Microglia Remodelling and Neuroinflammation Parallel Neuronal Hyperactivation Following Acute Organophosphate Poisoning

Abstract

Organophosphate (OP) compounds include highly toxic chemicals widely used both as pesticides and as warfare nerve agents. Existing countermeasures are lifesaving, but do not alleviate all long-term neurological sequelae, making OP poisoning a public health concern worldwide and the search for fully efficient antidotes an urgent need. OPs cause irreversible acetylcholinesterase (AChE) inhibition, inducing the so-called cholinergic syndrome characterized by peripheral manifestations and seizures associated with permanent psychomotor deficits. Besides immediate neurotoxicity, recent data have also identified neuroinflammation and microglia activation as two processes that likely play an important, albeit poorly understood, role in the physiopathology of OP intoxication and its long-term consequences. To gain insight into the response of microglia to OP poisoning, we used a previously described model of diisopropylfluorophosphate (DFP) intoxication of zebrafish larvae. This model reproduces almost all the defects seen in poisoned humans and preclinical models, including AChE inhibition, neuronal epileptiform hyperexcitation, and increased neuronal death. Here, we investigated in vivo the consequences of acute DFP exposure on microglia morphology and behaviour, and on the expression of a set of pro- and anti-inflammatory cytokines. We also used a genetic method of microglial ablation to evaluate the role in the OP-induced neuropathology. We first showed that DFP intoxication rapidly induced deep microglial phenotypic remodelling resembling that seen in M1-type activated macrophages and characterized by an amoeboid morphology, reduced branching, and increased mobility. DFP intoxication also caused massive expression of genes encoding pro-inflammatory cytokines Il1β, Tnfα, Il8, and to a lesser extent, immuno-modulatory cytokine Il4, suggesting complex microglial reprogramming that included neuroinflammatory activities. Finally, microglia-depleted larvae were instrumental in showing that microglia were major actors in DFP-induced neuroinflammation and, more importantly, that OP-induced neuronal hyperactivation was markedly reduced in larvae fully devoid of microglia. DFP poisoning rapidly triggered massive microglia-mediated neuroinflammation, probably as a result of DFP-induced neuronal hyperexcitation, which in turn further exacerbated neuronal activation. Microglia are thus a relevant therapeutic target, and identifying substances reducing microglial activation could add efficacy to existing OP antidote cocktails.

A Pediatric Rat Model of Organophosphate-Induced Refractory Status Epilepticus: Characterization of Long-Term Epileptic Seizure Activity, Neurologic Dysfunction and Neurodegeneration

“…We observed that DFP-exposed animals exhibited poor cognitive performance, shown by significant deficits in object recognition and spatial memory. Consistent with our study, other adult models of acute OP intoxication have also reported positive correlations between hippocampal neurodegeneration and cognitive impairment ( De Araujo Furtado et al, 2012 , Reddy et al, 2020a , b ; Somkhit et al, 2022 ). Inflammation signals in the hippocampus and amygdala, indicated by microglial activation, have also been proposed as a contributing factor to cognitive dysfunction as seen in an array of brain diseases, including epilepsy and Parkinson’s disease ( Guignet et al, 2020 ; Zhang et al, 2021 ; Somkhit et al, 2022 ; López-Meraz and Álvarez-Croda, 2023 ).…”

Microglia Mitigate Neuronal Activation in a Zebrafish Model of Dravet Syndrome

“…In close agreement with data gained from both animal models and human patients [50], microglia displayed significant morphological differences in scn1Lab-KD larvae when compared to those in their not-injected siblings. In the mutant context, we observed a decreased number of "branched" microglia and an increased percentage of cells showing an "amoeboid" morphology, the latter resembling M1-type pro-inflammatory "activated" microglia observed following brain injuries or in various neuronal disease situations, including epilepsy [18], but also, as we previously showed, following intoxication by neurotoxic organophosphates [37,51]. Interestingly, although DS larvae showed an increased number of microglia with an "activated" phenotype, the proportion of "transitional" microglia was similar in the two genetic contexts, and a small, but significant, percentage (10%) of "branched" (resting) microglia was still observed in scn1Lab-KD individuals.…”

“…Cluster analysis, based on microglia morphology parameters, was performed as described in [37]. Briefly, five morphological parameters were quantified for each microglial cell: sphericity (S), number of processes (NP), total process length (TL), surface area (A), and volume (V).…”

“…Quantification of neuronal calcium transients was performed as previously described [31,37]. Briefly, 4 dpf Tg[Huc:GCaMP5G] scn1Lab morphant and Tg[mpeg1:mCherryF] control larvae were paralyzed using pancuronium bromide, agar-mounted in the center of a recording chamber, and placed under a Leica SP8 laser scanning confocal microscope equipped with a 20×/0.75 multi-immersion objective.…”

“…To further evaluate the extent of microglia morphology changes in scn1Lab-KD larvae compared to that seen in their non-injected sibling controls, we performed a cluster analysis of these cells according to five morphological parameters (sphericity, branch number, branch length, surface area, and volume), leading to three cell clusters, as described in [37] (see Materials and Methods). The first comprised low-sphericity microglia with numerous elongated branches and high surface area and volume, which were referred to below as "branched" microglia.…”