Microglial ablation and lipopolysaccharide preconditioning affects pilocarpine-induced seizures in mice
Introduction
Microglia activation constitutes a major mechanism of self-defense against brain injury, infection and disease. The role undertaken by activated microglia is complicated, since they have been implicated as both neuroprotective and neurotoxic (Schwartz et al., 2006, Streit et al., 1999). Extensive microglial activation (microgliosis) and reactive astrogliosis occur in the brain parenchyma of individuals with recurrent seizure episodes and in animal models of epilepsy (Beach et al., 1995, Drage et al., 2002). In the pilocarpine mouse seizure model, microgliosis persists for at least 30 days following seizure induction and correlates with local neuronal death (Borges et al., 2003, Borges et al., 2006). Microglia in the hippocampus may contribute to the re-occurrence of spontaneous seizures that take place days after the original epileptic event (Coulter, 2001, Pierce et al., 2005) by facilitating aberrant migration of newborn neurons (Yang et al., 2010). Changes in glial function could perhaps be a component of metabolic abnormalities which are observed in epileptic patients (Altay et al., 2005, Lamusuo et al., 2001, Magistretti and Pellerin, 1996, Vielhaber et al., 2003) and rodent seizure models (Goffin et al., 2009, Kornblum et al., 2000, Mirrione et al., 2006, Mirrione et al., 2007). The question remains however, whether microgliosis is a consequence of recurrent seizure episodes, a bystander to neuronal damage, or if microglia could directly contribute to seizure symptoms, sensitivity, or threshold. One approach to investigate this question is to modulate the microglia activation state concurrently with seizures.
Recent studies have suggested that preconditioning microglia with bacterial lipopolysaccharide (LPS) can be protective in rodent seizure models (Akarsu et al., 2006, Arican et al., 2006, Dmowska et al., 2010, Sayyah et al., 2003b), a concept which has been critically investigated in ischemia (Marsh et al., 2009b, Rosenzweig et al., 2004). The mechanism of protection in ischemia has been shown to involve tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and reprogramming of the cellular response to excitotoxic insults (Marsh et al., 2009a, Saha et al., 2009), but the potential for protection in epilepsy is controversial and less understood. In one study, LPS administration prior to pentylenetetrazole (PTZ)-induced seizures, was beneficial by increasing plasma levels of nitric oxide (NO) and interleukin-6 (IL-6), which reduced blood brain barrier (BBB) permeability (Arican et al., 2006). A time course of LPS delivery prior to seizure induction with PTZ demonstrated complex effects, where LPS administered 4 h before PTZ-induced seizures was pro-epileptic, but when given 18 h before, it conferred anticonvulsant effects attributable to the expression of cycloxygenases-1 (COX-1) and -2 (Akarsu et al., 2006). LPS was inhibitory in a kindling model of seizures but only when it was administered daily for 16 days (Sayyah et al., 2003b). Neuronal protection from cell death was observed after LPS preconditioning 72 h prior to seizure induction (Dmowska et al., 2010). Several other studies have supported either no effect (Yuhas et al., 2002) or a pro-convulsant role for LPS when given 1–2 h prior to seizures (Sankar et al., 2007, Sayyah et al., 2003a). It is clear from these studies that both positive and negative effects are induced by LPS that appear to be time and dose dependent, thus warranting further investigation. In the present study, we have focused on the effects of LPS when given 24 h prior to pilocarpine injection, as in the protocol used by Akarsu and colleagues (2006), in order to explore the role of preconditioned microglia in seizure induction. This dose and timing of LPS administration did not result in promotion of seizures.
We previously showed that the threshold to experimentally induced seizures, and neuronal survival following intra-hippocampal kainate injections is affected by the levels of the protease tissue plasminogen activator (tPA) (Carroll et al., 1994, Mirrione et al., 2007, Tsirka et al., 1995, Wu et al., 2000, Zhang et al., 2005), which during excitiotoxic events, can activate microglia and exacerbate neuronal injury (Gravanis and Tsirka 2005, Rogove et al., 1999, Siao and Tsirka, 2002). Based on this evidence, we hypothesized that microglial activation could influence the threshold and/or sensitivity to pilocarpine-induced seizures and downstream pathology. To explore this, we evaluated microglia during acute seizures using a recently developed transgenic model of conditional microglia and macrophage ablation (Heppner et al., 2005). In these mice, administration of the prodrug gancyclovir (GCV) targets cells which express the herpes simplex virus thymidine kinase (HSVTK) suicide gene under the microglia/macrophage CD11b promoter, thereby causing specific ablation following DNA replication failure. We evaluated behavioral seizure symptoms, histology, and in-vivo metabolic activity (which measures brain activity on a systems level), following ablation of microglia/macrophages with and without LPS preconditioning. Our results demonstrate a significant modulatory role for preconditioned hippocampal microglia in mitigating acute seizure induction.
Section snippets
Animals and genotyping
Transgenic animals expressing thymidine kinase (TK) in cells of monocytic lineage, including microglia and macrophages, were generated (CD11b-HSVTK+/−) (Heppner et al., 2005). C57Bl/6 wild-type (wt) males were bred with females heterozygous for the transgene (CD11b-HSVTK+/−) due to male sterility, and offspring were genotyped by PCR with Taq DNA polymerase in 17 mM MgCl2 PCR buffer with the following primers: 5′-GACTTCCGTGGCTTCTTGCTGC-3′ and 5′-GTGCTGGCATTACAGGCGTGAG-3′. The PCR conditions were
Reduction of the extent of excitotoxic cell death in the absence of activated microglia
Experimental neuronal death in the hippocampus is accompanied by microglia/macrophage activation, an event that also contributes to the progression of cell death. We have previously shown that delay of microglia activation protects neurons from cell death (Rogove and Tsirka, 1998). As a proof of principle using transgenic microglia/macrophage ablation, we assessed if neuroprotection would be obtained in excitotoxin-challenged CD11b-HSVTK+/− mice. We delivered gancyclovir (GCV) unilaterally to
Discussion
Striking differences in acute seizure symptoms were observed between Cd11b-HSVTK−/− and Cd11b-HSVTK+/− mice when LPS preconditioning was combined with unilateral hippocampal microglia ablation. The seizure score differences were consistent at two doses of pilocarpine (260 mg/kg and 280 mg/kg). In this experimental paradigm, the only quantifiable cellular difference between Cd11b-HSVTK−/− and Cd11b-HSVTK+/− mice (in groups V and VI) was the numbers of activated microglia/macrophages in the
Conflict of interest statement
The authors declare no competing financial interests.
Acknowledgments
This work was supported by National Institutes of Health, RO1NS42168 and American Heart Association-Established Investigator Award (to S.E.T.), R01NS046006 and DFG SFB TRR43 and Exc 25 to F.L.H., National Institute on Drug Abuse (DA15041) and the Department of Energy (to S.L.D), and National Science Foundation Integrative Graduate Education and Research Traineeship, Minerals, Metals, Metalloids, and Toxicity (to S.E.T. and M.M.M). The authors would like to thank Drs. Joanna Fowler, Marian
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