Neuronal mdr-1 gene expression after experimental focal hypoxia: A new obstacle for neuroprotection?

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Abstract

Neuronal damage after stroke-associated brain hypoxia is a leading cause of long-term disability and death. The refractoriness to therapeutic strategies for neuroprotection after 3 h post brain ischemia is poorly understood. P-glycoprotein (P-gp), the multidrug resistance gene (MDR-1) product is normally expressed at blood–brain-barrier. P-gp neuronal expression has been demonstrated in refractory epilepsy and after brain ischemia. In this report we investigated the hypoxia-induced neuronal P-gp expression after local injection of CoCl2 (1–200 mM) in the fronto-parietal cortex of male adult rats (Bregma − 1.30 mm) by stereotaxic surgery. P-gp immunostaining of brain slides was analyzed using specific monoclonal antibodies and double immunolabeling was done with specific astrocytic and neuronal markers. Five days after injection of 1 mM CoCl2, P-gp expression surrounding the lesion site was observed in neurons, astrocytic end-foot on capillary blood vessels and endothelial cells on blood vessels. Higher CoCl2 doses (200 mM) resulted in additional P-gp immunostaining of the entire astrocytic and neuronal cytoplasm. Electron microscopy (EM) studies showed alterations in neurons as early as 6 h after the CoCl2 injection. P-gp expression in hypoxic neurons and astrocytic end-foot could potentially impair of drugs access to the brain parenchyma thus suggesting the presence of two P-gp-based pumping systems (one in astrocytes and other in the hypoxic neurons) that are able to behave as a previously unnoticed obstacle for pharmacological strategies of neuroprotection.

Introduction

Stroke is the second leading cause of death and the first leading cause of serious long term disability [1], [2]. In normal conditions, neurons sustain neurotransmission and prevent that calcium and other ions of reaching toxic levels by using active transport to regulate the internal and external electrolyte milieu. The acute ischemia induces molecular time-dependent cascade events, initiated by decreased energy production and finally inducing neuronal death. The excitotoxicity due to over-stimulation of glutamate receptors, excessive intraneuronal accumulation of sodium, chloride, and calcium ions and the mitochondrial injury are the main players in the neuronal death [1], [3], [4].

Current medical strategies are focused on the blood flow restoration to recover normal oxygen supply as soon as possible after ischemia. Neuroprotective experimental strategies based on the interruption or slowdown of the mentioned cell-damage or death-cascade have been tested in clinical studies without success. Thus, at present the unique available treatment in humans is the tPA therapy related with the first stratagem, which is only recommended if it is initiated within 3 h after the onset of symptoms. Neuroprotection is not observed after this very short therapeutic window. This lack of effectiveness could be related with the biological consequences for brain, and particularly for neurons exposed longer time than 3 h to low or null oxygen level [5], [6], [7].

The ischemic penumbra surrounding the core is an area of reduced perfusion in which cells are still viable. These cells are subjected to various active processes in which several mechanisms are involved in their demise or their survival. Spontaneous reperfusion usually occurs after cerebral ischemia [8] and it may reverse the ischemic damage when occurring early enough (e.g., transient ischemic attacks) in concordance with the mentioned therapeutic window. However, it usually takes place at a much later time points, when most penumbral cells are already suffering the secondary damage [3]. These changes on behavior and phenotype of still alive brain parenchyma could be related with the lack of neuroprotective effects of the drugs.

In concert with this wide spectrum of biochemical modifications, and additionally to the brain–blood barrier (BBB) restrictive function, brain parenchyma cells can develop, upregulate or overexpress several selective and/or nonselective mechanisms that are able to prevent the intracellular drug accumulation [9], [10].

P-glycoprotein (P-gp), a well-known member of the ATP binding cassette transporters superfamily, is the product of the multiple drug resistance 1 (MDR-1) gene. P-gp is a transmembrane ATP-dependent efflux pump of cytotoxic compounds that confers refractory phenotype to expressive cells [11]. P-gp is normally expressed on the apical luminal surface of secretor cells in the small intestine, the colon and the proximal tubules of the kidney. In normal brain, P-glycoprotein is expressed in the endothelial cells of the BBB [12] supporting the concept of a protective role for MDR-1-type proteins in the removal of potentially toxic xeno- and endobiotics. We have previously demonstrated that, secondary to seizures or ischemic stress in rats, brain expression of P-gp is upregulated from normally non-expressive cells as astrocytes and neurons [13], [14], [15].

Several reports indicate that cobalt chloride (CoCl2) stabilizes or induce the accumulation of hypoxia-induced factor 1α (HIF-1α) [16], [17], a transcription factor that up-regulates several genes [18] including the mdr-1 gene which produces the P-gp protein [19]. CoCl2 has been widely used as a hypoxia-mimicking agent in both in vivo and in vitro studies [20], [21]. Cobalt is essential for human health because of its critical role in the synthesis of vitamin B12 [22], however, excess exposure of cobalt can lead to tissue and cellular toxicity [23].

Our hypothesis is that persistent hypoxic stress (as the one induced by CoCl2 intracerebral administration) could trigger several molecular and signaling pathways and upregulate P-gp expression. In accordance with another report on retinal degeneration by CoCl2 [23], we have shown that the intracortical injection of CoCl2 induces a localized brain damage similar to that found in other models of focal ischemia [24].

Since CoCl2 stabilizes active the form of HIF-1α [25], and mdr-1 gene is sensitive to HIF-1α, we investigated the pattern of P-gp expression in cerebral cortex after the injection of CoCl2 in the brain parenchyma.

Section snippets

Materials and methods

Mouse monoclonal anti-MDR1 (P-gp, clone C494, Signet), polyclonal rabbit anti GFAP (Glial Fibrillary Acidic Protein, Dako) and polyclonal rabbit anti NSE (Neuron Specific Enolase, Sigma) were used. Secondary biotinylated antibodies and streptavidin peroxidase complex (Extravidin) for immunohistochemistry studies were purchased from Sigma (USA). Fluorescent Fab2 anti-mouse or anti-rabbit were purchased from Immunotech (France). All other chemical substances were of analytical grade.

Results

The protocol for the CoCl2 injection used in this study resulted in a minimal physical disruption of the vasculature overlying part of the prefrontal cortex. CoCl2 induced a necrotic infarct restricted to the injection site and a penumbra area was observed in the surrounding tissue. The depth of the lesions was constant and controlled being the needle track easily observed both in the animals injected with saline solution or in those injected with CoCl2. The effect of the CoCl2 injection was

Discussion

Hypoxia response triggers the activation of the transcription factor HIF-1α leading to up-regulation of genes that are expressed in most cell types, such as those encoding glucose transporters, glycolytic enzymes, and vascular endothelial growth factor, as well as genes that are expressed in a cell type-specific manner, such as erythropoietin, inducible nitric oxide synthase, and insulin-like growth factor II [29].

The hypoxia responsive pathway is also specifically stimulated by exposure to CoCl

Acknowledgements

Supported by grants UBACYT M-072, CONICET PIP5034 (to A.B.) and PIP6063 (to A.J.R). A.J.R. and A.B. are researchers from CONICET (Argentina). We thank Mrs. Emerita Jorge Vilela de Bianchieri for her expert technical assistance with the EM studies, Dr. C. Gibson (Leicester University, UK) for the TTC protocol and Dr. Juana M. Pasquini and Dr. Laura Pasquini for the Olympus UV microscope.

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    Present address: Department of Cell Biology and Neuroscience, University of California Riverside, USA.

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