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The bisphosphonate clodronate depletes microglial cells in excitotoxically injured organotypic hippocampal slice cultures

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Abstract

The bisphosphonate clodronate, clinically used in the treatment of osteoporosis, is known to deplete cells of the monocytic lineage. Using an in vitro model of excitotoxic damage in organotypic hippocampal slice cultures (OHSC), we investigated whether clodronate can also prevent microglial activation that occurs in CNS pathologies. Lesioning of OHSC was performed by application of 50 μM N-methyl-d-aspartate (NMDA) for 4 h after 6 days in vitro (div). Treatment of lesioned OHSC with clodronate (1000, 100, or 10 μg/ml) resulted in an almost complete abrogation of the microglial reaction after 3 further div: Confocal laser scanning microscopy showed that the number of Griffonia simplicifolia isolectin B4-labeled (IB4+) microglial cells in the dentate gyrus (DG) was reduced to 4.25% compared with OHSC treated with NMDA alone. Continuous treatment with clodronate (100 or 10 μg/ml) of lesioned OHSC for 9 days resulted in a further reduction in the number of microglial cells (reduction to 2.72%). The number of degenerating, propidium iodide-labeled (PI+) neurons in lesioned OHSC that received clodronate treatment between 6 and 9 div was not significantly different from OHSC treated with NMDA alone. However, the number of PI+ neurons in lesioned OHSC that received continuous clodronate treatment for 9 div was significantly higher when compared to NMDA-lesioned OHSC. In summary, clodronate is able to reduce microglial activation induced by excitotoxic neuronal injury. Our results demonstrate that clodronate is a useful tool in the investigation of neuron–glia interactions because it induces an efficient depletion of microglial cells that are activated after excitotoxic CNS injury.

Introduction

In the adult CNS microglial cells display a ramified morphology with numerous branched cellular processes and a relatively small soma 39, 45. These cells are considered “resting” cells with many characteristics of immunologically dormant tissue macrophages (Czapiga and Colton, 1999). Microglial cells quickly respond to all kinds of CNS pathologies and play an important role in diseases or injuries 15, 29. As a consequence of CNS lesions, microglial cells are rapidly activated. This activation is characterized by the morphological transformation of ramified cells into cells that display large, amoeboid somata with few pseudopodia 8, 23, 38. Through the secretion of neurotoxic or neuroprotective agents, stimulated macrophages and microglial cells react to CNS pathologies and determine the pattern and degree of functional recovery (Moore and Thanos, 1996).

The bisphosphonate clodronate is clinically used in the treatment of osteoporosis, Paget’s disease, and hypercalcaemia in hyperparathyroidism or metastatic bone disease (Russell and Rogers, 1999). Bisphosphonates inhibit the activation of osteoclasts by inducing osteoclast apoptosis (Benford et al., 2001) and thus by inhibiting bone resorption (Frith et al., 2001). Apart from its effects on osteoclasts, clodronate has been shown to inhibit peripheral macrophage function and to induce apoptotic death in peritoneal macrophages (Van Rooijen et al., 1996). Consequently, clodronate has been used in different experimental approaches to investigate effects of macrophage depletion. The intravenous injection of liposome-encapsulated clodronate has been shown to reduce the severity of LPS-induced neurodegeneration (Zito et al., 2001), and clodronate treatment reduced parenchymal macrophage invasion (Koennecke et al., 1999).

The intention of our study was to investigate whether clodronate also exerts effects on the macrophages residing in the CNS, i.e., microglial cells. The organotypic hippocampal slice culture (OHSC) was chosen as the experimental model because it contains resident microglial cells only and an influx of monocytes from the blood stream does not occur in this in vitro preparation.

Several in vitro lesions have been performed in the OHSC, e.g., excitotoxic injury by application of N-methyl-d-aspartate (NMDA) or perforant path transection. Treatment with NMDA has been found to cause a massive loss of granule cells in the dentate gyrus (DG), followed by the rapid accumulation of activated, amoeboid microglial cells at sites of neuronal injury (Heppner et al., 1998). Lesioning of OHSC by NMDA application was performed after 6 days in vitro (div), because all microglial cells in the inner layers of the slice culture are inactive at this time (Hailer et al., 1996). In this study, OHSC lesioned with NMDA alone were compared with OHSC that received additional clodronate treatment after or prior to lesioning. Microglial cells were stained with FITC-conjugated Griffonia simplicifolia isolectin B4 (IB4) 44, 46, and their number and distribution were determined by confocal laser scanning microscopy. To quantify the amount of neuronal injury, the number of propidium iodide-labeled (PI+), degenerating neurons was determined in the granule cell layer of the dentate gyrus (Fig. 1B). Additional staining with hematoxylin/IB4 (H/IB4) was used to assess the hippocampal cytoarchitecture.

Section snippets

Materials and methods

To prepare OHSC, 8-day-old Wistar rats were decapitated and their brains were dissected under sterile conditions (Gähwiler et al., 1997). The frontal pole and the cerebellum were removed and the brains were placed in minimal essential medium (MEM, Gibco BRL Life Technologies, Eggenstein, Germany), containing 1% glutamine (Gibco) at 4°C. Approximately 1 mm of the ventral surface was removed and 350-μm-thick slices were prepared using a sliding vibratome (Vibratome 1000 Classic, St. Louis, MO,

Group A: unlesioned OHSC cultured for 9 days contain ramified microglial cells and show an excellent neuronal preservation

DAB-labeled IB4 preparations of unlesioned control OHSC showed that few microglial cells were distributed in the different layers of the DG, mainly in the molecular and plexiform layers. The GCL contained a very small number of IB4+ microglial cells. The majority of these microglial cells possessed branched, usually tender cytoplasmic processes that extended in all directions. The morphological appearance of microglial cells was thus consistent with the ramified phenotype, indicating a resting

Discussion

The role of microglia is of special interest in neuronal injury and axonal regeneration. As a consequence of CNS lesions such as stroke or trauma, the phenotype of microglial cells changes from a ramified into an amoeboid form 48, 49. More importantly, activated microglial cells secrete a cocktail of factors that have been characterized as neurotoxic, such as proinflammatory cytokines, NO (Bal-Price and Brown, 2001) and proteases (Lokensgard et al., 2001). Such activated microglial cells are

Acknowledgements

This study was supported by the Stiftung Friedrichsheim, the Manja und Ernst Mordhorst-Stiftung, the Paul und Ursula Klein-Stiftung, and the Medical Faculty of the Johann Wolfgang Goethe-University. The authors gratefully acknowledge the expert technical assistance by Mrs. Nadine Roser-Bloh and Mr. Shawn Leslie.

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