Study of the oxidative stress in a rat model of chronic brain hypoperfusion

Abstract

A multiple analysis of the cerebral oxidative stress was performed on a physiological model of dementia accomplished by three-vessel occlusion in aged rats. The forward rate constant of creatine kinase, k_for, was studied by saturation transfer ³¹P magnetic resonance spectroscopy in adult and aged rat brain during chronic hypoperfusion. In addition, free radicals in aging rat brain homogenates before and/or after occlusion were investigated by spin-trapping electron paramagnetic resonance spectroscopy (EPR). Finally, biochemical measurements of oxidative phosphorylation parameters in the above physiological model were performed. The significant reduction of k_for in rat brain compared to controls 2 and 10 weeks after occlusion indicates a disorder in brain energy metabolism. This result is consistent with the decrease of the coefficient of oxidative phosphorylation (ADP:O), and the oxidative phosphorylation rate measured in vitro on brain mitochondria. The EPR study showed a significant increase of the ascorbyl free radical concentration in this animal model. Application of α-phenyl-N-tert-butylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin traps revealed formation of highly reactive hydroxyl radical (OH) trapped in DMSO as the CH₃ adduct. It was concluded that the ascorbate as a major antioxidant in brain seems to be useful in monitoring chronic cerebral hypoperfusion.

Introduction

For more than 30 years, Alzheimer's disease (AD) has been classified and managed as a neurodegenerative disorder. However, it was recently proposed that sporadic (nongenetic) AD is a vascular disease (de la Torre, 2002a, de la Torre, 2002b, review, Pratico and Delanty, 2000). This conclusion is based on common overlap of clinical AD and cognitive symptoms of vascular dementia. It is well known, that AD is heterogeneous and multifactorial nature, likely resulting from diverse presence or vascular risk factors or indicators of vascular disease.

In the present work a chronic pathophysiological animal model of dementia was used (Kašparová et al., 2000) for investigation of oxidative stress by means of two physical techniques—phosphorus-31 magnetic resonance spectroscopy (³¹P MRS) and electron paramagnetic resonance (EPR) spectroscopy. We supposed that the characteristic pathology of AD involves microvascular degeneration and chronic cerebrovascular hypoperfusion as has recently been reported (de la Torre, 1999, de la Torre, 2002a, de la Torre, 2002b). For an animal model of AD in rats it is proposed that two factors must be present before cognitive dysfunction and neurodegeneration is expressed in the AD brain: advanced aging and presence of a condition that lowers cerebral perfusion (de la Torre, 1999, de la Torre and Stefano, 2000). The model involves subjecting animals—aged rats to chronic cerebrovascular hypoperfusion for 2–10 or more weeks. With respect to reduced cerebral blood flow, it should be noted that prolonged brain ischemia could produce β-amyloid peptid precursor (APP) that was found increased in the hippocampus of AD brains, and which has been implicated in formation of senile plaques. A β-amyloid peptid-associated free radical model for neuronal death in AD brain has evolved from many observations. In this model, β-amyloid peptid-associated free radicals initiate lipid peroxidation and protein oxidation (Butterfield et al., 1999, review; Arivazhagan et al., 2002, Behl and Moosmann, 2002, Kaufmann et al., 2002). Thus, we supposed that EPR technique could be useful in investigation of free radical production in the above animal model of chronic cerebral hypoperfusion. Direct neurotoxicity of β-amyloid peptid can be related to its ability to associate with plasma membrane (Mattson et al., 1992), to induce Ca²⁺ influx and disrupt cell membrane functions (Mattson et al., 1992), to induce lipid peroxidation, to lead to protein oxidation (Bruce-Keller et al., 1998), to enhance glutamate toxicity (Mattson et al., 1992) and, in addition, to decrease the activity of several oxidative sensitive enzymes, including creatine kinase (Butterflied, 1997). Creatine kinase (CK) plays a central role in energy transfer in cells with highly energy flux or requirements and it is highly susceptible to oxidative inactivation (McCord and Russell, 1988). Dysfunction of the creatine kinase system under AD conditions has also recently been reported (Yatin et al., 1999, Askenov et al., 2000; David et al., 1998; Burbaeva et al., 1999, review). The causes of lower brain isoform creatine kinase (BB-CK) levels in the cell cytosol of the postmortem brain in mental pathology are discussed (Burbaeva et al., 1999, review). BB-CK, a member of the CK gene family, is a predominantly cytosolic CK isoform in the brain and plays a key role in regulation of the ATP level in neural cells (Askenov et al., 2000). It can be expected that activity of the creatine kinase reaction in the brain in vivo is significantly changed under condition of the above animal model of dementia or during oxidative stress. Therefore, we studied reaction kinetics of a reversible exchange of the phosphate group in the creatine kinase reaction catalyzed by CK:

PCr²⁻ + MgADP⁻ + H⁺ ⇔ MgATP²⁻ + Cr

in the adult and aged rat brains under conditions of severe hypoperfusion, using saturation transfer in vivo ³¹P MRS technique (Mlynárik et al., 1998, Kašparová et al., 2000).

CK in brain exists in multiple forms, and the total enzyme activity in brain is high enough to ensure near equilibrium of the CK reaction, hence the CK flux is comparable to the ATP consumption (Erecinska and Silver, 1989). Thus, if the brain CK reaction is inhibited during modeled hypoperfusion and the ATP turnover correlates with the flux through the CK reaction, then we should be able to monitor cerebral effects of chronic hypoperfusion or oxidative stress in the rats by ³¹P MRS saturation transfer experiments in vivo.

The purpose of our studies was to elucidate a relationship between the pseudo-first order rate constant, k_for, of the CK forward reaction (PCr ⇒ ATP) in aged rat brain under conditions of severe chronic cerebrovasular hypoperfusion, and the degree of the oxidative damage of brain cells via free radicals production monitored by means of the EPR spectroscopy. Therefore, after ³¹P MRS measurements, the rat brains were subjected to biochemical analysis focused on mitochondrial oxidative phosphorylation parameters, and the data were also correlated with the free radicals EPR investigations. In AD, a possible mechanism, by which impaired electron transport chain function leads to cell death is a decrease in production of ATP, and an increase in production of free radicals or reactive oxygen species (Beal, 1995, Butterflied, 1997). Isoenzymes CK can be inactivated by hydrogen peroxide and by superoxide (Suzuki et al., 1992). No data relating CK system and free radicals production are presently available for animal model of dementia.