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Intrinsic Inflammation Is a Potential Anti-Epileptogenic Target in the Organotypic Hippocampal Slice Model.

ABSTRACT: Understanding the mechanisms of epileptogenesis is essential to develop novel drugs that could prevent or modify the disease. Neuroinflammation has been proposed as a promising target for therapeutic interventions to inhibit the epileptogenic process that evolves from traumatic brain injury. However, it remains unclear whether cytokine-related pathways, particularly TNF? signaling, have a critical role in the development of epilepsy. In this study, we investigated the role of innate inflammation in an in vitro model of post-traumatic epileptogenesis. We combined organotypic hippocampal slice cultures, representing an in vitro model of post-traumatic epilepsy, with multi-electrode array recordings to directly monitor the development of epileptiform activity and to examine the concomitant changes in cytokine release, cell death, and glial cell activation. We report that synchronized ictal- and interictal-like activities spontaneously evolve in this culture. Dynamic changes in the release of the pro-inflammatory cytokines IL-1?, TNF?, and IL-6 were observed throughout the culture period (3 to 21 days in vitro) with persistent activation of microglia and astrocytes. We found that neutralizing TNF? with a polyclonal antibody significantly reduced ictal discharges, and this effect lasted for 1 week after antibody washout. Neither phenytoin nor an anti-IL-6 polyclonal antibody was efficacious in inhibiting the development of epileptiform activity. Our data show a sustained effect of the anti-TNF? antibody on the ictal progression in organotypic hippocampal slice cultures supporting the critical role of inflammatory mediators in epilepsy and establishing a proof-of-principle evidence for the utility of this preparation to test the therapeutic effects of anti-inflammatory treatments.

SUBMITTER: Chong SA

PROVIDER: S-EPMC5935638 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Intrinsic Inflammation Is a Potential Anti-Epileptogenic Target in the Organotypic Hippocampal Slice Model.

Chong Seon-Ah SA Balosso Silvia S Vandenplas Catherine C Szczesny Gregory G Hanon Etienne E Claes Kasper K Van Damme Xavier X Danis Bénédicte B Van Eyll Jonathan J Wolff Christian C Vezzani Annamaria A Kaminski Rafal M RM Niespodziany Isabelle I

Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 20180401 2

Understanding the mechanisms of epileptogenesis is essential to develop novel drugs that could prevent or modify the disease. Neuroinflammation has been proposed as a promising target for therapeutic interventions to inhibit the epileptogenic process that evolves from traumatic brain injury. However, it remains unclear whether cytokine-related pathways, particularly TNFα signaling, have a critical role in the development of epilepsy. In this study, we investigated the role of innate inflammation ...[more]

PMID: 29464573

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Project description:In organotypic hippocampal slice cultures, principal neurons form aberrant excitatory connections with other principal cells in response to slicing induced deafferentation, similar to mechanisms underlying epileptogenesis in posttraumatic epilepsy. To investigate the consequences of this synaptogenesis, the authors recorded field-potential activity from area CA3 during perfusion with the complete growth medium used during incubation. At 7 days in vitro, slice cultures only displayed multiunit activity. At 14 days in vitro, the majority displayed population bursts reminiscent of interictal-like spikes, but sustained synchronous activity was rare. Band-pass filtering of interictal discharges revealed fast ripple-like complexes, similar to in vivo recordings. Spontaneous ictal-like activity became progressively more prevalent with age: at 21 days in vitro, 50% of organotypic hippocampal slice cultures displayed long-lasting, ictal-like discharges that could be suppressed by phenytoin, whereas interictal activity was not suppressed. The fraction of cultures displaying ictal events continually increased with incubation time. Quantification of population spike activity throughout epileptogenesis using automatic detection and clustering algorithms confirmed the appearance of interictal-like activity before ictal-like discharges and also revealed high-frequency pathologic multiunit activity in slice cultures at 14 to 17 days in vitro. These experiments indicate that interictal-like spikes precede the appearance of ictal-like activity in a reduced in vitro preparation. Epileptiform activity in cultures resembled in vivo epilepsy, including sensitivity to anticonvulsants and steadily increasing seizure incidence over time, although seizure frequency and rate of epileptogenesis were higher in vitro. Organotypic hippocampal slice cultures comprise a useful model system for investigating mechanisms of epileptogenesis as well as developing antiepileptic and antiepileptogenic drugs.

Project description:Neurogenesis in the adult hippocampus has become an intensively investigated research topic, as it is essential for proper hippocampal function and considered to bear therapeutic potential for the replacement of pathologically lost neurons. On the other hand, neurogenesis itself is frequently affected by CNS insults. To identify processes leading to the disturbance of neurogenesis, we made use of organotypic hippocampal slice cultures (OHSC), which, for unknown reasons, lose their neurogenic potential during cultivation. In the present study, we show by BrdU/Prox1 double-immunostaining that the generation of new granule cells drops by 90% during the first week of cultivation. Monitoring neurogenesis dynamically in OHSC from POMC-eGFP mice, in which immature granule cells are endogenously labeled, revealed a gradual decay of the eGFP signal, reaching 10% of initial values within 7 days of cultivation. Accordingly, reverse transcription quantitative polymerase chain reaction analysis showed the downregulation of the neurogenesis-related genes doublecortin and Hes5, a crucial target of the stem cell-maintaining Notch signaling pathway. In parallel, we demonstrate a strong and long-lasting activation of astrocytes and microglial cells, both, morphologically and on the level of gene expression. Enhancement of astroglial activation by treating OHSC with ciliary neurotrophic factor accelerated the loss of neurogenesis, whereas treatment with indomethacin or an antagonist of the purinergic P2Y12 receptor exhibited potent protective effects on the neurogenic outcome. Therefore, we conclude that OHSC rapidly lose their neurogenic capacity due to persistent inflammatory processes taking place after the slice preparation. As inflammation is also considered to affect neurogenesis in many CNS pathologies, OHSC appear as a useful tool to study this interplay and its molecular basis. Furthermore, we propose that modification of glial activation might bear the therapeutic potential of enabling neurogenesis under neuropathological conditions.

Project description:Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.

Project description:BACKGROUND:Pyrethroids, such as bifenthrin (BF), are among the most widely used class of insecticides that pose serious risks to human and wildlife health. Pyrethroids are proposed to affect astrocytic functions and to cause neuron injury in the central nervous system (CNS). Microglia are key cells involved in innate immune responses in the CNS, and microglia activation has been linked to inflammation and neurotoxicity. However, little information is known about the effects of BF-induced toxicity in primary microglial cells as well as in organotypic hippocampal slice cultures (OHSCs). METHODS:Oxidative stress and inflammatory responses induced by BF were evaluated in primary microglial cells and OHSCs incubated with different concentrations of BF (1-20 ?M) for 4 and 24 h. mRNA and protein synthesis of cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), nuclear erythroid-2 like factor-2 (Nrf-2), and microsomal prostaglandin synthase-1 (mPGES-1) was also studied by qPCR and Western blot. Cell viability was analyzed by MTT-tetrazolio (MTT) and lactate dehydrogenase (LDH) assays. Neurotoxicity in OHSCs was analyzed by propidium iodide (PI) staining and confocal microscopy. RESULTS:Exposure of microglial cells to BF for 24 h resulted in a dose-dependent reduction in the number of viable cells. At sub-cytotoxic concentrations, BF increased reactive oxygen species (ROS), TNF-alpha synthesis, and prostaglandin E2 (PGE2) production, at both 4- and 24-h time points, respectively. Furthermore, BF incubation decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and increased lipid peroxidation, protein oxidation, and H2O2 formation. In addition, BF significantly induced protein synthesis and mRNA expression of oxidative and inflammatory mediators after 4 and 24 h, including Nrf-2, COX-2, mPGES-1, and nuclear factor kappaB (NF-kappaB). A 24-h exposure of OHSCs to BF also increased neuronal death compared to untreated controls. Furthermore, depletion of microglia from OHSCs potently enhanced neuronal death induced by BF. CONCLUSIONS:Overall, BF exhibited cytotoxic effects in primary microglial cells, accompanied by the induction of various inflammatory and oxidative stress markers including the Nrf-2/COX-2/mPGES-1/NF-kappaB pathways. Moreover, the study provided evidence that BF induced neuronal death in OHSCs and suggests that microglia exert a protective function against BF toxicity.

Dataset Information

Intrinsic Inflammation Is a Potential Anti-Epileptogenic Target in the Organotypic Hippocampal Slice Model.

Publications

Intrinsic Inflammation Is a Potential Anti-Epileptogenic Target in the Organotypic Hippocampal Slice Model.

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