanoids: prostaglandins. leukotrienes, and other derivatives of carbon-20 unsaturated fatty acids. J. Neurochem. 38: 1. 1982. 29. Wolfe. L. S., H. M. Pappius. Arachidonic acid metabolites in cerebral ischemia and brain injury, pp. 223-23 1 in: Cerebral Ischemia (A. Bes, P Braquet. R. Paoletti and B. K. Siesjo. Eds), Elsevier. 1984. 30. Moskowitz. M. A., K. J. Kiwak. K. Hekimian. L. Levine. Synthesis of compounds with properties of leukotrienes C, and Da in gerbil brains after ischemia and reperfusion. Science 224: 886. 1984. 31. Ghan, P. H. Role of oxygen radicals in brain injury and edema. p 89 in: Cellular Antioxidant Defense Mechanisms. Volume III. (C. K. Chan. Ed). CRC Press, Boca Raton, Florida, 1988. 32. Siesjo, B. K.. C. D. Agardh, B. Finn. Free radicals and brain damage. Cerebrovascular and Brain Metab. Rev. 1: 165, 1989. 33. Ginsberg. M. D.. B. D. Watson. R. Busto, S. Yoshida. R. Prado, H. Nahayama, M. Ikeda. W. D. Dietrich, M. Y. T. Globus. Peroxidative damage to cell membranes following cerebral ischemia: a cause of ischemic brain injury? Neurochem. Pathol. 9: 171, 1988. 34. Chan. P. H., R. A. Fishman. J. Caronna, J. W. Schmidley, G. Prioleau. J. Lee. Induction of brain edema following intracerebral injection of arachidonic acid. Ann. Neurol. 13: 625, 1983. 35. Salomon. R.G.: Miller. D.B.: Raychaudhuri. S.R.: Avasthi. K.: Lal. K.: Levison. B.S. Asymmetric total synthesis of levuglandin EZ. J. Am. Chem. Sot. 106: 8296,1984. 36. Fishman, R. A. Brain edema. N. Engl. J. Med. 293: 706, 1975. 37 Martin, N. A., J. W. Schmidley. S. L. Wissig, R. A. Fishman, P. H. Chan. Cerebral injury and blood brain barrier disruption induced by arachidonic acid: histopathology. Ann. Neurol.. 12: 110, 1982. 38 Chan, P. H., Fishman, R. A. Brain edema: induction in cortical slices by polyunsaturated fatty acids. Science 201: 358, 1978. 39. Hallenbeck, J. M.. T. W. Furlow, Jr. Prostaglandin Ir and indomethacin prevent impairment of post-ischemic brain reperfusion in yhr dog. Stroke, 10: 629. 1979. 40. Branston. N. M.. M. Bayham, R. J. Harris, A. Watson, L. Symon. Indomethacin increases ischemic brain oedema in the primate. J. Cerebr. Blood Flow Metabol.. I : 143 (suppl. 1), 1981. 41. Freeman, B. A., J. D. Crapo. Biology of disease: free radicals and tissue injury. Lab. Invest. 47: 412, 1982. 42. Flower, R. J.. H. S. Chung. D. W. Cushman. Quantitative determination of prostaglandins and malondialdehyde formed by arachidonate oxygenase system of bovine seminal vesicles. Prostaglandins. 4: 325, 1973. 43. Iannotti. F., A. Crockard. G. Ladds, and L. Symon. Are prostaglandins involved in experimental ischemic edema in gerbils? Stroke, 12: 301, 1981.
Fatty Acids
t1992)
41. 105-I IO
Brain Tissue Injury and Blood-Brain Barrier Opening Induced by Injection of LGE2 or PGE2 J. W. Schmidley, J. Dadson, R. S. Iyer* and R. G. Salomon” Deparrments of Neurology and *Chemistry, Case Western Reserve University, Cleveland, Ohio 441062699, USA (Reprint requests to JWS)
Room S404.3395 Scranton Road
ABSTRACT.
The hypothesis that the accumulation of prostaglandin (PG)E, during reperfusion of severely ischemic tissue contributes to a breakdown in the blood-brain barrier (BBB) was expanded to include a parallel role for levuglandins(LGs), y-ketoaldehydes produced by rearrangement of PGH2. LGEz was shown to be more potent than PGEz in causing breakdown of the BBB when injected intrahemispherically. Brain tissue necrosis was clearly evident with total doses of levuglandin as low as 100 nmole.
cerebral ischemia offer a potential environment for generation of LGs. In severe cerebral ischemia, free fatty acids (FFAs), in particular arachidonic acid (C2&, accumulate to levels many times normal. This increase is largest, in ,both absolute and relative terms for CZo:.. Thus, 0.4 mM concentrations of unesterified CZoZ4 are generated in the brain after 30 min of cerebral ischemia (17-20). During recirculation, levels of FFAs decline, but in both absolute and relative terms, the decline is greatest for C2a+ This fall in [email protected] is associated with a burst of eicosanoid synthesis (21-23). It has been proposed that superoxide radical produced during PG synthesis (24, 25) the PGs themselves (26, 27) other cyclooxygenase products (21, 22, 28, 29), or leukotrienes (30) may be responsible for reperfusion injury following cerebral ischemia (31-33). C20:4and other polyunsaturated fatty acids (PUFAs) have also been implicated directly (34). Furthermore, since synthesis of known eicosanoids accounts for only a fraction of FFAs released during ischemia, it is possible that LGs may also be generated, and participate in this process. Since there is as yet no reliable way of detecting either the LGs, of their protein adducts in normal of diseased tissues, we took an alternative, albeit indirect, approach, and examined the effects of these newly discovered molecules on brain parenchyma and the BBB following intracerebral injection.
INTRODUCTION In 1977, model studies (1) suggested that the endoperoxide intermediated of the cyclooxygenase cascade, prostaglandin (PG) H,, should yield products in addition to the PC&, prostacyclin (PGJ,) thromboxanes (TXs), malondialdehyde (MDA), and hydroxyheptadecatrienoic acid (HHT). In 1982, the production of novel y-ketoaldehydes was detected in the physiological aqueous environment of PGH;? biosynthesis (2, 3). These vinylogous P-hydroxy carbonyl compounds were designated the levuglandins (LGs) (Fig. 1). LGEz (a 9, lo-secoprostaglandin) and LGDp (a 10, I I-secoprostaglandin) both readily eliminate HZ0 to form anhydro derivatives, anLGEz and anLGD, (4). Subsequent studies showed that anLGDz selectively and reversibly binds to PGD2 and PGF2 receptors in the rat uterus (5) while a&GE2 binds to PGE, and weakly to PGD, receptors in this tissue (6). This discovery of biologically significant activity suggests that LGs may act to modulate or modify prostaglandin activities at the local level. The recent observation that the LGs avidly and covalently bind proteins (7) crosslinking and polymerizing them (8), suggest that they are the ‘unidentified metabolites’ of PGH, which covalently bind to proteins (916). This may explain the difficulty encountered in detecting the LGs in biological systems, and also offers a potential mechanism whereby they might cause tissue damage in ischemia or other disease processes. The conditions which exist during reperfusion following severe
MATERIALS AND METHODS Adult rats of either sex were anesthetized with pentobarbital, 50 mg per kg, given intraperitoneally. The scalp
Date received 4 November 199 1 Date accepted 3 1 March 1992 105
106
Prostaglandins Leukotrienes and Essential Fatty Acids
PGD 2
PGE 2
0
TXA2 +
MDA +
HHT
LGE 2 AnLGE2 OHC\,~\*~~CHWOH
)/+,%%I LG&
was infiltrated with 2% lidocaine, and the cranium exposed. A burr hole was drilled, and experimental or control solution was then infused slowly into the brain through a stereotaxically placed (5 mm below the dura) 26 gauge needle, over 10 min. Both the total dose of eicosanoid and volume of solution injected were chosen to be less than those used in a previous study (34) which injected solutions containing 2.5 pm01 of CZo14 in 500 ~1 of Krebs Ringer buffer. Since the bioconversion of Czo:. is not a practical source of supply for LGs, LGE2 was prepared by an asymmetric total synthesis as reported previously (35). Solutions (50 ~1) injected in the present study contained either 0.1 M pH 7.4 phosphate-buffered saline (PBS-control), or LGE2 (0.5 of 1.25 pmol) in (0.1 M PBS, of PGE2 (1.25 ymol) in 0.1 M PBS. Following infusion, the needle was withdrawn, the burr hole occluded with bone wax, and the scalp sutured. The animals were returned to their cages, and allowed to recover. One h before decapitation, they were reanesthetized, and 1 ml of 2% Evans Blue was injected via internal jugular vein. 4 or 24 h after intracerebral injection, the animals were killed by decapitation. The brains were removed and sectioned in a coronal plane at the level of the needle entry site, which was easily identified on the surface of the hemisphere. The slice (3 mm thick) anterior to the plane of section was used for de-
-H20- ‘j&+./W,, AnLGD2
termination of water content; the slice (5 mm in thickness) immediately posterior to the plane of section was weighed, rinsed thoroughly in normal saline, and placed in 5 ml of 100% formamide at 60 “C for determination of Evans Blue concentration. Water content was determined by first weighing the fresh brain slices, then drying them to constant weight at 100 “C. Water content was calculated using the formula (wet weight - dry weight)/wet weight x 100. After extraction in formamide for 24 h, the Evans Blue content of the tissue was determined calorimetrically using a Varian Cary 2300 UV-Visible NIR spectrophotometer by monitoring the absorption at 620 nm. For histological examination, other animals were anesthetized and injected in a similar manner, and after 3 days, the brains fixed by vascular perfusion with, or immersion in, 4% buffered parafotmaldehyde, embedded in paraffin, sectioned and stained with methylene blue for microscopic examination. For the histological studies, animals were injected with 50~1 of experimental solution containing 0.1, 0.5, of 2.5 pm01 of LGE2 in 0.1 M PBS, or with the PBS control solution, T-tests were used for statistical analysis, which was accomplished using the ABSTAT program (Anderson Bell, Parker, CO). P values co.05 were considered significant; except as noted, the p values reported were for the two-tailed t-test.
Brain Tissue Injury and Blood-Brain
Barrier Opening Induced by Injection of LGE, or F’GE,
107
Fig. 2 Light micrograph of lesion induced by intraparenchymal injection of 100 nmol of LGE;. Normal neuropil at lower left, pale. edematous area of lesion at upper right; with cellular reaction running diagonally from upper Ieft to lower right along border of lesion. Final magnification 175x.
RESULTS All the animals recovered consciousness at approximately the same time after surgery. Those injected with higher doses of LGE2 (0.5 or 2.5 pmol) were less active than those injected with lower doses of LGE2, PGE2 or controls.
Histopathological observations The histopathological
findings were similar in the animals injected with 0.1, 0.5, or 2.5 pm01 of LGE*. The only difference was in the size of the lesion; animals injected with the higher dose of LGE;? had very large lesions, occupying approximately one-third to one-half of the cross-sectional area of the hemisphere, and easily visible to the unaided eye. The lesions in the other animals were smaller, but otherwise similar. Even the lowest dose (100 nmol) caused lesions which were readily apparent at 175x magnification (Fig. 2). They consisted of a central area of pallor and loss of normal cellular constituents, with hypercellular margins. The cellular infiltrate consisted largely of lipid-laden macrophages. The lesions were sharply demarcated from the surrounding brain; the only detectable abnormality in neurons of adjoining brain was a change in the nucleolar morphology. In neurons immediately adjacent to the area of necrosis, the nucleoli were rod-like, rather than round, as in normal neurons. There was no obvious vascular damage of intravascular thrombosis. In two animals the LG solution entered the ventricle, and caused extensive denudation of the ependyma and choroid plexus epithelium (Fig. 3). The latter structure and the subependyma1 regions were also infiltrated with cells, leading to obliteration of normal architecture. Hemispheres injected with control solution showed no abnormality
beyond the area immediately needle penetration.
adjacent
to the site of
Evans Blue Intrahemispheric injection of either 0.5 of 1.25 pm01 of LGE,, or 1.25 pm01 of PGE,, caused marked extravasation of Evans Blue compared with hemispheres injected with the control solution. This was most marked for the higher dose of LGE,, and was significantly greater than that produced by the same amount of PGEz or the lower dose of LGE2 which were equivalent. Evans Blue was also increased in the contralateral hemisphere, although to a lesser extent, probably because of spread of the fluid across the corpus callosum (see Discussion). The amount of Evans Blue extravasation 4 h after intrahemispheric injection of LGEz was significantly less than that found 24 h after injection. Injection of the control solution alone caused a significant extravasation of Evans Blue in comparison to the noninjected hemisphere, probably as a result of needle trauma. This was somewhat less marked at 24 h compared with 4 h and, although greater than the noninjected hemisphere at 24 h, was significantly less than the values obtained following injection of LGE, of PGE, (Table 1).
Water content The water content of hemispheres injected with control solution, or with LGE, of PGE,, was significantly greater than that in the contralateral hemisphere, both at 4 h and at 24 h. The water content of hemispheres injected with LGE, of PGE, was not significantly different from that of hemispheres injected with control solution (Table 2).
108
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Fig. 3 Light micrograph of changes induced in ependyma, choroid plexus and periventricular neuropil by presence of LG& in CSF. Animal had been injected with 0.5 pm01 of LGE*, some of which presumably entered the ventricle. Left panel shows denudation of ependyma (E) with cellular infiltration of subjacent neuropil and of choroid plexus (C); right panel shows normal structures for comparison.
DISCUSSION The experiments described demonstrate that LGE2 causes tissue necrosis, accompanied by breakdown of the blood-brain barrier (BBB), when injected into the substance of the cerebral hemisphere. This effect was dose-dependent, as demonstrated by both the histological and the biochemical studies. The finding of increased Table 1
Extravasation
of Evans Blue, /@lo0
mg wet weight brain
N’
Injected Mean (& SD)
Non-injected Mean (*SD)
24 hour Control
14
LGE, 0.5 pm01
17
0.291 (H.245) 0.864” (ti.427)
LGE, 1.25 ,umol
11
0.507d (fl.266) I .2.57 a.d (&0.520) 2.409”. b. c, d. e (kl.057) 1.5O84d (ti.411)
(M.888) 1.219” (kO.321)
0.670 (~.lll) 1.255d* (f0.684)
0.534 (ti.053) 1.055” (ti.420)
PGE, 1.25 pm01
11
4 hour Control
4
LGE, 1.25 ~01
11
, ,862”
b, c. e
Evans Blue content of injected regions, and the same areas of noninjected contralateral hemispheres at 4 and 24 h following injection of control solution, LGE, or PGl$ ‘Number of animals. “indicates value for region was significantly > that for same region in brain injected with control solution; b indicates that value for the region was significantly > than same region in brain injected with LGE, 10 mM; c indicates that the value for the region was significantly > that for same region of brain injected with PGE, 25 mM; d indicates that the value for the injected hemisphere was significantly > that for noninjected hemisphere; c indicates that the value at 24 h is significantly > the value at 4 h for same region injected with the same solution. *indicates p value of 0.04 for one-tailed t-test, p of 0.08 for two-tailed t-test; all other p values were co.05 for two-tailed t-test.
Evans Blue in the contralateral hemisphere is probably explained by the tendency of the extravasated proteinrich fluid to follow white matter tracts such as the corpus callosum (36). We were unable to demonstrate an increase in brain water, despite the obvious tissue damage. It is possible that brain water had already increased and then was decreasing by 4 h, when the first determinations of brain water were made, whereas the Evans Blue extravasation was slower to develop, and to resolve. During reperfusion of severely ischemic gerbil brain, edema, as judged by specific gravity, increases to a maximum after about 1 h of reperfusion and then decreases, whereas extravasation of Evans Blue increases steadily in parallel with the concentration of PGE, generated during reperfusion over 3 h (27). Alternatively, it is possible that this measure of brain edema is merely less sensitive than histologic evaluation of Evans Blue extravasation. Table 2
Percent Hz0 N+
Injected Mean (+ SD)
Non-injected Mean (&SD)
24 hour Control
14
LGEz 0.5 ~01
21
LGE, 1.25 ~01
11
PG& 1.25 pm01
11
79.886d (? 1.567) 79.616d (f 1.847) 80.023d (fl.199) 79.307d (_+I,905)
79.121 (* 1.548) 78.7381 (_+1.664) 78.669 (kO.995) 78.878 (_+1.805)
4 hour Control
4
LG& 1.25 ~01
11
79.890” (fi.422) 79.573d (kO.859)
79.180 (+o. 105) 79.056 (f0.761)
Percent water determinations for injected regions, and the same areas of noninjected contralateral hemisphere at 4 and 24h. Superscripts as in legend for Table 1. The only statistical significance was as noted.
Brain Tissue Injury and Blood-Brain
The histologic changes observed were nonspecific but, in general, quite similar to those caused by injection of CZo14and other PUFAs in somewhat larger doses (2.5 pmol) and solution volumes (500 pL) into cerebral hemisphere (34) or ventricle (37). The increase in BBB permeability evoked by the higher dose of LGE? was proportionally greater than that evoked by C2,,+ The effects of Cz0:4 injection on BBB permeability were assayed using the radioactive serum albumin technique, rather than the Evans Blue technique, nonetheless, the amount of Evans Blue extravasation caused by intrahemispheric injection of 1.25 pm01 of LGE-,, was nearly 5 times that induced by intrahemispheric injection of control solution. This is comparatively greater than the 3- to 3.5fold increase in radioactive serum albumin space, from roughly 2% in animals injected with buffer control, to approximately 6.7% in animals injected with 2.5 pm01 of CZti4 (34). Given the negative results obtained by Chan and Fishman (38) for PGE2 with the in vitro brain slice model, it is surprising that PGE, had an effect following intrahemispheric injection. It is possible that higher local concentrations of PGE2 were responsible; in their in vitro incubation system, Chan and Fishman used 0.5 mM concentrations of PGs and PUFAs. BBB permeability. of course, could not be assessed in vitro. To summarize, the experiments reported establish that LGE,, injected in amounts ranging from 100 nmol to 2.5 pm01 total dose, is capable of causing brain tissue necrosis and, in doses greater than 500 nmol, breakdown of the BBB as manifested by extravasation of Evans Blue. It is well established that in cerebral ischemia free fatty acids, in particular the PUFAs, accumulate rapidly. The most striking increases occur for C& (17-20). With recirculation and resupply of oxygen, there is prompt synthesis of both cyclooxygenase and lipoxygenase products, and a sharp decline in free CZ0:4concentration. Yet eicosanoid synthesis can account for only a small percentage of the total CR4 released during ischemia. Studies of cyclooxygenase and lipoxygenase products in ischemic/reperfused brain generally find concentrations in the ng/gm range (21-23, 30, 40), whereas the PUFAs are released in amounts up to hundreds of ,ug/gm of wet weight tissue ( 17-20). Undoubtedly, reacylation, and possibly peroxidative damage, also account for some of the decrease in FFAs which occurs upon reperfusion (33). It is plausible, although as yet unproven, that a tiny fraction of the massive quantities of C,,:, released during severe ischemia is converted to LGs during the burst of cyclooxygenase-promoted oxidative metabolism that occurs in the reperfused brain, and that this contributes to tissue necrosis, edema, and increased vascular permeability. Several factors may suppress enzymatic conversions of PGH? and consequently favor rearrangement to LGs. For example, it is possible the superoxide radical, which may be a byproduct of cyclooxygenase activity, acts to inhibit PG12 synthetase. Similarly, high concentrations of Co+ which are typical during &hernia,
Barrier Opening Induced by Injection of LGE, or PGEz
109
inhibit PGE isomerase (42) and therefore make it more likely that PGHZ will yield other end products including the LGs. Since the extravasation of Evans Blue, indicative of the breakdown of the BBB, coincides with PGE, accumulation during reperfusion through severely ischemic tissue, it was postulated that PGE, may cause egress of Evans Blue by increasing vascular permeability (27). Our demonstration that LGE, is more potent than PGE, in causing breakdown of the BBB when injected intrahemispherically provides presumptive evidence that LGs may also contribute to BBB damage associated with repefusion through severely ischemic tissue. Since the LGs must be produced from PGH2, complete inhibition of cyclooxygenase should prevent their production, as well as that of the other PGs and TXs. Observations on the effects of cyclooxygenase inhibitors such as indomethacin and aspirin on cerebral ischemia have been contradictory, with some studies showing protection (22, 40) but others failing to do so (41). However, it appears that the effects of these inhibitors depend on the severity of the ischemia. Thus, inhibition of the cyclooxygenase pathway by indomethacin does not affect edema formation upon reperfusion after moderate ischemia but does after severe ischemia (43). Further definition of the role, if any, of LGs in ischemic injury to the central nervous system will depend on detecting them in ischemic or ischemic/reperfused tissues; this, in turn, will depend on development of a relatively sensitive assay. The development of an immunoassay for this purpose has not, thus far, been possible, probably because of the tendency of the LGs to covalently bind proteins. The exact mechanisms whereby the LGs contribute to tissue damage will, of course, need extensive further investigation. One potential mechanism, which also explains the difficulty with detecting them in vitro by usual chemical methods, is their remarkable tendency to covalently bind to, and crosslink proteins. The potential role of protein cross-linking agents in the damage associated with cerebral ischemia has already been extensively discussed in the context of free radical mechanism (41), but may apply equally to the damage caused by the LGs. We believe that the results reported in this paper represent an encouraging first step in the investigation of the properties of these newly discovered eicosanoid products.
Acknowledgement This research was supported and NS22524 (to JWS).
by NIH grants GM21249-12
(to RGS)
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