Changes in reactive oxygen species (ROS) production in rat brain during global perinatal asphyxia: an ESR study

doi:10.1016/S0006-8993(01)02781-0

Brain Research

Volume 914, Issues 1–2, 28 September 2001, Pages 204-207

https://doi.org/10.1016/S0006-8993(01)02781-0 Get rights and content

Abstract

A large body of evidence suggests that the production of reactive oxygen species (ROS) can play an important role in ischemic neuronal injury. However any studies has been performed in hypoxic conditions. In the present experiments we studied using electron spin resonance (ESR) techniques the ROS release in neostriatum of newborn rats subjected to acute perinatal asphyxia (PA) followed by various periods of reoxygenation. Pregnant rats’ uteri still containing foetuses were taken out and subjected to PA by immersion in a 37°C water bath during the following periods of time: 5, 10, 15, 19 and 20 min. After performing PA, animals were recovered and ROS measured after 0, 5, 15, 30 or 60 min of reoxygenation. Then, pups were sacrificed, their neostriatum removed and homogenised with N-tert.-butyl-α-phenylnitrone (PBN) and diethylenetriamine-pentacetic acid (DPTA) in phosphate-buffered saline (PBS) and the formed complexes were extracted with ethyl acetate an analysed using an X-band ESR spectrometer. A significant release of ROS was detected at 19 and 20 min of PA after 5 min of reoxygenation. These data provide strong evidence that ROS could be involved in neuronal damage during PA.

Section snippets

Induction of asphyxia

Forty-four Sprague–Dawley pregnant rats were anaesthetised on the last day of gestation with an intraperitoneal injection of 28% (w/v) chloral hydrate (0.1 ml/100 g body weight) (Merck). Uterus horns, containing the foetuses were taken out by hysterectomy, detached and placed in a water bath at 37°C for 5, 10, 15, 19 or 20 min of PA. Following PA, the uterus horns were rapidly opened and the pups removed and stimulated to breathe on a heating pad by cleaning off the delivery fluids and by

ROS detection

At the end of reoxygenation the animals were decapitated, and the neostriatum was removed, weighed and homogenized in 300 μl of a spin trap solution containing 100 mM of N-tert.-butyl-α-phenylnitrone (PBN) and 2 mM diethylenetriamine-pentacetic acid (DPTA) in phosphate-buffered saline (PBS). After homogenization, 500 μl of ethyl acetate were added, vortexed for 30 s and centrifuged at 5000 rpm 2 min for phase separation. The ethylacetate phase was transferred to a flat quartz cell for ESR study

Statistical methods

Data from the experimental and control groups were statistically evaluated using the Instat/PC program (Graft Software, 1993). The height of central peak of ESR signals was measured. Mean and standard deviation (SD) were calculated at each time interval. One-way analysis of variance (ANOVA) and subsequently the Newman–Keuls test was used to analyse the differences in mean and SD between different times of PA and control groups, considering significant a P value less than 0.05.

Results

Because the survival drops to less of 3% after 20 min of PA [10] we could not obtain any animal to study this point of the PA time course. Controls, 5, 10 and 15 min of PA failed to produce an ESR signal at any reoxygenation interval (0–60 min) in all of the animals analysed. ESR signal began to be evident in neostriatal samples after 19 min PA with 5 min of reoxygenation (Fig. 1) reaching the maximum ESR signal at 20 min PA (Fig. 1). Statistical study comparing these two groups showed that the

Acknowledgements

This study was supported by grants from the Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET), and from the Universidad de Buenos Aires (UBA).

References (21)

F. Capani et al.
Ultrastructural changes in nitric oxide synthase immunoreactivity in the brain of rats subjected to perinatal asphyxia: neuroprotective effects of cold treatment
Brain Res.
(1997)
U. Dirnagl et al.
Pathobiology of ischaemic stroke: an integrated view
Trends Neurosci.
(1999)
J. Garthwaite
Glutamate, nitric oxide and cell–cell signalling in the nervous system
Trends Neurosci.
(1991)
H. Hara et al.
Mechanism and pathogenesis of ischemia-induced neuronal damage
Prog. Neurobiol.
(1993)
H.A. Kontos
Oxygen radicals in CNS damage
Chem. Biol. Interact.
(1989)
C.F. Loidl et al.
Long-term effects on basal ganglia neurotransmitter systems studied with microdialysis in rat
Neurosci. Lett.
(1994)
C.F. Loidl et al.
Hypothermia during or after severe perinatral asphyxia prevents increase in cyclic-related nitric oxide levels in the newborn rat striatum
Brain Res.
(1998)
J.M. McCord et al.
Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex
FEBS Lett.
(1978)
J.W. Phillis et al.
Oxypurinol attenuates hydroxyl radical production during ischemia/reperfusion injury of the rat cerebral cortex: and ESR study
Brain Res.
(1993)
J.W. Phillis
A ‘radical’ view of cerebral ischemic injury
Prog. Neurobiol.
(1994)

There are more references available in the full text version of this article.

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Short communicationChanges in reactive oxygen species (ROS) production in rat brain during global perinatal asphyxia: an ESR study

Abstract

Section snippets

Induction of asphyxia

ROS detection

Statistical methods

Results

Acknowledgements

Brain Res.

Trends Neurosci.

Trends Neurosci.

Prog. Neurobiol.

Chem. Biol. Interact.

Neurosci. Lett.

Brain Res.

FEBS Lett.

Brain Res.

Prog. Neurobiol.

Short communication
Changes in reactive oxygen species (ROS) production in rat brain during global perinatal asphyxia: an ESR study