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Neurochemical Research, Vol. 30, No. 5, May 2005 ( 2005), pp.

625631
DOI: 10.1007/s11064-005-2750-9

Catalase Activity and Thiobarbituric Acid Reactive


Substances (TBARS) Production in a Rat Model of Diuse
Axonal Injury. Eect of Gadolinium and Amiloride
Alejandro Santos,1,2,4 Nuno Borges,2 Antonio Cerejo,3 Antonio Sarmento,1
and Isabel Azevedo1
(Accepted April 21, 2005)

This study evaluated the eect of mechanogated membrane ion channel blockers on brain
catalase (CAT) activity and thiobarbituric acid reactive substances (TBARS) production after
traumatic brain injury (TBI). A weight drop trauma model was used. Controls were shamoperated and received no weight drop. Gadolinium (GAD) or amiloride (AMI) were
administered to control and experimental rats (30 min after TBI). Brain CAT activity and
TBARS production were measured. When blood vessels were washed out with saline perfusion
brain CAT activity signicantly increased up to 6 h after trauma, decreasing signicantly by
24 h; GAD or AMI administration preserved CAT activity 24 h after TBI. TBARS production
increased after trauma, this eect being signicantly reversed by GAD or AMI administration.
GAD signicantly decreased TBARS production in control animals. Mechanogated membrane
ion channels may be involved in the genesis of the ionic disruption leading to oxidative stress and
other secondary injury processes in head trauma.
KEY WORDS: Head trauma; rat; catalase; TBARS; mechanogated membrane ion channel blockers.

In the United States, Australia, France and Spain the


incidence of death from head injury is reported as 20
30 per 100,000, whereas in the United Kingdom the
rate is approximately two thirds lower at 7 per
100,000 (3,4). This major health problem has no
current eective pharmacological interventions, most
eort being focused on supportive measures (5).
Traumatic injury to the central nervous system is
regarded as the initial step in a series of biochemical
and physiopathological events that may have as a
consequence irreversible tissue damage. Several factors are involved in this secondary injury process
including ion changes, excitatory amino acids release,
formation of reactive oxygen species and metabolic
energy perturbations (6,7).
Data obtained with animal models of traumatic brain injury (TBI) show that high levels of

INTRODUCTION
Traumatic injuries are the leading cause of mortality
in individuals aged 144 years, and brain injury signicantly contributes to the outcome in nearly one-half of all
deaths from trauma. Most survivors of severe injury are
permanently disabled to a lesser or greater degree (1,2).

Servico de Bioqu mica (U38-FCT) Faculdade de Medicina,


Universidade do Porto, 4200-319, Porto, Portugal.
Faculdade de Ciencias da Nutricao e Alimentacao da Universidade do Porto, 4200-465, Porto, Portugal.
Servico de Neurocirurgia, Hospital de S. Joao, Alameda Prof.
Hernani Monteiro, 4200, Porto.
Address reprint requests to: Alejandro Santos, Servico de
Bioqu mica (U38-FCT) Faculdade de Medicina. Universidade
do Porto. 4200-319 Porto, Portugal. Tel: 351-22-5508838; Fax:
351-22-5508838; E-mail: alejandr@med.up.pt

625
0364-3190/05/05000625/0 2005 Springer Science+Business Media, Inc.

626
excitatory amino acids (EEA), through the activation of N-methyl-D-aspartate (NMDA) receptor/
ion channels (811), increase intracellular Ca2+
concentration. Maintenance of Ca2+ homeostasis is
critical after injury to avoid the triggering of potentially harmful biochemical and metabolic cascades
(12). After brain injury, reactive oxygen and nitrogen
species may be generated through several dierent
cellular pathways, including Ca2+ activation of
phospholipases, nitric oxide synthase, xanthine oxidase, and the Fenton and HaberWeiss reactions by
inammatory cells (13).
During trauma the brain is submitted to
mechanical deformations resulting in diffuse axonal
injury (DAI). This is believed to be the most common
pathology associated with TBI (14) and the main
cause for a poor clinical outcome in the absence of
detectable intracranial lesions (15).
In 1994, Marmarou and co-workers developed
an experimental head injury model in the rodent
capable of producing diffuse brain injury without
focal lesions (16,17).
Recent research suggests that traumatic deformation of axons is responsible for triggering abnormal ionic inuxes through several channels. There is
evidence to support that some of the channels activated after trauma are mechanosensitive (18,19).
Mechanosensitive ion channels are ion channels
whose gating can be altered by mechanical forces.
Mechanical stress is subsequently transformed into
an electrical or chemical response (20). Gadolinium
(GAD) is considered, with amiloride (AMI) and
gentamicin, a relatively selective blocker of mechanosensitive membrane ion channels (21).
Lipid peroxidation in brain leads to the production of substances with biological activity (e.g. isoprostanes). This phenomenon occurs to such an extent
that the capability to control lipid peroxidation and
minimize the biological effects of its by-products
seems to be an important route for TBI treatment
(22). Thiobarbituric acid reactive substances
(TBARS) determination has been used as a marker of
lipid peroxidation in rat brain homogenates (23). The
abnormal production of oxygen derived free radicals
such as superoxide, hydroxyl, and reduced O2 intermediate hydrogen peroxide (H2O2) has been presented as an important step in the chain of events
between cerebral blood ow reduction and neuronal
damage, mainly in the reperfusion period. Catalase
(CAT) and glutathione peroxidase, acting in concert
with superoxide dismutase, constitute the major defense enzymes against superoxide radicals (24,25).

Santos, Borges, Cerejo, Sarmento, and Azevedo

Considering the role of post-traumatic intracellular ionic disruption in the generation of free radicals,
we proposed to investigate the effects of GAD and
AMI administration after TBI on brain CAT activity
and TBARS production.

EXPERIMENTAL PROCEDURE
Diuse Axonal Injury Model. The Marmarous rat closed head
trauma model was used (17). Briey, 340360 g male Wistar rats
(Harlan, Barcelona, Spain), kept under standard laboratory conditions, were anaesthetised i.p. with a solution containing diazepam
(6 mg kg)1, Bialzepam, Bial, Portugal), ketamine (60 mg kg)1;
Ketalar, Pzer (Parke-Davis, Portugal) and atropine sulphate
(0.5 mg kg)1, B. Braun, Portugal), and allowed to breathe spontaneously. After a midline incision in the scalp, a metallic disc was
xed to the skull surface with dental cement. The animal was
placed in a prone position on a foam bed and a 450 g weight was
dropped through a Plexiglas tube from a 2-m height. The rat body
temperature was controlled until recovery from anaesthesia by a
thermostatic heating pad coupled to a rectal thermal probe. In an
initial set of experiments non-perfused rat brains were analysed as
to CAT activity 24 h after TBI; these tissues were placed in ice cold
saline while the external visible vessels were removed. In additional
experiments the animals were anaesthetised 1, 6, 12 or 24 h after
trauma, a cold saline perfusion was performed to wash out the
remaining blood and the brains were carefully removed. All samples were run immediately or stored at )80 C until further
processing.
All procedures were carried out in accordance with EU
guidelines for animal experiments.
Catalase Activity. CAT activity was evaluated in whole brain
homogenates. Tissue samples were homogenized in an ice-cold
isotonic 0.01 mol/l sodium phosphate buer (pH 7.4) and centrifuged for 5 min at 12,000 g at 4 C. Enzyme activity was assayed in
the supernatant by the method of Aebi, which involves monitoring
the disappearance of hydrogen peroxide (H2O2) at 240 nm (26).
Measurements were performed in triplicate. Protein concentrations were estimated by the Bradford method (27). CAT activity
was calculated as units per milligram of protein and expressed as
percent of control.
Gadolinium Treatment. Thirty minutes after trauma gadolinium treated rats (TBIGAD) were administered gadopentetate
dimeglumine (GAD; 70 mg kg)1 i.v.; Magnevist, Schering Lusitana, Portugal). The GAD treated saline perfused samples for CAT
activity were collected 6 h (TBIGAD 6 h) or 24 h (TBIGAD 24 h)
after trauma and non-perfused GAD treated samples were collected 24 h after trauma (TBIGAD). The non-treated traumatised
groups received saline i.v. (TBI). Control groups (CONT) were
sham operated and received no weight drop. Gadolinium control
groups (CONTGAD) were sham operated, received no weight drop
but were administered GAD (70 mg kg)1 i.v.).
Amiloride Treatment. Thirty minutes after trauma amiloride
treated rats (TBIAMI) were administered amiloride (AMI;
20 mg kg)1 i.p.; Sigma, Spain). The AMI treated saline perfused
samples for CAT activity were collected 6 h (TBIAMI 6 h) or 24 h
(TBIAMI 24 h) after trauma and non-perfused AMI treated samples were collected 24 h after trauma (TBIAMI). The non-treated
traumatised groups received saline i.p. (TBI). Control groups

Catalase Activity in Head Trauma


(CONT) were sham operated and received no weight drop. Amiloride control groups (CONTAMI) were sham operated, received
no weight drop but were administered AMI (20 mg kg)1 i.p.).
TBARS Determination. Lipid peroxidation in brain homogenates was determined by the formation of thiobarbituric acidreactive substances (TBARS) as described by Buege and Aust
(1978) (28). TBARS determination was performed 24 h after
trauma in animals perfused with cold saline.
Statistical Analysis. Statistical analysis of the results was
performed using the NewmanKeuls test. The dierences were
recognized as statistically signicant when P<0.05.

RESULTS
Brain CAT activity values in non-perfused animals (meanSEM) are presented in Fig. 1 (expressed
as percentage of control). TBI increased CAT activity, this eect being reversed by GAD or AMI
administered thirty minutes after TBI. Both compounds showed no signicant eect on CAT activity
when administered to non-traumatised control rats.
CAT activity values in perfused animals
(meanSEM) are presented in Fig. 2 (expressed as
percentage of control). Trauma signicantly increased
CAT activity up to 6 h after TBI, this eect being not
reversed by GAD or AMI administered 30 min after
TBI. Twelve hours after trauma CAT activity had
declined to values not dierent from control values and
by 24 h it was signicantly lower than control values; in
animals administered GAD or AMI 30 min after TBI
brain CAT activity 24 h after trauma was not dierent
from that of control animals.
Thiobarbituric acid reactive substances (TBARS)
values (meanSEM), expressed as nmol malondialdehyde equivalents/g brain wet weight, are presented
in Fig. 3. TBI signicantly increased TBARS production, this eect being reversed by GAD or AMI
administered 30 min after TBI. GAD reduced TBARS
production in non-traumatised control animals. AMI
showed no signicant eect on TBARS production
when administered to non-traumatised control rats.
DISCUSSION
In mammalian cells, peroxides are continuously
generated during aerobic metabolism. The continuous
detoxication of peroxides is essential to prevent the
oxidation of cellular components by peroxides or peroxide derived reactive oxygen species. The brain seems to
be more susceptible to oxidative damage than other organs. The reasons for that lie on a great content of highly
peroxidizable fatty acids, on the fact that cells of this

627
organ use 20% of the oxygen consumed by the body
although they constitute only 2% of the body weight and
on a relative scarcity of protective antioxidant enzymes
(24,29). The respiratory chain of mitochondria is the
main source of intracellular superoxide. Several
antioxidant enzymes constitute the primary line of
defence against intracellularly generated reactive
oxygen species. Superoxide dismutase (SOD) catalyses the dismutation of superoxide radicals producing
hydrogen peroxide. The activity of this enzyme is one
of the most important defences against superoxide
radicals (30). The activity of SOD requires a concerted action with two other enzymes: glutathione
peroxidase (GSH-Px) and CAT, since they are the
only enzymes scavenging hydroperoxides (25).
It has been shown in animal models that the role
of oxygen radical damage is greatest immediately
after trauma. Wei and co-workers, using a model of
experimental uid percussion brain injury in cats,
showed that previous treatment with SOD and CAT
prevented the arteriolar loss of normal reactivity that
occurs immediately after trauma (31). Furthermore,
Muir and co-workers demonstrated that SOD improved the cerebral perfusion after uid percussion
brain injury in rats (32).
CAT is a peroxisomal diffusion controlled enzyme, being particularly effective when clearing high
concentrations of H2O2. The expression of CAT has
been demonstrated in all brain cell types in vitro (33)
and in vivo (34,35). This suggests an important
function in the H2O2 metabolism in this organ, in
spite of CAT activity in brain being two orders of
magnitude lower than in kidney or liver (36).
In an initial set of experiments we evaluated
CAT activity 24 h after trauma. A marked increase in
activity that could be prevented by GAD or AMI
administration was observed. Gadolinium or AMI
administration to control animals had no signicant
effect on the activity of the enzyme.
We later found out that these values didnt reect the enzyme activity of brain tissue. The variation
in CAT activity observed in these animals was
probably related with the brain hyperaemia produced
by TBI. As these animals had not been perfused with
saline, a signicant amount of erythrocyte CAT was
probably present in brain vessels. As CAT activity in
blood is several orders of magnitude higher than that
in brain tissue, hyperaemia can have a dramatic effect
on total activity in non-perfused brain. As a matter of
fact, an increase in cerebral blood ow after trauma
has been described in experimental and clinical settings (37,38). Most interestingly, in our experimental

628

Santos, Borges, Cerejo, Sarmento, and Azevedo

Brain Catalase Activity


24 h after TBI
(non perfused animals)

450%

400%
350%
300%
250%

**

**

200%
150%
100%
50%
0%
Control

Control AMI

Control GAD

TBI

TBIGAD

TBIAMI

n=6

n=3

n=3

n=11

n=6

n=6

Fig. 1. Non-perfused animals brain catalase (CAT) activity expressed as percentage of control (meanSEM). TBI signicantly increased
CAT activity (*, P<0.05, NewmanKeuls test), this eect being reversed by GAD or AMI administered 30 min after TBI (**, P<0.05,
NewmanKeuls test). Both compounds showed no signicant eect on CAT activity when administered to non-traumatised control rats.

Brain Catalase activity


perfused animals
(% of control)
*

160%
140%
120%
**

100%
80%
60%
40%
20%
0%
ControlD

TBI 1 h

TBI 6 h

TBIGA 6 h

TBIAMI 6h

TBI 12 h

TBI 24 h

TBIGAD 24 h TBIAMI 24 h

Fig. 2. Saline perfused animals brain catalase (CAT) activity values expressed as percentage of control (meanSEM). TBI signicantly
increased CAT activity up to 6 h after trauma (*, P<0.05, NewmanKeuls test), this eect being not reversed by GAD or AMI administered
30 min after TBI (**,. 24 h after trauma CAT activity was signicantly lower than that of controls, P<0.05, NewmanKeuls test) this eect
being reversed by GAD or AMI administered 30 min after trauma. Both drugs showed no signicant eect on CAT activity when administered to non-traumatised control rats.

conditions GAD or AMI administration after trauma


prevented this increase in CAT activity.
All subsequent experiments included a saline
perfusion operation before collecting tissue to evaluate brain CAT activity. Brain tissue was collected 1,
6, 12 and 24 h after trauma. The results showed a
signicant increase in CAT activity up to 6 h, a decline to control values by 12 h and by 24 h activity it
was signicantly lower than control. GAD or AMI
treatment had apparently no effect on the increase in
CAT activity 6 h after TBI. However, both drugs
were able to prevent the drop in CAT activity 24 h
after TBI. We hypothesize that the decrease in CAT
activity observed 24 h after TBI is probably due to
peroxide-dependent autoinactivation of CAT by
conversion of the active CAT complex I into the

inactive complexes II or III as described in cultured


astrocytes (39).
We also know from the results of Hoffman and
co-workers that astrocyte lipid peroxidation generates isoprostanes in response to trauma or oxygen
radicals (22).
The rationale for our work was based on the
hypothesis that these drugs would act by blocking the
mechanogated membrane ion channels, attenuating
the intracellular ionic disruption that occurs after
trauma. This in turn could prevent (at least partially)
the cascade of metabolic events leading to oxidative
damage. This is also the reason for administering
these drugs as soon as possible. Additionally previous
results from our group showed that these drugs could
prevent some events related to secondary injury after

Catalase Activity in Head Trauma

629

TBARS
24 h after TBI
(perfused animals)

nmol/ g brain wet weight

35

*
30
25

**
20

**

15
10
5
0

Control

n=6

Control AMI Control GAD

n=3

n=3

TBI

n=11

TBI GAD

n=6

TBI AMI

n=6

Fig. 3. Thiobarbituric acid reactive substances (TBARS) values expressed as nmol malondialdehyde equivalents/g brain wet weight
(meanSEM). TBI signicantly increased TBARS production measured 24 h after trauma (*, P<0.05, NewmanKeuls test), this eect being
reversed by GAD or AMI administered thirty minutes after TBI (**, P<0.05, NewmanKeuls test). GAD reduced TBARS production in
non-traumatised control animals (#, P<0.05, NewmanKeuls test). AMI showed no signicant eect on TBARS production when administered to non-traumatised control rats.

brain trauma, namely mitochondrial swelling and


blood brain barrier disruption (40,41).
Many signal transduction pathways depend on
Ca2+, and cells must have a tight regulation of its
intracellular levels. Several intracellular organelles
and proteins bind Ca2+, thus buering free Ca2+
levels or triggering second-messenger pathways.
Chronic activation of NMDA receptors by glutamate
is toxic to cultured neurons. The extensive Ca2+ entry accompanying receptor activation is largely
accumulated by mitochondria, with resultant eects
in mitochondrial membrane potential, ATP synthesis,
glycolysis, reactive oxygen species generation and
ultimately failure of cytoplasmic Ca2+ homeostasis
and cell death (42).
However, the disruption in ionic homeostasis
after TBI may also be due to other factors. In fact,
recent research suggests that traumatic deformation
of axons is responsible for an abnormal inux of
sodium via mechanically sensitive Na+ channels.
This subsequently triggers an increase in intracellular
Ca2+ through opening of voltage sensitive Ca2+
channels (VGCC) and reversal of the Na+Ca2+
exchanger (19).
Other authors, working with rat sensory neurons, concluded that mechanical stimulation induced
a Ca2+ inux through mechanosensitive Ca2+
channels. This increase was blocked by gadolinium
(18), a substance considered, with AMI and gentamicin, as a relatively selective blocker of mechanosensitve cation channels (21).

We also found that GAD or AMI administration 30 min after TBI signicantly lowered TBARS
production in comparison with non-treated TBI
animals. Whereas amiloride administration to control
animals didnt change TBAR production in comparison with non-treated controls, GAD signicantly
decreased TBAR in control, i.e. non-traumatised
animals.
Zhong and co-workers produced a possible
explanation for the higher effect of GAD in the
reduction of TBAR production. These authors, using
a model of liver reperfusion injury, described that the
administration of gadolinium chloride (GdCl3, 20mg/kg-body weight) to control animals signicantly
reduced malondialdehyde production (TBARS
method) versus non-treated controls. This eect was
attributed to the fact that GdCl3 is a selective inhibitor of Kuper cells (43). When activated these cells
release toxic mediators that eventually lead to lipid
peroxidation (44). In the central nervous system,
microglia are considered as resident macrophages
that under normal conditions display a limited
phagocytotic or endocytotic activity (45).
It is known that TBI triggers inammatory and
immune responses including the activation of microglia and the recruitment of circulating phagocytes.
This inammatory/immune response is thought to be
a repair mechanism of injured tissue. However an
excessive response can produce further damage
through, e.g. phagocyte derived radical oxygen and
nitrogen species (46). Considering all this data we can

630
argue that the more pronounced decrease in TBARS
production observed in GAD treated animals may be
due to both the inhibition of macrophages and the
attenuation of the ionic disruption after trauma.

Santos, Borges, Cerejo, Sarmento, and Azevedo

10.

11.

CONCLUSIONS
12.

Our results indicate that mechanosensitive ion


channel blockers prevent hyperaemia, decrease lipid
peroxidation and preserve CAT activity after TBI.
This is suggestive of the involvement of mechanogated
membrane ion channels in the genesis of the ionic
disruption that leads to oxidative stress and other
secondary injury processes after trauma. Furthermore,
it is noteworthy that if normalisation of CAT activity
and TBAR production by MMICB represents a benecial effect on brain trauma, our results demonstrate
that this pharmacological approach may be useful in a
30 min time window after trauma.

13.
14.

15.
16.

17.

18.

ACKNOWLEDGMENT
Supported by FCT and Programa Ciencia, Tecnologia e
Inovacao do Quadro Comunitario de Apoio (POCTI and
FEDER).

19.

20.
21.

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