Microglia attenuate the kainic acid‐induced death of hippocampal neurons in slice cultures

Abstract

Background Status epilepticus‐induced hippocampal neuronal death, astrogliosis, and the activation of microglia are common pathological changes in mesial temporal lobe epilepsy (mTLE) with resistance to antiepileptic drugs. Neuronal death in mTLE gradually progresses and is involved in the aggravation of epilepsy and the impairment of hippocampus‐dependent memory. Thus, clarifying the cellular mechanisms by which neurons are protected in mTLE will significantly contribute to the treatment of epilepsy. Here, mainly using hippocampal slice cultures with or without the pharmacological depletion of microglia, we directly examined whether microglia, the resident immune cells of the brain that can act either neurotoxically or in a neuroprotective manner, accelerate or attenuate kainic acid (KA)‐induced neuronal death in vitro. Methods Hippocampal slice cultures were treated with KA to induce neuronal death in vitro. Clodronate‐containing liposomes or PLX3397 was used to deplete microglia in hippocampal slice cultures, and the effect on KA‐induced neuronal death was immunohistochemically assessed. Results The loss of microglia significantly promoted a decrease in neuronal density in KA‐treated hippocampal slice cultures. Conclusion Our results suggest that microglia are neuroprotective against KA‐induced neuronal death in slice cultures.

Organotypic Brain Slice Culture Microglia Exhibit Molecular Similarity to Acutely-Isolated Adult Microglia and Provide a Platform to Study Neuroinflammation

“…Similarities in cellular composition and architecture between the in vitro organotypic brain slice cultures and the in vivo setting offer advantages to model complex brain physiology and pathophysiology even though a detailed characterization of the cellular and molecular features of the brain slice culture system was lacking as it pertains to microglia properties in this experimental system. These presumed similarities prompted many studies using variants of the brain slice culture platform to study pathogenic mechanisms involved in stroke, epilepsy, neurodegenerative proteinopathies, and responses to specific neuroinflammatory stimuli (Bernardino, 2005 ; Vinet et al, 2012 ; Ziemka-Nałecz et al, 2013 ; Olajide et al, 2014 ; Hellwig et al, 2015 ; Gerlach et al, 2016 ; Masuch et al, 2016a , b ; Bhatia et al, 2017 ; Saliba et al, 2017 ; Yousif et al, 2018 ; Araki et al, 2019 ; Croft et al, 2019a ; Grabiec et al, 2019 ; Richter et al, 2019 ; Sheppard et al, 2019 ). Revisiting these studies in light of our results may reveal new insights into the role of microglia in these settings.…”

“…As this was observed at timepoints of 2 weeks and beyond, this would coincide with our observed expression of neurodegeneration-associated genes and provide an opportunity to study the role of microglia in this process. Kainic acid, AMPA and NMDA agonist induced excitotoxicity in brain slices have been used to model epilepsy with some studies using timepoints at 7–9 days in culture (Vinet et al, 2012 ; Masuch et al, 2016a , b ; Araki et al, 2019 ; Grabiec et al, 2019 ) while others used later timepoints at 2–3 weeks (Bernardino, 2005 ; Grabiec et al, 2019 ). As neonatal microglia have been shown to mediated synaptic pruning with relevance to epilepsy (Andoh et al, 2019 ), we would wonder whether the neonatal/inflammatory phenotype of microglia observed prior to 2 weeks would alter their contribution to excitotoxicity and differ from the contribution of microglia at later timepoints where they exhibit more mature, and eventually neurodegenerative, features.…”

“…Primary microglia cultured on conventional tissue culture plates represent a simple alternative to study response to disease-relevant stimuli, however gene expression profiling identified an epigenetically-driven loss of microglia identity gene expression within hours of transfer to culture plates (Gosselin et al, 2017 ). In contrast organotypic brain slice cultures derived from primary mouse tissue preserve tissue architecture important for microglia physiological phenotypes and offer the advantage of a tissue-relevant context that have yielded important insights into neuroinflammatory processes (Czapiga and Colton, 1999 ; Bernardino, 2005 ; Lossi et al, 2009 ; Vinet et al, 2012 ; Hellwig et al, 2015 ; Masuch et al, 2016a ; Saliba et al, 2017 ; Yousif et al, 2018 ; Araki et al, 2019 ; Croft et al, 2019a ; Richter et al, 2019 ; Sheppard et al, 2019 ). Analogous studies with rat organotypic brain slice cultures have also provided valuable insights regarding the role of microglia during inflammation (Mitrasinovic et al, 2001 ; Schermer and Humpel, 2002 ; Ding and Li, 2008 ; Hall et al, 2009 ; Benakis et al, 2010 ; Jung et al, 2013 ; Nam et al, 2013 ; Lutz et al, 2015 ; Ramírez-Sánchez et al, 2015 ; Papageorgiou et al, 2016 ; Chong et al, 2018 ; Magalhães et al, 2018 ).…”

Table_5.XLSX

“…Similarities in cellular composition and architecture between the in vitro organotypic brain slice cultures and the in vivo setting offer advantages to model complex brain physiology and pathophysiology even though a detailed characterization of the cellular and molecular features of the brain slice culture system was lacking as it pertains to microglia properties in this experimental system. These presumed similarities prompted many studies using variants of the brain slice culture platform to study pathogenic mechanisms involved in stroke, epilepsy, neurodegenerative proteinopathies, and responses to specific neuroinflammatory stimuli (Bernardino, 2005;Vinet et al, 2012;Ziemka-Nałecz et al, 2013;Olajide et al, 2014;Hellwig et al, 2015;Gerlach et al, 2016;Masuch et al, 2016a,b;Bhatia et al, 2017;Saliba et al, 2017;Yousif et al, 2018;Araki et al, 2019;Croft et al, 2019a;Grabiec et al, 2019;Richter et al, 2019;Sheppard et al, 2019). Revisiting these studies in light of our results may reveal new insights into the role of microglia in these settings.…”

“…As this was observed at timepoints of 2 weeks and beyond, this would coincide with our observed expression of neurodegeneration-associated genes and provide an opportunity to study the role of microglia in this process. Kainic acid, AMPA and NMDA agonist induced excitotoxicity in brain slices have been used to model epilepsy with some studies using timepoints at 7-9 days in culture (Vinet et al, 2012;Masuch et al, 2016a,b;Araki et al, 2019;Grabiec et al, 2019) while others used later timepoints at 2-3 weeks (Bernardino, 2005;Grabiec et al, 2019). As neonatal microglia have been shown to mediated synaptic pruning with relevance to epilepsy (Andoh et al, 2019), we would wonder whether the neonatal/inflammatory phenotype of microglia observed prior to 2 weeks would alter their contribution to excitotoxicity and differ from the contribution of microglia at later timepoints where they exhibit more mature, and eventually neurodegenerative, features.…”