Opposing actions of hippocampus TNFα receptors on limbic seizure susceptibility
Highlights
► Limbic seizures increase type 1 TNF receptors, but decrease type 2 TNF receptors. ► TNF overexpression increases microglial activation and BBB compromise. ► Chronic activation of type 1 TNF receptors increases seizure sensitivity. ► Chronic activation of type 2 TNF receptors decreases seizure sensitivity. ► Blockade of endogenous TNF significantly reduces seizure-induced cell death.
Introduction
Over the last decade, both clinical and basic research has directly linked neuroinflammatory events with epilepsy (Vezzani et al., in press). Clinically, tissues resected from refractory temporal lobe epileptics exhibit a number of neuroinflammatory changes, such as increased IL-1β levels (Ravizza et al., 2008), upregulated TNF-α receptor 1 (TNFR1) signaling pathways (Yamamoto et al., 2006), increased CCL2 protein concentrations (Choi et al., 2009, Wu et al., 2008), increased CSF levels of IL-6 (Lehtimaki et al., 2004) and activated astrocytes (Bordey and Sontheimer, 1998). Additional neuropathological consequences include the presence of a compromised blood–brain barrier (BBB) and activated microglia (Hufnagel et al., 2003). Although these chronic neuropathologies inevitably contribute to temporal lobe seizures, studies also have established direct in vivo seizure interactions for a wide range of neuroimmune modulators from TNF-α and IL1-β to toll-like receptor-4 and high mobility group box-1 (Dedeurwaerdere et al., 2012).
A substantial literature suggests a direct role for TNF-α in seizure sensitivity, primarily from basic research studies that utilized acute manipulation of TNF-α function or transgenic mouse models. Balosso et al. (2005) found that acute, selective activation of TNFR1 by human TNF-α exerted no influence on hippocampal seizure sensitivity, but activation of both TNFR1 and TNFR2 by mouse TNF-α strongly inhibited seizure activity. Similarly, mice deficient in TNFR2 or both TNFR2 and TNFR1 exhibited increased seizure sensitivity, while astrocyte overexpression of TNF-α attenuated seizure sensitivity (Balosso et al., 2005). From these findings the authors concluded that activation of TNFR2 attenuates seizure activity, while activation of TNFR1 did not influence seizure sensitivity. In contrast, Probert et al. (1995) reported that neuronal overexpression of TNF-α caused seizures and premature death. Clearly, these studies provided substantial insight into TNF-α interactions with seizures, but acute TNF-α manipulations do not faithfully recapitulate the clinical finding of chronic TNFα exposure. Furthermore, with transgenic animal models the results may be influenced by the likely developmental adaptation.
Adeno-associated virus (AAV) vectors provide the opportunity to mimic the chronic state of elevated TNF-α in vivo and determine the influence of this chronic activation on in vivo seizures. AAV vectors can non-toxically transduce cells in the CNS, and in most cases the transduction is predominantly neuronal (McCown, 2010). Since AAV vectors alone do not evoke significant neuroinflammation, gene expression would take place in the absence of any vector influence on neuroimmune function (Gorbatyuk et al., 2008, Wang et al., 2010). Thus, chronic vector-mediated TNF-α expression in the hippocampus should more closely mimic conditions found in tissue resected from intractable temporal lobe epileptics. The following studies characterized the influence of chronic TNF-α exposure, as well as the interactions between seizures and TNFR1 or TNFR2. The results demonstrate clear, opposing receptor-dependent actions of chronic TNF-α, and provide future directions for treating the pathology associated with temporal lobe epilepsy.
Section snippets
Animals
The animals were male Sprague–Dawley rats (Charles River, Morrisville, NC, USA) weighing between 275 and 300 g. The rats were maintained in a 12-h light–dark cycle and had free access to water and food. All care and procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and received prior approval by the University of North Carolina Institutional Animal Care and Usage Committee.
Amygdala kainic acid delivery and time course tissue collection
For intracranial kainic acid infusions, rats were
Generalized limbic seizures differentially alter TNFRs and evoke immune responses in the hippocampus
TNFR1-like immunoreactivity and TNFR2-like immunoreactivity were found in neuronal cell layers of the normal hippocampus, but 24 h after limbic seizure activity was evoked by amygdala kainic acid infusion, the hippocampus exhibited an apparent increase in TNFR1-like immunoreactivity and an apparent decrease in TNFR2-like immunoreactivity (Figs. 1A,B). These observations were validated by Western blot analysis (Figs. 1C,D) where hippocampal TNFR1 levels increased significantly as compared to
Discussion
Over the past decade conclusive evidence indicates that not only do seizures cause long-term neuroimmune consequences but that some immune factors can produce pro-convulsive actions (Balosso et al., 2005, Crespel et al., 2002, Ravizza et al., 2008, Yamamoto et al., 2006). Given the chronic elevation of TNF-α activity in resected temporal lobe epileptics (Yamamoto et al., 2006), we investigated the interplay of limbic seizures and hippocampal TNF-α activity. Overall, the studies show that limbic
Conclusions
Our findings show that after limbic seizures, hippocampal TNFR1 protein expression increases and TNFR2 protein expression decreases, a change that should favor a pro-convulsant state. In addition, chronic hippocampal TNFR1 activation induces an increased seizure propensity in two limbic seizure models. Chronic activation of TNFR1/2, however, exerts anti-seizure properties, even in the context of presumptive, simultaneous activation of TNFR1. With regard to endogenous TNF-α, vector mediated
Funding
This work was supported by the National Institutes of Health (NS035633 to T.J.M; F32NS070356 to M.S.W).
Acknowledgments
The authors would like to thank Dr. Josh Grieger and Steve Soltys of the UNC Gene Therapy Center Vector Core for their advice regarding vector production and purification. We also thank Dr. Chengwen Li and Swati Yadav of the Samulski Lab (UNC), Kirk McNaughton of the UNC Cell and Molecular Physiology Histology Research Core Facility, and Michael Chua and Dr. Neal Kramarcy of the UNC Hooker Microscopy core facility for technical assistance.
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