Microglia remodelling and neuroinflammation parallel neuronal hyperactivation following acute organophosphate poisoning

doi:10.21203/rs.3.rs-1662275/v1

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Research Article

Microglia remodelling and neuroinflammation parallel neuronal hyperactivation following acute organophosphate poisoning

https://doi.org/10.21203/rs.3.rs-1662275/v1

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posted 31 May, 2022

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Background

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.

Methods

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.

Results

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 pro-inflammatory cytokines Il1β and Il8, and, to a lesser extent, immuno-modulatory cytokine Il4, suggesting complex microglial reprogramming that included neuroinflammatory activities. Last, 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.

Conclusions

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.

Microglia

Organophosphate poisoning

Microglia in vivo imaging

Diisopropylfluorophosphate (DFP)

Zebrafish

Cytokines

Inflammation

Organophosphates (OPs) are a family of organic compounds that includes highly toxic chemicals widely used as pesticides, and, to a lesser extent, warfare nerve agents, making OP poisoning a major public health issue worldwide. In recent years, several million intoxications have been reported annually, causing more than 200,000 deaths, primarily suicides [1, 2]. OPs bind covalently to acetylcholinesterase (AChE) and irreversibly inhibit its activity, inducing a large accumulation of acetylcholine (ACh) at cholinergic synapses and so hyperactivation of acetylcholine receptors (AChR), resulting in the so-called cholinergic syndrome. At the neuronal level, AChR overstimulation causes a massive hyperexcitation of cholinergic neurons with large glutamate release and excitotoxicity, and eventually neuronal death [3]. In poisoned humans and preclinical models, acute OP exposure induces peripheral manifestations and impacts the central nervous system, with seizures that may worsen into status epilepticus (SE), a life threat if not quickly treated [4]. Besides direct neurotoxicity, OP poisoning can trigger long-term neuronal disorders, such as OP-induced delayed neuropathy (OPIDN), a complex syndrome associating cognitive and psychomotor deficits [5]. Existing countermeasures combine the AChR antagonist atropine with an AChE reactivator oxime such as pralidoxime (2-PAM), and a γ-aminobutyric acid (GABA) receptor agonist of the benzodiazepine family, such as diazepam. However, while such antidote cocktails do mitigate the acute toxicity of OPs, they must be delivered in the very first minutes after exposure and they do not alleviate all long-term neurological deficits [6]. More potent countermeasures with extended therapeutic and temporal windows are thus needed.

Besides neuronal hyperexcitation, data from preclinical models, mostly rodents, have shown that exposure to OPs rapidly induces massive, sustained brain inflammation [7–9] a harmful condition that may be at least partly responsible for the long-term psychomotor comorbidities observed in intoxicated patients and animal models [10]. Specifically, it has been clearly established that neuroinflammation creates an environment that is not conducive to the maintenance of brain homeostasis and can promote epileptogenesis [11]. In line with these findings, Gonzales et al. [12] recently showed that microglial cells, the brain-resident macrophages, which are key actors in brain inflammation, likely play an important role in the cognitive deficits observed 1-month post-acute OP intoxication in juvenile rats. Importantly, at the therapeutic level, the evidence suggests that agents able to mitigate the OP-induced neuroinflammation could be a promising additional treatment in future antidote cocktails to relieve both the immediate effects of OPs and their long-term consequences.

As an animal model suitable for both toxicological investigations, but also medium- to high-throughput drug screening, we previously described a zebrafish model of acute OP intoxication using diisopropylfluorophosphate (DFP), a prototypic OP structurally similar to the G-class nerve agent sarin and widely used in toxicological research due to its moderate toxicity and low volatility [10, 13]. This zebrafish model of acute DFP poisoning reproduces almost all the major neuropathological defects seen in exposed humans and preclinical models, including AChE inhibition, massive neuronal excitation leading to epileptiform activity, an imbalance of glutamatergic/GABAergic synaptic activity, and increased neuronal death [14].

Here, using in vivo imaging and transgenic lines encoding fluorescent reporter proteins, we first showed that acute DFP poisoning caused deep microglia remodelling resembling M-1 type macrophage activation, combined with massive expression of pro-inflammatory cytokines. Importantly, not only DFP-induced inflammation but also neuronal hyperactivation were significantly mitigated in larvae fully devoid of microglia, suggesting that a neuroinflammatory environment may increase the excitability of neuronal networks. Our findings therefore suggest that microglia are a novel therapeutic target to identify compounds mitigating OP-induced neuroinflammation, and that they could be used to improve the efficacy of existing antidote cocktails.

Animals. Zebrafish were kept at 26–28°C in a 14 h light/10 h dark cycle. Larvae were collected by natural spawning and raised in E3 medium at 28.5°C [15]. To inhibit embryo pigmentation, 0.003% 1-phenyl-2-thiourea (PTU) was added at 1 day post-fertilization (dpf). The Tg[HuC:GCaMP5G] transgenic line [16], a gift from Dr. George Debrégeas (Laboratoire Jean Perrin, Paris), were raised in our facility. All animal experiments were conducted at the French National Institute of Health and Medical Research (Inserm) UMR 1141 in Paris in accordance with European Union guidelines for the handling of laboratory animals (https://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm). They were approved by the Direction Départementale de la Protection des Populations de Paris and the French Animal Ethics Committee under reference No. 2012-15/676 − 0069.

Drug treatment. Diisopropylfluorophosphate (DFP) was purchased from Sigma Aldrich. A stock solution (5.46 mM), made in DMSO and stored at − 20°C, was diluted extemporaneously to 15 µM in 1% DMSO/E3 medium. Control zebrafish larvae were treated with 1% DMSO/E3 medium.

RNA isolation and quantitative RT-PCR. For RNA isolation, whole larvae or dissected brains were homogenized using a syringe equipped with a 26G needle (7 larvae or 10 brains per sample) using the RNA XS Plus kit (Qiagen, Hilden, Germany). Following RNA quantification with a Nanodrop 2000 (ThermoScientific) and RNA integrity assessment using denaturing gel electrophoresis, total RNAs (1 µg) were reverse-transcribed into cDNAs using the iScript cDNA Synthesis Kit (Bio-Rad, Germany) and qPCRs were performed using iQ SYBR Green Supermix (Bio-Rad, Germany). Samples were run in triplicate and expression levels of the studied genes were normalised to that of the tbp gene. The primers (Eurofins Genomics, Ebersberg, Germany) used are listed below.

Live imaging of microglial cells. The transgenic line Tg[mpeg1:mCherryF] was used for all confocal live imaging experiments. 5 dpf larvae were mounted in 1.2% low melting-point agarose containing 300 µM pancuronium bromide (PB) (Sigma) to paralyse the larvae, and either 1% DMSO or 15 µM DFP. They were then imaged for 6 h using a Leica SP8 confocal scanning laser microscope equipped with either a Leica 20x/0.75 multi-immersion objective or an Olympus 40x/1.1 water objective.

Analysis and quantification of microglial morphological parameters. For all microglia analysed (327 cells in control and 294 in DFP-treated larvae), six morphological parameters were determined using the Imaris software (Bitplane): sphericity (Sp), calculated as the ratio of the measured surface area of a cell (A_c) to the surface area of a sphere of the same volume (V_c), ranging from 0 (fully disorganised shape) to 1 (perfect sphere), surface area (S), volume (V), mean number of branches (NB), mean branch length (ML), and total branch length (TL).

Clustering Of Microglial Cells Into Morphologically Homogeneous Populations

Among the six morphological parameters defined above, we selected five that changed significantly after 6 h of exposure to 15 µM DFP: Sp, S, NB, ML, and TL. We then defined ranges for each one to characterise the microglial populations. For branched "resting" microglial cells, the ranges were: Sp \(\le\)0.6, S \(\ge\) 2400 µm², NB \(\ge\) 6, ML \(\ge\) 19 µm, and TL \(\ge\) 90 µm. For amoeboid “activated” microglial cells, the ranges were: Sp \(\ge\) 0.8, S \(\le\)1500 µm², NB \(\le\) 2, ML \(\le\) 7 µm, and TL \(\le\)20 µm. All microglia in at least three of these five ranges were identified as "activated" or "resting"; the remaining cells were classified as "intermediate" microglia.

Statistics. Statistical analyses were performed using GraphPad Prism 8.4.3.686 (https://www.graphpad.com/scientific-software/prism/). Data were first challenged for normality using the Shapiro-Wilk test. Data with a normal distribution were analysed with a two-tailed unpaired t-test with or without Welch’s correction depending on the variance difference of each sample. Data not showing a normal distribution were analysed using a two-tailed Mann-Whitney test. All graphs show mean ± SEM.

DFP exposure induced dramatic phenotypic remodelling of microglia

To investigate the consequences of acute DFP poisoning on the physiology of microglia, we first made use of our well-established zebrafish model of DFP-intoxication [14] and the zebrafish transgenic line Tg[mpeg1:mCherryF], which enables live imaging of these cells in embryos from 3 days post-fertilization (dpf) onward [17] (Fig. 1A). In close agreement with previous data [18, 19], in 5 dpf larvae, almost all microglial cells showed a highly branched morphology, characteristic of the so-called “resting” state, with cells showing several long branches (Fig. 1B–B''). In contrast, in age-matched individuals exposed for 6 h to 15 µM DFP, microglial cells displayed a much more rounded morphology and a marked decrease in both the number and length of their branches (Fig. 1C–C'').

Precise measurements of several morphological parameters using the Imaris software (Bitplane) confirmed that DFP poisoning caused deep morphological changes of microglial cells. Their mean sphericity (Sp) (see Materials and methods) was significantly increased (0.79 ± 0.01 vs 0.63 ± 0.01, p < 0.0001) (Fig. 1D) and their average surface area (S) was decreased in DFP-treated larvae compared to that observed in controls (840 µm² ± 20 vs 1118 µm² ± 27, p < 0.0001) (Fig. 1E). In contrast, the average volume (V) of the cells was roughly similar in DFP-treated and control individuals (1654 µm³ ± 56 vs 1724 µm³ ± 58, p = 0.64) (Fig. 1F). In DFP-exposed larvae, microglia also displayed a reduced number of branches (NB) (0.6 ± 0.03 vs 4.5 ± 0.1, p < 0.0001) (Fig. 1G), a decreased mean branch length (ML) (17 µm ± 0.1 vs 21 µm ± 0.1, p < 0.001) (Fig. 1H), and thus a decreased mean total branch length (TL) (12.1 µm ± 1.3 vs 80.0 µm ± 2.1, p < 0.0001), compared to those in controls (Fig. 1I). A 3D Sholl analysis, a method used to quantify both the extent and complexity of cell branches (Imaris software, Bitplane), fully confirmed that DFP exposure induced major microglial phenotypic changes, with cells showing a decrease in both branch number and length (Fig. 1J).

To further characterize the consequences of acute DFP poisoning on microglial morphological changes, we performed a cluster analysis of these cells in control and DFPtreated larvae, based on the five morphological parameters that significantly changed following DFP exposure, namely Sp, S, NB, ML and TL, as indicated above (see Materials and methods). Results showed that microglial cells could be clustered into three distinct populations in controls (Fig. 1K). The largest cluster comprised 47.1% of the cells and included microglia showing highly branched morphology and low sphericity, likely corresponding to “resting” microglia. The smallest cluster, corresponding to 14.7% of the cells, represented microglia with a low process number and a high sphericity, likely corresponding to M1-type “activated” microglia. The third cluster, which contained 38.2% of the cells, comprised microglia displaying both an intermediate branch number and an intermediate sphericity. We refer to these cells as “intermediate” microglia. In contrast, only two main clusters were observed in DFP-treated larvae (Fig. 1K). The larger one, which contained 73.1% of the cells, included microglia resembling “activated” microglia. The other cluster, comprising 25% of the cells, contained microglia showing the “intermediate” phenotype as defined above. It is of note that fewer than 2% of the microglia showed a "resting" phenotype in DFP-exposed larvae, compared to 47% in controls, suggesting that DFP caused a massive, brain-wide microglial activation.

Because the above data indicate that microglia displayed deep morphological changes after 6 h of acute DFP exposure, we next investigated the dynamics of these changes using real-time confocal imaging on live 5 dpf Tg[mpeg1:mCherryF] larvae during 6 h of exposure to 15 µM DFP. In agreement with our previous data [18], prior to DFP addition, microglia were highly branched with several long processes that permanently scan their environment and neighbour cells, and during the first 2 h of DFP exposure, no significant morphological changes could be detected (Supplementary Fig. 1 and supplementary Video 1). In contrast, from 2.5 h of exposure, clear remodelling of the cells was observed (Supplementary Fig. 1 and supplementary Video 1), which included an increased sphericity and a decrease in the length of the processes. After 3.5 h of exposure, almost all the cells displayed a phenotype resembling “activated” microglia (Supplementary Fig. 1 and supplementary Video 1).

Visual examination of supplementary Video 2 suggested that DFP poisoning markedly increased the distance travelled by microglial cell bodies. To confirm this observation, we tracked the distances travelled by each cell body during an approximately 30-minute period (Supplementary Fig. 2A), before exposure (Supplementary Fig. 2B and Supplementary Video 1) and after 5.5 h of DFP exposure (Supplementary Fig. 2C and Supplementary Video 2). Measurements of track lengths indicated that microglial cell bodies moved more in DFP‑treated larvae, with a 0.86 ± 0.27 µm/minute increase compared to controls (p < 0.001) (Supplementary Fig. 2D).

DFP exposure induced microglia-mediated overexpression of inflammatory cytokines

Microglia phenotypic changes observed in DFP-exposed larvae were highly reminiscent of those of "activated“ M1-type microglia observed in human epileptic brains [20], but also in DFP-exposed rats [21].

To confirm that the remodelling of microglia observed in DFP-treated larvae does reflect an M1-like inflammatory type of activation of these cells, we next investigated, by qRT-PCR analysis of whole-body RNAs, the expression levels of transcripts encoding a set of pro‑inflammatory (Il1β and Il8) and immuno-modulatory cytokines (Il4), before and at different time points during a 6-hour exposure to either 1% DMSO or 15 µM DFP. In close agreement with the results obtained in preclinical models of DFP intoxication [22–24], our data first revealed a massive expression of il1β (fold change (fc): 413 ± 67, p < 0.0001) and il8 (fc: 44 ± 6, p < 0.0005), together with a significantly increased expression of il4 transcripts (fc: 2.9 ± 0.4, p < 0.001) after 6 h of DFP exposure (Fig. 2A). Moreover, precise timing of the expression of these RNAs during DFP exposure indicated that significantly increased expression of il8 (fc: 1.6 ± 0.2, p < 0.05) and il1β (fc: 10.4 ± 2.4, p < 0.01) was observed after 1 h and 2 h of exposure, respectively, prior to increasing gradually after 3 h (fc: 3.3 ± 0.7, p < 0.001 and fc: 18.5 ± 3, p < 0.001), 4 h (fc: 26 ± 9, p < 0.001 and fc: 217 ± 28, p < 0.001) and 5 h of exposure (fc: 82 ± 9.7, p < 0.001 and fc: 125 ± 15.79, p < 0.001) (Fig. 2). In contrast, increased expression of il4 transcripts was delayed compared to that of il1β and il8, starting after 5 h of exposure to DFP (fc: 5.3 ± 0.56, p < 0.001) (Fig. 2).

To verify that the massive overexpression of pro-inflammatory cytokines observed in DFP-exposed larvae did reflect brain inflammation, we next investigated, by qRT-PCR analysis of RNAs extracted from dissected brains, the expression levels of transcripts encoding the same set of cytokines (Il1β, iI8, and Il4), in larvae exposed for 6 h to either DMSO or DFP. Results confirmed that a 6-h DFP exposure induced massive expression of il1β (fc: 189 ± 37, p < 0.01) and il8 (fc: 42 ± 19, p < 0.05), and increased expression of il4 transcripts (fc: 2.9 ± 0.7, p < 0.05) in the brain of exposed larvae (Fig. 3), confirming that DFP exposure induced bona fide brain inflammation.

Kinetics of neuronal activity in DFP-exposed larvae

We previously showed that larvae exposed to 15 µM DFP displayed massive neuronal hyperactivation from 1 h to 1.5 h of exposure to DFP [14]. To further investigate this point, we studied, by qRT-PCR analysis of RNAs extracted from larvae at different time points of DFP exposure, the temporal expression profile of fosab, an immediate early gene (IEG) whose expression is an early, sensitive marker of neuronal activation, especially epileptiform seizures [25]. A significantly increased expression of fosab transcripts was detected in larvae exposed to DFP for 1 h, (fc: 2.89 ± 0.4, p > 0.01), which then gradually increased over the next 5 h (fc: 21.7 ± 2.9, p < 0.001) (Fig. 4). This result suggests, in agreement with our previous calcium imaging data and the results in rodent models [8,14,23] that neuronal hyperactivation in zebrafish larvae is an early consequence of DFP poisoning that starts as early as 1 h post-exposure.

Inflammatory cytokines and neuronal activity in DFP-treated larvae without microglia

Two glial cell types mediate brain inflammation, including that induced by acute DFP exposure: microglial cells and astrocytes [26,27]. Accordingly, we next undertook to assess the role played by microglia in the neuroinflammatory process induced by DFP exposure. To this end, we analysed the expression levels of the same three cytokine RNAs in larvae fully devoid of microglia as the result of morpholino-oligonucleotide-mediated inactivation of the pU.1 gene, hereafter referred to as pU.1 morphants [28]. Results indicated that il1β and il8 transcripts were still overexpressed in pU.1 morphants exposed for 6 h to DFP, albeit at markedly lower levels than observed in their wild-type counterparts (fc: 134 ± 44.3 vs 413 ± 94, p < 0.01, and fc: 15.7 ± 4.7 vs 44 ± 6, p < 0.001 respectively), suggesting that while microglial cells are important players in DFP-induced neuroinflammation, other cells are also involved in the process. In contrast, no overexpression of il4 was detected in pU.1 morphants exposed to DFP (fc: 1.2 ± 0.2, p = 0,81) (Fig. 5), supporting the hypothesis that microglia were the main cell type overexpressing this cytokine following DFP exposure.

DFP-induced neuronal hyperactivation was markedly reduced in larvae without microglia

It has long been known that brain inflammation creates an environment that favours neuronal hyperexcitation and epileptogenesis [29]. Therefore, we next set out to evaluate the consequences of microglia activation and subsequent inflammation on the neuropathological processes induced by DFP poisoning. For this purpose, we used pU.1 morphants lacking microglia and showing reduced inflammatory response to DFP to study the consequences of the absence of microglia on DFP-induced neuronal activation as revealed by fosab transcript expression.

The results indicated that fosab RNAs were still overexpressed in pU.1 morphants exposed to DFP albeit at significantly reduced levels when compared to that observed in their wild-type counterparts (fc: 7.3 ± 1.5 vs 22.2 ± 2.9, p < 0.001) (Fig. 6), suggesting that DFP-induced neuronal hyperactivation was markedly reduced in larvae without microglia.

The first important finding of the present study is that acute DFP poisoning in zebrafish larvae rapidly triggered the activation of microglial cells and the synthesis of inflammatory mediators. Here we used in vivo imaging of microglia to describe the dynamics of microglia/macrophage activation after DFP exposure. These microglial morphological changes comprised a rounding of the cells and a decrease in both the number and length of their branches, associated with an increased distance travelled by microglial cell bodies. Interestingly, our cluster analysis further revealed the extent of this remodelling with the population of resting microglia, which decreased from 47.1–2% after 6 h of exposure, while that of activated microglia increased from 14.7–73.1% over the same period. Such phenotypic remodelling of microglia/macrophages has already been described and is characteristic of cells committed to an M1-like macrophage activation observed in different brain injury situations, including OP poisoning and various forms of epilepsy [20, 21]. We previously showed that zebrafish larvae exposed for 6 h to DFP displayed a marked neuronal hyperexcitation likely due to a shift in the synaptic excitation/inhibition balance towards an excitatory state associated with an increased number of apoptotic neurons in the brain [14]. The deep remodelling of microglia observed in DFP-exposed larvae might thus reflect an increase in the phagocytic capacities of these cells to cope with the increasing neuronal death and synaptic pruning. Moreover, consistent with the deep remodelling of microglia, we also showed that DFP induced a massive expression of inflammatory cytokines Il1β and Il8, confirming that DFP poisoning induced a massive and brain-wide activation of microglial cells towards an M1-like inflammatory phenotype. These results are in close agreement with the data from rodent models of DFP poisoning, which showed that DFP exposure triggers a robust neuroinflammatory response resulting from the activation of both microglia and astrocytes [10, 21, 23, 30–33].

In preclinical rodent models, several teams have reported that DFP poisoning triggers a neuroinflammatory response as early as 1–2 h post-exposure [23, 31], making this response an early event in the physiopathology of OP poisoning. Using real-time recording of microglial morphological changes in live transgenic Tg[mpeg1:mCherryF] larvae, we first showed here that phenotypic reprogramming could be detected after approximately 2.5 h of DFP exposure and was clearly seen after 3.5 h of exposure. This result was further refined by the qRT-PCR analysis of the expression of RNAs encoding inflammatory cytokine, which showed significant increases of il8 and il1β transcripts after 1 h and 2 h of DFP exposure, respectively. In the zebrafish DFP model, the increased expression of both il8 and fosab RNAs was detected after 1 h of exposure, making it difficult to establish a causal link between neuronal hyperactivation and neuroinflammation. Thus, as was shown in rodents, in the zebrafish acute DFP model, microglia-mediated neuroinflammation is an early event in OP poisoning that starts as early as 1 h post-exposure. Several authors have hypothesized that neuroinflammation of the brain observed after DFP exposure, and more generally OP poisoning, is the consequence of the excitotoxicity induced by the massive release of glutamate after the overstimulation of AChRs in brain neurons. Data have shown that the severity of seizures in rodents acutely exposed to DFP is positively correlated with the neuroinflammatory response [34]. Moreover, administration of either a low dose of anaesthetic urethane or diazepam to rats during early stages of DFP intoxication, 1 h or 10 minutes respectively, markedly mitigated not only neuronal hyperactivation, but also microglia activation and astrogliosis [30, 32]. More generally, it has long been known that epileptic seizures trigger a marked neuroinflammatory response that includes the differentiation of microglial cells towards an activated state and the production of inflammatory mediators [35, 36]. In human patients with pharmaco-resistant epilepsy, postmortem analysis of brain tissues revealed a significant microglial activation, which was directly correlated with seizure severity [20, 36]. Thus, although the hypothesis of a direct effect of DFP on microglia activation cannot be formally ruled out, the massive microglia-mediated inflammation observed in DFP-exposed larvae is likely caused, directly or indirectly, by the overactivation of cholinergic neuronal networks in the brain.

Using a genetic method, we produced larvae completely devoid of microglia and showed that they displayed a markedly reduced inflammatory response to DFP compared to that of their wild-type counterparts. In particular, we observed an approximately four-fold reduction in the expression levels of il1β and il8 transcripts, strong evidence that microglia play a major role in the inflammatory response following DFP poisoning. However, the inflammatory response observed in larvae without microglia, albeit reduced, also suggests that other cell types are involved in the process, likely activated astrocytes. Thus, as already described in DFP-exposed rats [21] and mice [37], our data suggest that DFP-induced neuroinflammation in zebrafish larvae is first mainly mediated by the activation of microglia. In contrast, the expression of RNAs encoding the immuno-modulatory Il4 cytokine, which was increased in the brain of larvae exposed for 5 h and 6 h to DFP, was not augmented in microglia-depleted individuals similarly exposed, suggesting that overexpression of this cytokine is mainly mediated by microglial cells in exposed larvae. This result also suggests that following DFP poisoning, microglia first respond through M1-like activation and synthesis of inflammatory mediators, followed, a few hours later, by the expression of regulatory mediators, possibly reflecting an attempt by these cells to restore brain homeostasis. Previous results had already shown that in addition to inflammatory substances, production of anti-inflammatory mediators is also increased in microglia after epileptic seizures, demonstrating that the microglial response to seizures is not limited to the classic M1-like pro-inflammatory activation [38]. However, it is not known whether the same or distinct microglial populations are involved in the two types of responses. Future work will need to clarify this point.

Using pU.1 morphants, which are devoid of microglia and displayed a significantly reduced inflammatory response to DFP intoxication, we showed that DFP-induced overexpression of fosab RNAs was significantly reduced in larvae without microglia. This result suggests that while neuronal activation was first a direct consequence of DFP-induced CNS AChR overstimulation, inflammatory mediators synthesized by activated microglia further exacerbated neuronal activation. Moreover, the relationship between epileptic seizures and brain inflammation is complex, since besides the neuronal activation-induced neuroinflammatory process mentioned above, inflammatory stimuli have also been identified as causative agents of epileptogenesis [39–42], suggesting a possible vicious circle involving the two processes. In particular, it has been shown that inflammatory Il1β is widely implicated in epileptogenesis [29, 35, 37] and, more importantly, that pharmacological inhibition of Il1β signaling with anakinra, a modified recombinant isoform of the human Il1R agonist, Il1Ra, was shown to drastically reduce seizure numbers in epileptic patients unresponsive to conventional anti-epileptic drugs [43–47]. In particular, data suggested that Il1β might play a role in the pathophysiology of epilepsy through increasing glutamatergic signalling [48] and N-methyl-d-aspartate (NMDA) receptor activity [49], and decreasing GABAergic transmission [50].

In conclusion, our results confirm that the zebrafish model of acute DFP poisoning precisely reproduces the key pathological features observed in rodent preclinical models, including AChE inhibition, epileptiform seizures, neuronal death, and microglia-mediated brain inflammation. In addition, we found that larvae lacking microglia displayed markedly reduced neuronal activation following DFP exposure, suggesting that microglia-mediated neuroinflammation further potentiates DFP-induced neuronal network hyperactivation. Microglia therefore appear as a key part of a vicious circle involving neuronal activation and neuroinflammation following DFP poisoning. These cells could thus be a therapeutic target to identify substances mitigating neuroinflammatory processes. They could thereby add to existing antidote cocktails and improve their efficacy.

DFP

Diisopropylfluorophosphate

Organophosphate

AChE

Acetylcholinesterase

ACh

Acetylcholine

AChR

Acetylcholine receptor

Status epilepticus

OPIDN

OPinduced delayed neuropathy

PTU

Phenyl thiourea

2-PAM

Pralidoxime

GABA

γaminobutyric acid

dpf

days post-fertilization

Sphericity

Surface area

Volume

Number of branches

Mean branch length

Total branch length

cDNA

complementary Deoxyribonucleic acid

qRT-PCR

quantitative Real-Time Reverse Transcription PCR

RNAs

Ribonucleic acids

IL1β

Interleukin-1ß

IL8

Interleukin-8

IL4

Interleukin 4

DMSO

Dimethyl sulfoxide

IEG

Immediate early gene. NMDA:Nmethyldaspartate.

Ethical Approval and Consent to participate

All animal experiments performed in this study were conducted at the French National Institute of Health and Medical Research (Inserm) UMR 1141 in Paris in accordance with European Union guidelines for the handling of laboratory animals and approved by the Direction Départementale de la Protection des Populations de Paris and the French Animal Ethics Committee.

Consent for publication

Not applicable.

Availability of data and materials

The data sets generated for this study are available on request to the corresponding author.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the French Agence Innovation Défense (AID), by the Institut National de la Santé et de la Recherche Médicale (INSERM), and the National Centre for Scientific Research (CNRS). Funding sources had no involvement in study design, collection, analysis or interpretation of data, nor decision to publish.

Authors' contributions

JS performed experiments, contributed to the analyses and interpretation of the data, and to the writing of the manuscript. CY co-led the project and contributed to the writing of the article. NSY led the project, designed the experimental research, and contributed to the interpretation of the data and writing of the manuscript.

Acknowledgements

We thank Christiane Romain and Olivier Bar (Inserm UMR 1141) for their technical assistance.

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No competing interests reported.

Somkhitetal.SupplFigure.1.tiff
Supplementary Fig. 1. Dynamics of microglia remodelling in DFP-exposed embryosSelected snapshot views of Video 1 showing the dynamics of microglia morphological changes in a living 5 dpf Tg[mpeg1:mCherryF] larva, before (0 h) and at different time points of a 6-hour exposure to 15 µM DFP. Coloured arrowheads indicate individual microglial cells. Scale bar represents 20 µm.
Somkhitetal.SupplFigure.2.tiff
Supplementary Fig. 2. DFP exposure increased the mobility of microgliaTimeline of treatment with red lines indicating the two 30-minute imaging periods. (B-C) Tracking of the cell body movements of microglia over a 30-minute period, before (B) and after 5.5 h of DFP exposure (C). The small coloured spheres correspond to the location of the cell body of individual cells at the start of the recordings. Scale bar represents 15 µm. (D) Mean distance travelled by microglial cells per minute, before and after DFP exposure (N = 5 larvae, n = 48 cells). Error bars on all graphs represent the standard error of the mean (SEM). Statistics: ***; p < 0.001.
Somkhitetal.SupplVideo1.avi
Supplementary Video 1: Microglial dynamics in 5 dpf larva exposed to 1% DMSO Time-lapse video of a dorsal view of the optic tectum of a 5 dpf larva exposed to 1% DMSO for 6 h. The video shows the last 45 minutes of the treatment, showing the dynamics of microglia, which mainly use their processes to monitor neighbouring cells. Time interval between frames: 40s. Video speed: 17 frames/s.
Somkhitetal.SupplVideo2.avi
Supplementary Video 2: Microglial dynamics in 5 dpf larva exposed to DFP.Time-lapse video of a dorsal view of the optic tectum of a 5 dpf larva exposed to 15 µM DFP for 6 h. The video shows the last 45 minutes of the treatment, showing the dynamics of microglia, which cell bodies appear to be more mobile than those of control larvae. Time interval between frames: 40s. Video speed: 17 frames/s.

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Microglia remodelling and neuroinflammation parallel neuronal hyperactivation following acute organophosphate poisoning

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DFP exposure induced dramatic phenotypic remodelling of microglia

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