Neuroprotective effects of erythropoietin in the rat hippocampus after pilocarpine-induced status epilepticus
Introduction
Erythropoietin (Epo) was originally described for its role in hematopoiesis, which consists of increasing red blood cells (Jelkmann, 1992) by protecting erythroid progenitors against apoptosis (Ghezzi and Brines, 2004). Epo and its receptor (Epo-R) are expressed in rodent and human brain, in cultured neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells (Marti et al., 1996, Juul et al., 1999, Chin et al., 2000, Siren et al., 2001, Hasselblatt et al., 2006), which has led to studies of additional biological roles of Epo. Exogenous administration of Epo revealed neuroprotective activity in vitro and in vivo in models of central and peripheral neuronal injury occurring in the contexts of trauma, stroke and inflammation (Juul et al., 2004, Campana and Myers, 2003, Maiese et al., 2004, Brines and Cerami, 2005).
Recombinant human Epo (rHuEpo)-administered peripherally crosses the blood–brain barrier (BBB) (Brines et al., 2000, Ehrenreich et al., 2004, Juul et al., 2004, Gorio et al., 2005). Studies in rodent models of temporal lobe epilepsy (TLE) revealed significant effects of rHuEpo in antagonizing the development of status epilepticus (SE), but did not determine whether rHuEpo was neuroprotective (Brines et al., 2000, Uzum et al., 2006). Interestingly, many of the programmed cell death pathways involved in animal models of TLE (Henshall and Simon, 2005) are those targeted by Epo (Maiese et al., 2004, Brines and Cerami, 2005).
Possible adverse effects of rHuEpo, such as increase in blood pressure, thrombosis, tumor expansion, and mortality (Maiese et al., 2004, Brines and Cerami, 2005) have spurred the development of Epo derivatives that are neuroprotective, but not erythropoietic (Leist et al., 2004). Carbamylated Epo (CEPO), which is one such derivatives, has a low affinity for Epo-R homodimer compared to native Epo, and is neuroprotective in vivo in the neocortex (Leist et al., 2004) and the spinal cord (Brines et al., 2004, Leist et al., 2004). Although it has been suggested that Epo-mediated neuroprotection occurs via Epo-R homodimers (Maiese et al., 2005), CEPO- and rHuEpo-mediated neuroprotection in the spinal cord may involve a heteroreceptor complex, consisting of a single Epo-R and a homodimer of the common β chain (βc) of the Granulocyte–Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin-3 (IL-3) and IL-5 receptors, as demonstrated in âc-knockout mice, which express normal levels of Epo-R (Brines et al., 2004, Genc et al., 2004, Brines and Cerami, 2005). Thus, available data suggest that several Epo binding sites may mediate neuroprotection, either through Epo-R homodimers or Epo-R/βc heteroreceptors.
Although exogenous Epo appears to be neuroprotective, little is know about the expression patterns of Epo system constituents under basal and induced conditions. Epo system expression has been investigated after cerebral ischemia (Bernaudin et al., 1999) and traumatic injury in the spinal cord (Grasso et al., 2005) and peripheral nervous system (Campana and Myers, 2001, Li et al., 2005). However, Epo system expression has not yet been assessed after chemically-induced SE, which initiates the process of epileptogenesis (Turski et al., 1983).
In this study, we have investigated the expression of Epo system constituents (Epo, Epo-R and the βc) both in basal conditions and in response to pilocarpine-induced SE (Pilo-SE). Our results show that Epo-R is expressed constitutively by most neurons of the hippocampus, but rarely by astrocytes. Following Pilo-SE, we report the transient induction of Epo in hippocampal astrocytes and a long-lasting induction of Epo-R. We also report that constitutive expression of βc subunit is low in the hippocampus compared to the spinal cord, whereas βc subunit expression dramatically increases after Pilo-SE in reactive microglia. Finally, we demonstrate that administration of rHuEpo following Pilo-SE significantly protects hippocampal neurons, suggesting that rHuEpo may be both neuroprotective and anti-epileptogenic.
Section snippets
Procedures and methods
All animal procedures were in compliance with the guidelines of the European Union (directive 86/609), taken in the French law (decree 87/848) regulating animal experimentation. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Animals
Sprague–Dawley male rats (Harlan, France) were used throughout the experiments. They arrived at 5 weeks old in approved facilities, and housed at 21 ± 1°C under diurnal lighting conditions (lights on from 06:00 to 18:00). They were maintained in groups of 5 in plastic cages with free access to food and water. After 2-week acclimatization, rats (180–200 g) underwent Pilo-SE as described below, and were then housed individually to support recovery until sacrificed. Control rats were housed in
Reverse Transcriptase real time Polymerase Chain Reaction (RT-real time PCR)
Total RNAs were extracted with Tri-reagent LS (Euromedex) and genomic DNA was removed after DNase I digestion (RNAse Free DNAse Set, Qiagen). After column purification (RNeasy kit, Qiagen) and prior to reverse transcription, total RNA from all samples were shown to be free of genomic DNA contamination by a PCR amplification of the exon V of the gene encoding brain-derived neurotrophic factor (BDNF) (see below for details). Messenger RNAs, contained in 500 ng of hippocampal total RNAs, were then
Basal expression and distribution of both Epo-R and Epo in the hippocampus
In control rats, 983 ± 133 copies of Epo-R cDNA and 224 ± 55 copies of Epo cDNA were quantified by real time PCR following reverse transcription of 500 ng of hippocampal total RNA.
Colorimetric immunolabeling showed that Epo-R was expressed in the principal cell layers of the dentate gyrus and Ammon’s horn (Fig. 1A). Dual fluorescent labeling of Epo-R together with specific markers of either neurons (NeuN), astrocytes (GFAP) or microglial cells (OX-42) demonstrated that Epo-R was expressed primarily
Discussion
The original findings of this study include description of Epo, Epo-R, and common β chain (βc) expression in the rat hippocampus under basal and induced conditions. Epo-R protein is constitutively expressed in a large majority of neurons, while Epo and βc protein expression are much more discrete, and restricted to specific neuronal populations. Few astrocytes constitutively express Epo and Epo-R in every area of the hippocampus. Following Pilo-SE, Epo and Epo-R are induced in numerous
Acknowledgments
This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS) and the Université Claude Bernard Lyon 1 (UCBL1). We thank B. Smatti, D. Ressnikoff and Y. Tourneur from the Centre Commun de Quantimétrie (CCQ) of the UCBL1 for their excellent technical assistance in confocal microscopy studies.
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