Elsevier

Brain Research

Volume 816, Issue 1, 16 January 1999, Pages 124-130
Brain Research

Research report
Exogenous creatine delays anoxic depolarization and protects from hypoxic damage: dose–effect relationship

https://doi.org/10.1016/S0006-8993(98)01131-7Get rights and content

Abstract

Incubation of hippocampal slices with different concentrations of creatine (0.5, 1, 10, 25 mM) results in a dose-dependent increase in intracellular phosphocreatine (PCr). Electrophysiological evidence suggests that this effect can protect neurons from anoxic damage by delaying the depletion of ATP during oxygen deprivation. In this paper we show that incubation of brain slices with varying doses of creatine increases intracellular phosphocreatine and delays anoxic depolarization (AD) in a dose-dependent way. Specifically, addition to the incubation medium of 1 mM creatine significantly increased AD latency during hypoxia and prevented irreversible neuronal damage. Adding 0.5 mM creatine had no significant effect. Higher concentrations of creatine (up to 25 mM) did not provide any better protection. Our data also suggest a linear correlation between intracellular PCr and AD latency. These data report neural protection by exogenous creatine at concentrations lower than those usually reported in the literature.

Introduction

Incubating brain slices with high-concentration (25 mM) creatine increases the intracellular store of phosphocreatine (PCr) [9]. This is a high-energy compound that can phosphorylate ADP to ATP, thus replenishing the latter's stores even in the absence of oxygen. Accordingly, exogenous creatine in high concentration (25 mM) has been shown to protect in vitro brain slices from hypoxic damage [9]. Data from our laboratory have also confirmed that 25 mM exogenous creatine in brain slices delays anoxic depolarization (AD), an event that probably contributes to hypoxic damage [2]. At variance with brain slices data, one of us has reported that exogenous creatine does not protect neuronal cultures from hypoxic damage [7]. This negative finding could be easily explained by the immaturity (perinatal age) of neuronal cultures, in which creatine-kinase is not fully expressed [12], thus likely preventing the transformation of creatine to phosphocreatine. Very recently, increasing the dietary intake of exogenous creatine has been shown to protect from brain lesions induced by a metabolic inhibitor in vivo [13], a finding that could not be easily predicted because exogenous creatine does not increase intracellular stores of phosphocreatine in normal rats 6, 13. Taken together, these findings suggest that exogenous creatine, a treatment that has not been so far fully investigated, may be a useful tool in brain protection against hypoxic or ischemic damage. They also point to questions that are still unanswered about this promising treatment, such as the dose–effect relationship and the mechanism of protection. In the present study we tried to address some of those questions. Specifically, we (1) looked for confirmation of creatine efficacy in preventing irreversible damage, (2) investigated the efficacy of lower doses of creatine in such prevention as gauged by neurophysiological parameters, and (3) tried to correlate delay of AD with phosphocreatine content to asses whether or not this damaging event may be caused by ATP depletion.

Section snippets

Slices preparation and incubation

This followed previous published work from our laboratory [2]. Sprague–Dawley female rats (155–190 g) were anesthetized with chloroform and decapitated. The left hippocampus was dissected free and cut in 600-μm-thick transversal slices. This thickness was chosen because it provides an amount of tissue sufficient to allow optimal extracellular electrical recordings (personal observations). All dissection procedures were done under ice-cold artificial cerebrospinal fluid (ACSF, see below). Slices

Results

In a first set of experiments we could correlate creatine treatment with protection against hypoxic damage, as gauged by either one of the two electrophysiological parameters we used. In treated slices recovery of CA1 population spike after hypoxia was observed (Fig. 1) significantly more often than in controls (Fig. 2). Concerning the dose-dependency of this effect, 0.5 mM creatine did not improve the recovery rate, while 1 mM caused a recovery indistinguishable from that of higher

Discussion

Our results show protection by creatine against hypoxic damage. This finding confirms similar observations previously made in brain slices 2, 9, 10and in vivo [13]and indirectly supports the idea that previous negative results in neuronal cultures [7]may be due to the limited expression of the creatine-phospho-kinase enzyme in neurons of perinatal age [12]. In the brain slice study that we report, exogenous creatine dose-dependently increased the intracellular store of phosphocreatine (Fig. 6)

Acknowledgements

We thank Dr. Aroldo Cupello and Mrs. Paola Asselle for their help in measuring tissue proteins. This work was partially funded by grant BIO4-97-2307 from the Commission of the European Communities, DG XII, and by a grant from the University of Genova for the scientific training of R.R.

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