Neurobiology of Aging 20 (1999) 695– 697 Influence of aging on thromboxane A2 and prostacyclin levels in rat hippocampal brain slices૾ F. Marmola,*, P. Puig-Parelladaa, J. Sancheza, R. Trullasb a Unitat de Farmacologia.IDIBAPS.(Institut d’Investigacions Biomediques August Pi I Sunyer) Facultat de Medicina, Universitat de Barcelona, ` Casanova 143, 08036 Barcelona, Spain b Unitat de Neurobiologia, IIBB, CSIC/IDIBAPS, Barcelona, Spain Received 20 April 1999; received in revised form 10 August 1999; accepted 30 August 1999 Abstract We have investigated the influence of age (3, 18, 24 months) on Thromboxane A2 (TXA2) and Prostacyclin (PGI2) levels in hippocampal slices from F344/NHSD rats. A significant increase in TXA2 and PGI2 levels was observed in 18 and 24 months old compared to 3 months old animals. A significant reduction in the ratio TXA2/PGI2 produced by a higher increase in PGI2 was observed in 24 month old animals. The reduction in the TXA2/PGI2 ratio has been related to vasodilatory and antiaggregating effects that may contribute to protect the brain against neuronal damage. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Aging; Thromboxane; Prostacyclin; Hippocampus 1. Introduction A massive release of fatty acids and specially of arachidonic acid from membrane phospholipids has been associated with different acute cerebral pathologies; among them transitory ischemia, hypoglucemia, convulsions and trauma [1]. However, it is not known whether the normal process of aging is associated with alterations of arachidonic acid metabolism. In the present experiments, we have investigated modulation of the levels of arachidonic acid metabolites, Thromboxane (TXA2) and Prostacyclin (PGI2), in the hippocampus during aging. Arachidonic acid metabolites are produced in brain by both neurons and glia [14]. The functional roles of TXA2 and PGI2 in brain have been mainly related to their effects on brain microcirculation and aging has been associated with both morphological and functional changes of the vascular endothelium [5]. TXA2 induces vasoconstriction and platelet aggregation [15] which may cause a postischemic neuronal injury [9]. PGI2 has opposite effects on microvessel and platelets [15] and may ૾ Supported by CICYT grant SAF98 – 0063, Ministerio de Educacion ´ of Spain. * Corresponding author. Tel.: ϩ34-934-024-526; fax: ϩ34-934-035260. E-mail address: ppuig@medicina.ub.es (P. Puig-Parellada) provide neuroprotection in brain ischemia [11]. However the final effect (vasodilatation or vasoconstriction) depends rather on changes in the ratio of TXA2 to PGI2 than on their absolute levels. We now report that aging is associated with a reduction in the TXA2/PGI2 ratio in hippocampal slices from F344/ NHSD rats. 2. Methods The animals used in this study were male F344/NHSD RB rats from the National Institute on Aging (USA) of 3, 18, and 24 months of age. Rats were killed by decapitation, the brains removed and hippocampal tissue dissected on ice. Tissues were cross-chopped into 350 ϫ 350 m slices and plunged into Krebs buffer solution, pH 7.4. (mM: NaCl 117.7, KCl 4.69, CaCl2, 2.47, KH2PO4, 1.16, MgSO4, 1.18, glucose 9.9, NaHCO3, 24.3) continuously gassed with 95% O2 and 5% CO2 mixture and maintained at 37°C. The suspension was washed several times with Krebs buffer. Assays were performed in triplicate. Aliquots (0.5 mL) were incubated, at 37°C in a shaking water bath, for 10 min, with Krebs buffer. To stop the reaction, the tubes were placed immediately in a boiling bath for 10 min, cooled to 4°C and centrifuged (1500ϫ g for 15 min). The supernatant was stored at Ϫ20°C in the presence of indomethacin (200 M). 0197-4580/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 7 - 4 5 8 0 ( 9 9 ) 0 0 0 6 9 - X 696 F. Marmol et al. / Neurobiology of Aging 20 (1999) 695– 697 Fig. 1. (a) Levels of TXA2 and PGI2 in hippocampal brain slices from F-344 male rats of 3, 18 and 24 months of age. For TXA2 the number of animals (n) was 3 months old ϭ 20, 18 months old ϭ 8 and 24 months old ϭ 8. For PGI2, n was 3 months old ϭ 17, 18 months old ϭ 8 and 24 months old ϭ 6. Results are expressed as pg/mg protein. Each value represents mean Ϯ SEM. ***p Ͻ 0.001 versus 3 month-old rats. (b) Ratio of TXA2/PGI2 levels in hippocampal brain slices from F-344 male rats of 3, 18 and 24 months of age. The number of animals was 3 months old ϭ 16, 18 months old ϭ 8 and 24 months old ϭ 6. Each value represents mean Ϯ S.E.M. **p Ͻ 0.01 versus the other groups of age (3 and 18 month-old animals). Protein content was determined in the pellet in accordance with Lowry et al. [7]. Thromboxane B2 and 6-keto-PGF1a which are the stable products of TXA2 and PGI2 were determined from the supernatant using an enzyme immunoassay kit (Cayman Chemical). Data are expressed as mean Ϯ SEM. Comparisons F. Marmol et al. / Neurobiology of Aging 20 (1999) 695– 697 among groups were evaluated using one-way ANOVA and post-hoc comparisons using independent samples t tests. Animals were treated in compliance with national and international (European Economic Community Council Directive 86/609/EEC) laws and policies. 3. Results The effects of age on TXA2 levels in hippocampal slices are represented in Fig. 1a. A one-way ANOVA revealed a highly significant effect of age on TXA2 levels [F2.31 ϭ 16, p Ͻ 0.001]. A significant increase in TXA2 levels was observed, either at 18 months (p Ͻ 0.001) or at 24 months (p Ͻ 0.001) compared to animals of 3 months of age. The effects of age on PGI2 levels are presented in Fig. 1a. A one-way ANOVA revealed a highly significant effect of age on PGI2 levels [F2,28 ϭ 22, p Ͻ 0.001]. A significant increase in PGI2 was observed, either at 18 months (p Ͻ 0.001) or at 24 months (p Ͻ 0.001) compared to animals of 3 months of age. The relationship between TXA2 and PGI2 levels among the different age groups is represented in Fig. 1b. One-way ANOVA revealed a significant effect of age [F2,27 ϭ 8.7, p Ͻ 0.001]. The ratio TXA2 /PGI2 was significantly lower in hippocampal slices of 24 month animals when compared to both 3 month and 18 month groups (p Ͻ 0.01). 4. Discussion The present results show that there is a significant increase of TXA2 and PGI2 levels with age. Previous studies have shown that membrane arachidonic acid concentration is reduced in the hippocampus of 22 month old animals compared with a 4 month old group [8]. Likewise, carbachol-stimulated arachidonic acid release has been shown to be significantly reduced in synaptoneurosomes from aged brains [13]. These studies show that there is a decrease in brain membrane arachidonic acid levels with aging. Thus, the increase in TXA2 and PGI2 levels observed in hippocampal slices from 18 and 24 month old animals reported in the present studies can be explained by increased arachidonic acid metabolism rather than by enhanced arachidonic acid availability. Increases in TXA2 have been reported in neurodegenerative processes like ischemia, trauma, ischemia-reperfusion syndrome, etc. [1,2,10]. The increase in TXA2 and PGI2 observed in the present experiments suggests that hippocampal slices from aged animals exhibit a higher susceptibility to inflammatory stimuli [15]. However, in 24 months old animals there is a significant reduction in the ratio TXA2/PGI2, which is produced by a significantly higher increase in PGI2 in relation to TXA2. TXA2 reached a maximum increase at 18 months, but the maximum increase in PGI2 was not observed until 24 months. These results indicate that the activity of thromboxane synthase and prostacyclin synthase, the enzymes that metabolize prostaglandin H2 697 into TXA2 and PGI2 respectively, are differentially influenced by age. To our knowledge, this is the first indication of a maturational change in brain thromboxane synthase while prostacyclin synthase is also influenced by aging. The increase in PGI2, observed in very old animals, could represent a physiological mechanism to protect the brain against the neuronal damage produced by aging, since PGI2 is a potent vasodilator and antiaggregating agent. Physical exercise has also been shown to enhance PGI2 production [3,4,12]. It could be speculated that both aging and exercise may impose an oxidative stress to the organism [6]. 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