Impairment of dentate gyrus neuronal progenitor cell differentiation in a mouse model of temporal lobe epilepsy

Abstract

Unilateral intrahippocampal injection of kainic acid (KA) in adult mice induces an epileptic focus replicating major histopathological features of temporal lobe epilepsy (TLE). In this model, neurogenesis is impaired in the lesioned dentate gyrus, although cell proliferation transiently is increased bilaterally in the subgranular zone (SGZ). To investigate further the relationship between epileptogenesis and neurogenesis, we compared the differentiation of cells born shortly before and after KA injection. Immunohistochemical staining for doublecortin and PSA-NCAM, two markers of young neurons, revealed a rapid downregulation of both markers ipsilaterally, whereas they were increased transiently on the contralateral side. To determine whether KA treatment directly affects neural progenitors in the SGZ, dividing cells were prelabeled with 5′-bromo-2′deoxyuridine (BrdU) treatment before unilateral injection of KA. Double staining with the proliferation marker PCNA showed that prelabeled BrdU cells survived KA exposure and proliferated bilaterally. Unexpectedly, the neuronal differentiation of these cells, as assessed after 2 weeks with doublecortin and NeuN triple-staining, occurred to the same extent as on the contralateral side. Only 5% of pre-labeled BrdU cells were GFAP-positive within the lesion. Therefore, SGZ progenitor cells committed to a neuronal phenotype before KA treatment complete their differentiation despite the rapid down-regulation of doublecortin and PSA-NCAM. These findings suggest impaired fate commitment and/or early differentiation of proliferating cells in the lesioned dentate gyrus. Loss of neurogenesis in this TLE model likely reflects an irreversible alteration of the SGZ germinal niche during development of the epileptic focus and may therefore be relevant for human TLE.

Introduction

Temporal lobe epilepsy with hippocampal sclerosis (TLE-HS) is one of the most common forms of medically refractory epilepsies. Histopathologically, it is characterized by profound neuronal loss in CA1, CA3 and the hilus and a strong reactive astrogliosis (Babb and Najm, 2001). In almost 50% of the patients, the dentate gyrus (DG) is affected by dispersion and partial to severe loss of granule cells (Blumcke et al., 2002). These alterations are accompanied by profound reorganization of neuronal circuits underlying the generation of recurrent seizures. The demonstration that neurogenesis persists in the subgranular zone (SGZ) of human adult hippocampus (Eriksson et al., 1998) and the fact that multipotent progenitor cells can be isolated from resected tissue from epileptic patients (Moe et al., 2005) provide a strong stimulus to investigate whether neurogenesis is related to the pathophysiology of TLE.

Recent studies in patients with TLE-HS reported that cell proliferation (mitotic activity of progenitor cells) is markedly increased throughout the hippocampal formation, in particular in relation to granule cell dispersion (Thom et al., 2002, Crespel et al., 2005). Indirect evidence for neurogenesis (the process by which newborn cells differentiate into neurons) has also been suggested in a study of pediatric specimens with severe seizures (Blumcke et al., 2002). However, other studies observed decreased number of cells positive for polysialylated neural cell-adhesion molecule (PSA-NCAM), a marker of newborn neurons, in both adult and pediatric patients (Mathern et al., 2002, Pirttila et al., 2005). In animal models of TLE, it is well established that induction of acute seizures transiently increases the rate of neurogenesis in the SGZ (Bengzon et al., 1997, Parent et al., 1997, Gray and Sundstrom, 1998, Scott et al., 2000, Hattiangady et al., 2004, Jessberger et al., 2005). A major observation was the abnormal migration of some newly born granule cells into the CA3 area, where they form aberrant connections contributing to increased excitability of hippocampal networks (Scharfman et al., 2000). It remains unsettled, however, whether neurogenesis contributes to epileptogenesis and whether it is sustained in animals experiencing chronic recurrent seizures (Cha et al., 2004, Hattiangady et al., 2004, Mohapel et al., 2004, Jessberger et al., 2005).

Several factors have been identified that might interfere with neurogenesis in TLE. Stem and precursor cells in the SGZ rely on a neurogenic microenvironment and on specific cell–cell interactions that might be disrupted in epileptic tissue (Seki, 2003). Furthermore, inflammation, which often accompanies seizure-related brain injury, can be detrimental for neurogenesis (Ekdahl et al., 2003, Monje et al., 2003). Finally, the severity of the lesion induced upon prolonged experimental status epilepticus negatively influences long-term survival of newborn cells (Mohapel et al., 2004).

We have shown recently in a mouse model of TLE, characterized by a pronounced hypertrophy and dispersion of DG granule cells, that neurogenesis is suppressed selectively in the lesioned area, but not in the contralateral, unaffected hippocampus (Kralic et al., 2005). In this model, an epileptic focus develops unilaterally following intrahippocampal injection of kainic acid (KA) in adult mice, leading to chronic spontaneous recurrent seizures (SRS). KA treatment increased cell proliferation in the SGZ bilaterally. However, the vast majority of newly born cells in the injected DG were astrocytes when examined 4 weeks later. Thus, the neurogenic potential of the SGZ might be disrupted upon development of an epileptic focus. It is unclear, however, whether this effect is secondary to KA toxicity or whether it reflects an alteration of the germinal niche in the lesioned DG.

Here, we investigate further the relationship between epileptogenesis and neurogenesis in the KA mouse model of TLE. Cell proliferation and differentiation during 2 weeks following intrahippocampal KA injection were characterized immunohistochemically with proliferating cell nuclear antigen (PCNA), a marker of actively dividing cells, doublecortin (DCX) and PSA-NCAM, two markers of young neurons, and glial fibrillary acidic protein (GFAP), which labels astrocytes and radial glia/stem cells residing in the SGZ. Furthermore, cells proliferating in the SGZ shortly before KA administration were prelabeled with BrdU and their fate was monitored during the subsequent 2 weeks to determine how and when neurogenesis declines during development of the epileptic focus.