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.
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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].
In summary, the results reported in the present experiments show that aging is associated with an increase in
arachidonic acid metabolites, TXA2 and PGI2, and with a
reduction in TXA2/PGI2 ratio in 24 month old animals,
evoked by an increase in PGI2 that may contribute to protect
the brain against neuronal damage.
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