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(Stroke. 1996;27:514-519.)
© 1996 American Heart Association, Inc.

Articles

Platelet-Activating Factor in Brain Regions After Transient Ischemia in Gerbils

Keiji Nishida, MD, PhD S.P. Markey, PhD

From the Section on Analytical Biochemistry, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md.

Correspondence to Sanford P. Markey, PhD, Section on Analytical Biochemistry, Laboratory of Clinical Science, National Institute of Mental Health, Bldg 10, Room 3D40, 10 Center Dr MSC 1262, Bethesda, MD 20892-1262. E-mail s_markey@codon.nih.gov.

	Abstract

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Background and Purpose Platelet-activating factor (PAF) has been reported to be an active mediator in ischemic brain damage on the basis of indirect pharmacological data from PAF antagonists. The direct measurement of PAF in neuronal tissues has not been reported previously in analogous animal models. We have examined regional brain PAF concentration changes during the reperfusion period after ischemia in gerbils to obtain direct evidence for the involvement of PAF with ischemic brain damage and reported gas chromatography/mass spectrometry (GC/MS) methods of PAF quantitative analysis in brain tissues.

Methods After transient (10 minutes) ischemia followed by controlled periods (0 to 96 hours) of reperfusion and recovery, regional PAF concentrations were determined in gerbil brain tissue. Quantitative analysis of PAF in brain regions is performed using an electron-capture negative chemical ionization GC/MS method, modified for brain tissue.

Results The level of PAF was increased significantly and maximally in hippocampus (211%), cortex (168%), and thalamus (169%) after 1 hour of reperfusion. In contrast, there were no significant changes of PAF in any brain region from 6 hours to 96 hours after reperfusion.

Conclusions PAF is increased in gerbil brain in response to ischemia at early stages of reperfusion. PAF increases could contribute to the onset and progress of ischemic neuropathology.

Key Words: platelet-activating factor • spectrum analysis, mass • gerbils

	Introduction

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Platelet-activating factor is a powerful phospholipid mediator that has been associated with many acute pathological responses.^{1 2} Recently, it was demonstrated that PAF might contribute to the damage of the central nervous system after cerebral ischemia.² PAF decreases regional blood flow, increases blood-brain barrier permeability, activates neutrophils, and mediates vasoconstriction, so there are multiple mechanisms by which PAF may produce neural damage after cerebral ischemia. There is indirect evidence suggesting the involvement of PAF: administration of PAF antagonists reduces both the histological and functional damage after experimental cerebral ischemia.^{3 4} We examined regional brain PAF concentration changes during the reperfusion period after 10 minutes of bilateral carotid artery occlusion in gerbils to obtain direct evidence for the involvement of PAF and to determine the time course of PAF generation or concentration.

The direct measurement of PAF in neuronal tissues has not been reported previously in analogous animal models. We have modified GC/MS methods of PAF quantitative analysis^{5 6} for the present study. Measurements of PAF concentrations by bioassay or immunoassay methods lack the specificity and sensitivity required for quantitative regional brain concentration measurements.⁷ Furthermore, determination of PAF bioactivity is ambiguous because PAF homologues and analoguesexhibit variable PAF activities,⁸ and other lipid mediators present in tissue may influence platelet activation. The published GC/MS methods for PAF derive the required sensitivity by using ECNCI MS, and specificity for brain tissue is achieved in the present study with selective chromatographic separations.

	Materials and Methods

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Materials
DMAP, PFBz-Cl, and hydrofluoric acid were from Aldrich Chemical Co, and formic acid was from J.T. Baker Chemical Co. PAF, d₃ PAF, lyso-PAF, and d₄ lyso-PAF were from Biomol Research Laboratories Inc. Hexane and other chemicals of analytical grade were from Fisher Inc.

Animal Experiments
Eighty-nine female Mongolian gerbils (Meriones unguiculatus, Harlan Sprague-Dawley) weighing 50 to 60 g were used in these experiments. They were housed in Plexiglas cages for at least 7 days before study on a 12-hour dark/light cycle. Food and water were allowed ad libitum through the acclimatization and study periods. Eighty-four animals (all but 5 gerbils, which were used as controls) were anesthetized by inhalation of 2% halothane in 30% oxygen/70% nitrous oxide. A midline incision exposed both common carotid arteries. Bilateral common arteries were occluded for 10 minutes with Heifetz aneurysm clips.⁹ Anesthesia was discontinued at the same time as reperfusion was begun. The absence of carotid blood flow during the occlusion and the resumption of flow after removal of the clips were verified visually. Rectal temperature determined with a rectal probe (Harvard, Homeothermic System) was maintained at 37°C to 37.5°C in all animals with a thermostatic heating pad and infrared lamp during the occlusion. Rectal temperature in all animals was monitored before anesthesia, before and during ischemia, and up to 1 hour after discontinuation of anesthesia. Sham-operated animals were treated in the same manner except for occlusion of the bilateral common carotid arteries. Animal protocols were approved by the National Institute of Mental Health and National Institute of Neurological Disorders and Stroke animal care and use committees.

After the designated times of reperfusion, 53 ischemic animals and 31 sham-operated animals were decapitated. The brains were removed, and cerebral cortex, striatum, hippocampus, thalamus, and cerebellum were quickly put on ice. Immediately each prepared brain region was disrupted using a Polytron in 10 volumes of ethanol and water mixture (1:1 vol/vol) containing 0.18% formic acid for inactivation of endogenous acid-labile acetylhydrolase and 500 pg d₃ PAF and 50 ng d₄ lyso-PAF as internal standards for PAF and lyso-PAF assays. Homogenates were frozen at -80°C until analyzed.

PAF Extraction From Brain and Derivatization
Brain PAF and lyso-PAF extraction and derivatization conditions were modified from published methods as follows.^{5 6} Brain tissue homogenates containing d₃ PAF, d₄ lyso-PAF, and formic acid were centrifuged at 10 000g for 10 minutes at 4°C, and the supernatants from brain homogenates were applied to C-18–packed cartridges (100 mg C-18/1 mL, Varian Associates) previously conditioned with 5 mL methanol followed by 5 mL water. The cartridges were washed with 5 mL water followed by 5 mL methanol/water (1:1 vol/vol), and the PAF-containing fraction was eluted with 5 mL methanol. The methanol eluate was loaded onto a silica-packed cartridge (500 mg silica/2.8 mL, Varian), preconditioned with 5 mL ethanol, and then rinsed with 5 mL ethanol. PAF was eluted from silica-packed cartridges with 4 mL methanol/water (3:1 vol/vol), and that fraction was evaporated in a centrifugal vacuum dryer. The dry residue was hydrolyzed to diglyceride and extracted simultaneously by shaking with a mixture of 0.5 mL 49% hydrofluoric acid and 1 mL hexane for 3 hours at room temperature in a polyethylene tube. The hexane layer was evaporated to dryness under nitrogen stream, and the residue was derivatized with 25 µL 0.5% PFBz-Cl and 1% DMAP at 60°C for 15 minutes. The derivatized and dried diglyceride was redissolved in 30 µL decane admixed with 500 µL water. The top decane layer was transferred to injection vials for injection into the GC/MS instrument.

GC/MS of PAF and Lyso-PAF
Quantitative analyses of PAF and lyso-PAF were performed by ECNCI GC/MS as previously described^{5 6} with a Hewlett-Packard 5890 GC/5970 and 5989 quadrupole analyzer. Gas chromatography was performed on a 15-m DB-17 (J&W Scientific) capillary column of 0.25-mm ID and 0.25-µm film. The PFBz glycerides were analyzed by selected ion monitoring techniques to record ions specific for PAF (m/z, 552), d₃ PAF (m/z, 555), lyso-PAF (m/z, 704), and d₄ lyso-PAF (m/z, 708). The initial column temperature was 210°C (1 minute) and then was programmed to 235°C at 50°C/min. At this point, the temperature was increased to 243°C at 1°C/min and then to 300°C at 20°C/min. The injector temperature was 280°C, and the GC/MS interface line temperature was 310°C. Helium was used as a carrier gas, and 1 µL of the sample was injected in the splitless mode. The detection limit of the PAF assay was 25 pg per sample (ie, injection of 1 µL containing 1 pg) and that of lyso-PAF was 100 pg per sample.

Statistical Analyses
All values presented are mean±1 SEM. Results were analyzed by computerized statistical packages (SuperANOVA, Statview II). Each mean value was compared by one-way ANOVA and Fisher's protected least significant difference for multiple comparisons as the post hoc test. A value of P<.05 is reported as statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detection of PFBz Derivative of PAF
Selected ion chromatograms of hippocampal PAF and lyso-PAF extracts and corresponding procedural blanks are shown in Fig 1. The retention times of d₃ PAF (m/z, 555) and d₄ lyso-PAF are approximately 7.5 and 11.2 minutes, respectively, and these elute from the gas chromatograph approximately one scan before that of native PAF and lyso-PAF. The ion current signals for PAF (m/z, 552) and lyso-PAF (m/z, 704) from hippocampus (800 mg) of a sham-operated animal (1 hour after surgery) and of an ischemic animal (1 hour after reperfusion) are measurable and distinct from those of the procedural blanks. The abundance of PAF and lyso-PAF ion currents corresponds to 2.9 ng/g tissue and 0.87 µg/g tissue for the sham-operated and 6.0 ng/g tissue and 0.88 µg/g tissue for the ischemic animals, respectively.

View larger version (23K): [in this window] [in a new window]   Figure 1. ECNCI-selected ion monitoring chromatograms for PFBz derivatives of PAF (m/z, 552) and lyso-PAF (m/z, 704) and of d3 PAF (m/z, 555) and d4 lyso-PAF (m/z, 708) as internal standards are shown. The signal from sham-operated–animal (1 hour after sham operation) hippocampal PAF (a) and lyso-PAF (a) corresponds to approximately 2.9 ng/g tissue and 0.87 µg/g tissue, respectively; the signal from ischemic-animal (1 hour after ischemia) hippocampal PAF (b) and lyso-PAF (b) corresponds to approximately 6.0 ng/g tissue and 0.88 µg/g tissue, respectively, in contrast to that from the procedural blanks (c).

Effect of Transient Cerebral Ischemia on Regional Brain PAF and Lyso-PAF Concentrations
The regional brain PAF concentrations at 0, 15, and 30 minutes and 1, 3, 6, 24, 48, and 96 hours after 10 minutes of cerebral ischemia were investigated (Fig 2). At 30 minutes to 3 hours after ischemia, the concentrations of PAF were significantly increased in hippocampus and cortex and were maximal after 1 hour of reperfusion. Significant PAF concentration increases were observed 1 and 3 hours after reperfusion in thalamus also and after 3 hours in striatum. In contrast to significant increases of PAF concentrations acutely, there were no significant changes of PAF in any brain region from 6 hours to 96 hours after reperfusion. Notably, no significant changes in PAF concentration occurred at any time in the cerebellum, a region that does not become ischemic, because cerebellar blood supply was preserved in this model. In addition, no differences were observed between normal control gerbils and sham-operated gerbils in the designated times of reperfusion, whereas significant increases in PAF levels resulted from ischemic insult.

View larger version (18K): [in this window] [in a new window]   Figure 2. Time course of PAF changes in brain regions after reperfusion following 10 minutes of ischemia () or sham operation (). Each point represents the mean±SE for 5 to 7 ischemic animals and 3 to 4 sham-operated animals. C in the time scale indicates regional brain PAF levels of control gerbils. There is no significant difference in PAF levels for control gerbils compared with sham-operated gerbils at the different periods. *P<.05 compared with controls.

Lyso-PAF levels in hippocampus, cortex, thalamus, striatum, and cerebellum of control animals were 316-, 493-, 167-, 143-, and 279-fold higher than PAF levels in each brain region, respectively. At 1 hour after reperfusion, there was no significant difference in lyso-PAF levels between control and ischemic animals (Table), and these lyso-PAF levels did not change during the experimental period of reperfusion (data not shown).

View this table: [in this window] [in a new window]   Table 1. Lyso-PAF Levels in Gerbil Brain 1 Hour After Reperfusion Following 10 Minutes of Ischemia

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study reports PAF concentration changes in brain regions in gerbils submitted to 10 minutes of ischemia. It has been thought that PAF might contribute to the aggravation of ischemic brain injury on the basis of the neuroprotective effects of PAF antagonists in animal studies.^{4 10 11 12} It is known that 3.5-µmol/L concentrations of PAF produce neurodegeneration in experiments in vitro via increases of intracellular Ca²⁺ levels.¹³ Presumably, the amelioration of physiological,⁴ biochemical,^{4 10} behavioral,¹⁰ and histological^{11 12} changes in ischemic animals treated with PAF antagonists is due to inhibition of the pathological function of elevated PAF. In our studies, PAF concentration increases were observed in hippocampus, thalamus, and cortex at 1 and 3 hours after reperfusion, with the magnitude of the response as hippocampus>cortex>thalamus (Fig 2). In experimental ischemia models, pathological brain damage is localized principally in the hippocampus and cortex.¹⁴ Therefore, the degree of PAF elevation in each brain region is consistent with previously observed pathology. Because PAF stimulates production of tumor necrosis factor,^{15 16} interleukin-1, and leukotriene B₄ in macrophages,¹⁶ the increases in PAF may be correlated to inflammatory reactions occurring in the later phases of ischemia/reperfusion injury.

Arachidonic acid appears to play an important role in the regulation of PAF biosynthesis¹⁷ because PAF precursors contain more than 80% arachidonate at the C-2 position,¹⁸ and PAF synthesis is remarkably inhibited in arachidonic acid–depleted polymorphonuclear leukocytes.¹⁹ During occlusion, free arachidonic acid increases markedly in ischemic gerbil brain tissue, and then during reperfusion the level rapidly decreases.²⁰ Free arachidonic acid is metabolized through cyclooxygenase, lipoxygenase, or other metabolic pathways.²¹ Levels of cyclooxygenase products of arachidonic acid metabolism in brain are not changed during ischemia; however, these arachidonic acid metabolites increase rapidly during reperfusion (5 to 120 minutes).²² The relatively rapid production of PAF during the reperfusion period (1 to 3 hours) may be highly correlated to the rapid production of the arachidonic acid metabolites.

PAF is synthesized rapidly and locally by acetylation of lyso-PAF via activation of acetyltransferase. Lyso-PAF results from the activation of a specific phospholipase A₂ that removes the C-2–position fatty acid from alkylacyl-GPC.²³ The activation of these PAF synthetic enzymes depends on cell activation and requires the presence of Ca²⁺.¹ A high level of intracellular Ca²⁺ is a known trigger of ischemic neuronal cell death.^{24 25} Recently, the changes of intracellular Ca²⁺ levels in brain transient ischemic models have been reported. Silver and Erecinska²⁶ observed approximately 30-fold intracellular Ca²⁺ increases in rat hippocampus (CA1) soon after occlusion and during the immediate postischemic period, which normalized after 20 minutes to the control level. Greenberg et al²⁷ documented a similar intracellular Ca²⁺ change pattern in the cat transient ischemic model. PAF synthesis in the early stage of the reperfusion period (1 to 3 hours) can be stimulated markedly by influx of Ca²⁺, activating phospholipase A₂ and acetyltransferase by raising the intracellular Ca²⁺ concentration.²⁸ In contrast, 1-alkyl-2-acetyl-sn-glycerol phosphocholine transferase, which catalyzes the final step in the de novo route of PAF synthesis, is inhibited in vitro by Ca²⁺.²⁹ It is generally accepted that the remodeling route is stimulated by inflammatory agents but has low activity in resting cells, while the de novo route has highest activity in resting cells.³⁰ Thus, the production of PAF in the ischemic brain is expected to derive mainly from the remodeling route of PAF synthesis.

In stimulated cells, lyso-PAF is an obligatory intermediate for both biosynthesis and inactivation of PAF in its synthetic pathway. Lyso-PAF is a degradation product of PAF in both the de novo and remodeling routes but is a precursor only in the remodeling route. Lyso-PAF level changes in the brain as a precursor or a degradation product of PAF are likely after ischemic insult; however, the lyso-PAF pool is very large relative to PAF level changes, and no lyso-PAF concentration changes were detected in our studies (Table). In catabolic and anabolic pathways of lyso-PAF, hydrolysis of alkylacyl-GPC by phospholipase A₂, acetylation of lyso-PAF by acetyltransferase, and deacylation of PAF by acetylhydrolase are Ca²⁺-dependent, whereas reacylation of lyso-PAF by coenzyme A–dependent acyltransferase is inhibited by Ca²⁺.¹ Presumably, increased intracellular Ca²⁺ in ischemic brain affects the activities of those enzymes. However, the changes of those lyso-PAF metabolic enzymes are not sufficient to change the large lyso-PAF pool level.

The cellular origin of PAF synthesis after ischemia is not known. PAF is produced by appropriate stimulation in a variety of cells, including monocytes,³¹ macrophages,³² glial cells,³ and neurons.³³ Macrophage/monocyte infiltration is exhibited in areas of infarction 2 to 6 days after transient gerbil ischemia.³⁴ Moreover, although immunocytochemically the number of active microglia increased in rat hippocampus as early as 20 minutes after reperfusion, the strongest microglial reaction was observed 4 to 6 days after reperfusion of rat transient ischemia.³⁵ In our studies, significant PAF increases were not observed at 6 hours and later after ischemia, suggesting that the acute rise and subsequent normalization of PAF levels may not be due to trafficking macrophages, monocytes, or activated microglia. It is possible that PAF is synthesized by neurons; however, further studies are need to determine which cells are directly related to the rapid production of PAF after ischemic brain injury. Such questions may be explored in cell culture studies using the highly specific and sensitive GC/MS methods.

Although there have been questions raised in the literature as to whether PAF is related to the onset and progress of brain ischemic damage, the PAF changes in brain after ischemia have not been reported previously. Quantitative analysis of PAF with GC/MS using modifications of published procedures is feasible and permits accurate determinations in regional normal and ischemic gerbil brain. In this study, we show that there are significant PAF increases in the early reperfusion phase of ischemia by using GC/MS methods for PAF analysis and that the degree of regional increase in the brain was consistent with the vulnerability of each brain region for ischemic insult. Because the activities of PAF synthetic enzymes are regulated by Ca²⁺, rapid response of PAF production may be related to Ca²⁺ influx in the early reperfusion phase of ischemia.

	Selected Abbreviations and Acronyms

 

d3 = trideuterated d4 = tetradeuterated DMAP = dimethylaminopyridine ECNCI = electron-capture negative chemical ionization GC/MS = gas chromatography/mass spectrometry lyso-PAF = 1-O-hexadecyl-sn-glycero-3-phosphorylcholine PAF = platelet-activating factor; 1-O-hexadecyl-2-O-acetyl-sn-glycero-3-phosphorylcholine; alkylacetyl-GPC PFBz-Cl = pentafluorobenzoyl chloride

	Acknowledgments

 
We wish to thank Drs Hiroshi Abe and Thaddeus S. Nowak Jr in the Department of Neurology, University of Tennessee, Memphis, and Dr Kazuhiko Suyama in the Department of Neurosurgery, Nagasaki University (Japan), for helping us learn the surgical technique for transient ischemia in gerbils.

Received August 23, 1995; revision received December 5, 1995; accepted December 6, 1995.

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