Time-sequential Neuroinflammatory Response After Organophosphate-induced Status Epilepticus

doi:10.21203/rs.3.rs-72760/v1

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Time-sequential Neuroinflammatory Response After Organophosphate-induced Status Epilepticus

https://doi.org/10.21203/rs.3.rs-72760/v1

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Background: Organophosphate compounds (OPs), either pesticides or chemical warfare agents are characterized by irreversible cholinesterase inhibition. In addition to severe peripheral symptoms, high doses of OPs can lead to seizures and status epilepticus (SE). Long lasting seizure activity and subsequent neurodegeneration promote neuroinflammation leading to profound pathological alterations of the brain. The aim of this study was to characterize neuroinflammatory responses at key time points after DFP-induced SE.

Methods: SE was induced by diisopropylfluorophosphate (DFP) injection in male Swiss mice. Immunohistochemistry (IHC) analyses of microglial (Iba1) and astrocytic (GFAP) cell markers were studied at 1, 4, 24 h and 3 days post-SE in the hippocampus. In parallel, using RT-qPCR, microglial and astrocytic phenotype markers were quantified on isolated CD11B (microglia) and GLAST (astrocytes)-positive cells after DFP-induced SE.

Results: At earlier stages (1-4 h) after SE, although IHC analysis presented no modification, an increase in pro-inflammatory (M1-like) markers and A2-specific markers was observed in CD11B and GLAST-positive isolated cells respectively. Microglial cells sequentially expressed immuno-regulatory (M2b-like) and anti-inflammatory (M2a-like) at 4 h and 24 h of SE induction. At later stages (24 h and 3 days), microglial and astrocytic activations were visible by IHC and Iba1-positive cells were increased in number in DFP animals compared to controls. Finally, at these later stages, A1-specific markers were increased in isolated astrocytes.

Conclusions: Our work identified sequential microglial and astrocytic phenotype activations. Although the role of each phenotype in SE cerebral consequences requires further study, targeting specific markers at specific time point could be a beneficial strategy for OP-induced SE treatment.

Neurobiology of Disease

Astrocyte

microglia

cytokines

seizure

diisopropylfluorophosphate

organophosphates

organophosphorus

Exposure to organophosphate compounds (OPs), widely used as chemical warfare agents and pesticides, represent a threat for both civilian and military communities. In addition to severe peripheral symptoms, high doses of OPs can elicit seizures that rapidly progress to status epilepticus (SE). SE is a life-threatening neurological emergency, characterized by continuous seizures without full recovery resulting in later neuronal damage, cognitive alterations and epileptogenesis [1]. OPs act as potent irreversible inhibitors of both central and peripheral cholinesterases (ChEs), resulting in acetylcholine accumulation and therefore uncontrolled activation of cholinergic receptors. Centrally, excessive activation at muscarinic receptors by acetylcholine can initiate extended seizures [2] evolving to self-sustained SE with the additional involvement of glutamatergic and GABAergic systems imbalance. The first-line treatment of OP poisoning consists of the injection of a muscarinic antagonist (e.g. atropine sulfate) combined with an oxime acting mainly as a peripheral ChE re-activator (e.g. pralidoxime or obidoxime). Additionally, the administration of an anticonvulsant (a benzodiazepine or a prodrug like avizafone) is currently indicated in the early phase of OP poisoning in order to control seizures and SE. However, experimental data in line with clinical experience clearly show that approximately 30–40 min after the initial seizures, SE becomes refractory to benzodiazepines and classical anti-epileptic drugs [3]. In animal models, thirty minutes of unabated SE is also associated to severe seizure-related brain damage [4–7] and long-term consequences as epilepsy and cognitive decline [1].

Excitotoxic neuronal damage and seizure activity following SE development activate a neuroinflammatory response with marked microglia and astrocytes activations (7–10). Conversely, neuroinflammatory mechanisms have been implicated in SE-induced excitotoxic neuronal death, epileptogenesis and drug-resistance [8–11]. Reactive microglia and astrocytes can both have either protective or deleterious effects suggesting functional heterogeneity for both cell types. Based on in vitro experiments, microglia has been categorized into pro-inflammatory (M1-like), anti-inflammatory (M2a-like) and immuno-regulatory (M2b-like) subtypes [12–14]. Likewise, reactive astrocytes have also been recently differentiated in A1 and A2 phenotypes [15, 16]. Although the different subtypes are more likely a continuum with possibly numerous sub-classes, the different subtypes help our understanding of the complex neuroinflammatory mechanisms taking place after a neurological insult as SE.

We have recently developed and characterized a new convulsive mouse model of OP exposure using diisopropylfluorophosphate (DFP) [17]. In this model, DFP induces electrographic SE within 20 min and all seizing animals develop long-lasting SE. To study neuroinflammation in this model, we choose 4 key time points: 1 h after DFP injection corresponding to the onset of benzodiazepine resistance, 4 h corresponding to beginning of neuronal suffering observed by the marker FluoroJade C, 24 h corresponding to the peak of neurodegeneration and 3 days corresponding to early epileptogenesis.

Here, we show with histological techniques that DFP-induced SE is associated to microglia and astrocyte activations in the hippocampus. We choose to focus on the hippocampus given the crucial role of this structure in seizure development and epileptogenesis. We further characterize changes in microglial activation state with specific cell sorting followed by RT-qPCR, identifying sequential pro-inflammatory, immuno-modulatory and anti-inflammatory markers up-regulation beginning 1 hour after DFP exposure. Using the same technique, we observed that A2 astrocyte markers up-regulation precedes the A1 markers up-regulation. These data should help to understand microglial and astrocytic activations kinetics and adapt treatment paradigm in order to address the different phases of neuroinflammation and increase therapeutic impact.

Animals

Adult male Swiss mice (8 weeks, Janvier Labs, Le Genest-Saint-Isle, France) were housed four per cage with a 12 h/12 h light/dark cycle and food and water ad libitum. A period of 5–7 days of acclimation to housing conditions was observed. All animals were 9 weeks old when exposed to DFP. Animal experiments were approved by the ethics committee according to applicable French legislations, in compliance with the “3Rs” policy of reduction, refinement and replacement of animal use for scientific procedures (Directive 2010/63/UE, décret 2013 − 118, approval number 9642-2017040316305252).

Drugs

DFP (Sigma Aldrich, L'Isle d'Abeau Chesnes, France) was freshly prepared by diluting the initial concentrated solution (0,1 g/ml in isopropanol) in ice-cold 0.9% (w/v) saline. The oxime HI-6 (1-2-hydroxy-iminomethyl-1-pyridino-2-oxanopropane) dichloride was a generous gift of DRDC Suffield (Canada). Atropine sulfate (AS, Sigma Aldrich, L'Isle d'Abeau Chesnes, France) and HI-6 were diluted in 0.9% (w/v) saline. Veterinary Diazepam (TVM, Lempdes, France) was used undiluted.

DFP exposure

The day of the experiment, mice received an injection of DFP (3 LD50 = 9.93 mg/kg, LD50 calculated without HI-6 and AS post-treatment, 10 ml/kg, s.c.) followed by combined i.p. injection of HI-6 (50 mg/kg) and AS (3 mg/kg) at + 1 min of DFP injection. HI-6 and AS post-treatment is used to reduce respiratory distress and mortality. As previously shown [17], the OP exposure protocol used in this project caused reduced mortality. In this project, the mortality rate in the DFP treated animals was < 10%, death occurring in the first 30 minutes after DFP injection. Protocol used for drugs administration is depicted in Fig. 1.

Except for the 1 h experimental group, all animals received Diazepam (10 mg/kg, i.p.), eighty minutes after DFP injection, corresponding to 1 h long SE. Diazepam was used to minimize the impact of SE on animal welfare and to homogenize SE duration between animals. Control animals received all treatments after vehicle injection without DFP.

Based on our previous work using electrocorticography in parallel of behavioral signs evaluation of seizing vs nonseizing animals [17], in this work observation of intoxication signs after DFP exposure allow to identify SE animals.

Immunofluorescence

After deep anesthesia, animals were transcardially perfused with 10 ml of 0.9% NaCl/5 UI/ml heparin, followed by 50 ml of 4% paraformaldehyde (PFA) in phosphate buffer. After overnight post-fixation in 4% PFA, brains were cryoprotected 24 h in 30% sucrose and then frozen 1 min in -40 °C isopentane. Thirty µm coronal sections were immunolabeled with anti-Iba1 (1:1000, Wako) and anti-GFAP (1:1000, Millipore) antibodies overnight at room temperature. Brain sections were then washed in PBS and subsequently incubated for 1 h at room temperature in 1:500 TRITC anti-rabbit IgG and 1:500 Alexa Fluor 488 anti-mouse IgG (Invitrogen). For each animal, analyses were conducted on images acquired at 3 rostrocaudal levels separated by 180 µm centered on Fig. 48 of Franklin and Paxinos mouse brain atlas (3rd edition). For each image, using Fiji software, regions of interest were delimited in the CA1, CA3 and dentate gyrus hippocampal regions. The same regions of interest were used for Iba1 cells counting and Iba1 and GFAP immunodensity evaluations. The final results were expressed as % of positive area and % of mean control values for Iba1 and GFAP immunodensities and in cells number/mm² for Iba1 cells counts.

Neural tissue dissociation and magnetic activated cell sorting

Brains were collected (1 animal per sorting) for cell dissociation and microglial cells and astrocytes were enriched using a magnetic-bead-coupled antibody (anti-CD11B and anti-GLAST respectively) extraction technique (MACS), as previously described and according to the manufacturer's protocol ([18]; Miltenyi Biotec, Bergisch Gladbach, Germany). In brief, after removing the cerebellum and olfactory bulbs, each brain was dissociated using the neural tissue dissociation kit. From the resulting brain homogenates, microglial cells were enriched by MACS using anti-CD11B microbeads, and after elution, isolated cells were centrifuged for 10 min at 300 g and conserved at -80 °C. Astrocytes were enriched by MACS using anti-GLAST microbeads on the remaining unretained cells and collected similarly to microglial cells. Microglial cells and astrocytes were sorted 1 h, 4 h, 24 h and 3 d after vehicle/DFP injection in control (n = 7–8 /time point) and DFP (n = 12–16/time point) mice.

RNA Extraction and RT-qPCR

Total RNA from isolated microglial cells and astrocytes were extracted with NucleoSpin ® RNA Plus XS kit according to the manufacturer’s instructions (Macherey-Nagel, Hoerdt, France). Purified RNA quality and concentration were assessed by spectrophotometry using a NanoDropTM apparatus (Thermoscientific, Wilmington, DE). 200–400 ng of total RNA was subjected to reverse transcription using the IscriptTM cDNA synthesis kit (Bio-Rad, Marnes-la-Coquette, France). RT-qPCR was performed in triplicate for each sample on a CFX384 Real Time System (Bio-Rad), using SYBR Green Supermix (Bio-Rad) for 40 cycles. Amplification specificity was assessed by melting curve analysis. Primers were designed using Primer3 software and manufactured by Eurofins Genomics (Ebersberg, Germany). Primer sequences are summarized in Table S1. For expression analyses of CD11B-positive cells, for each sample, the expression of genes of interest was calculated relative to the expression of the reference gene TATA-box binding protein (Tbp). For expression analyses of GLAST-positive cells, for each sample, the expression of genes of interest was calculated relative to the expression of the reference gene ribosomal protein L13a (Rpl13a). The final results are presented as fold change over control mean values. Analyses were performed using Bio-Rad CFX Manager 3.0.

Statistical analysis

Data were analyzed using PRISM 7 software (GraphPad, San Diego, CA, U.S.A.). Data were expressed as means \(\pm\) standard errors of the mean (SEM). Statistical comparison to control group was performed using the Mann-Whitney test. Sample sizes are indicated in figures.

Microglial and astrocytic activations 1 hour after DFP exposure

To determine glial activation state at the time of SE drug refractoriness, we quantified microglial and astrocytic activations 1 h after DFP exposure, corresponding to 40 min of SE according to our previous report [17]. We first undertook an immunohistochemical (IHC) analysis of Iba1-positive microglia and GFAP-positive astrocytes. One hour after DFP injection, Iba1-positive cells number and Iba1 immunodensity were not statistically different in DFP SE animals and control animals in CA1, CA3 and DG (Fig. 2a-c). On the same animals, GFAP immunodensity was neither altered by SE (Fig. 2a and 2d). Given the intertwining of astrocyte processes, we were unable to quantified GFAP-positive cells number at any time point.

We further studied microglial and astrocytic activations using expression analysis of markers of polarized states. Gene expression analysis was performed after CD11B-positive (microglia/infiltrating macrophages) and GLAST-positive (astrocytes) cell sorting in DFP SE and control whole brains. In isolated CD11B-positive cells, quantitative RT-PCR experiments showed a differential increase of pro-inflammatory markers (Il1b, Tnf) but lack of immuno-regulatory (Il1rn, Il4ra, Socs3) and anti-inflammatory markers (Lgals3, Mrc1, Igf1, Il10) modulations (Fig. 2e). In GLAST-positive cells, quantitative RT-PCR experiments indicated an increase of A2-specific markers Ptgs2 and Ptx3, as well as Il6 cytokine mRNA but no A1-specific markers (Serping1, Ggta1, Amigo2, H2-D1) modifications (Fig. 2f).

Taken together, these findings indicate that 1 h after DFP injection, microglial cells very rapidly develop an activated state with a pro-inflammatory phenotype. Interestingly, in the same time, expression experiments on isolated astrocytes, allowed us to observe that astrocytes do not yet develop a neurotoxic A1-associated reactivity but an A2-associated reactivity associated to an Il6 mRNA up-regulation. This is supported by the lack of pro-inflammatory cytokines Ilb and Tnf mRNA up-regulations in GLAST-positive cells.

Microglial and astrocytic activations 4 hours after DFP exposure

Going forward, we next studied microglial and astrocytic activations 4 h after DFP injection. The IHC analysis showed no modifications of Iba1-positive cells number and immunodensity (Fig. 3a-3c). Similarly, GFAP-positive astrocyte immunodensity was not different in DFP SE animals compared to control animals (Fig. 3a and 3d).

While no modifications were visible in histology, RNA expression analysis allowed us to identify a combined pro-inflammatory and immuno-regulatory markers up-regulations in CD11B-positive microglia/infiltrated macrophages, associated to an increase of the anti-inflammatory marker, Lgals3 (Fig. 3e). In the same time, isolated GLAST-positive astrocytes developed a frank A2-associated phenotype with an up-regulation of Emp1, S100a10 and Cd109 A2-specific markers (Fig. 3f). As for the 1 h time point, this A2 activation state was associated to Il6 mRNA increase. No A1-specific markers were modified as Il1b and Tnf mRNA (Fig. 3f).

These findings indicate that 4 h after DFP injection, in absence of clear morphological modifications that could be visualized by IHC immunodensity, microglia develop in parallel to a pro-inflammatory phenotype an immuno-regulatory phenotype. These experiments could not evaluate if these 2 activated states were present in the same microglial population or in 2 different cell populations. At the same time point, astrocytic activation state did not change from the 1 h time point, astrocytes strengthen their A2 phenotype associated with an Il6 mRNA up-regulation. Interestingly, the A1 phenotype was also not observed at this time point.

Microglial and astrocytic activations 24 hours after DFP exposure

We have previously reported important neurodegeneration 24 h after DFP-induced SE [17]. At this time point, CA1, CA3 and DG hippocampal regions, as other brain regions, are affected by prominent neuronal damage. The IHC analysis uncovered an increase of Iba1 microglia/infiltrated macrophages cells number (Fig. 4a and 4b), as well as, an important increase of Iba1 immunodensity in CA1 and CA3 hippocampal regions (Fig. 4a and 4c). In these 2 regions, GFAP immunodensity was also increased (Fig. 4a and 4d). Conversely, also affected by brain damage at this time point [17], the DG region presented no elevation of Iba1, nor GFAP immunodensities.

These histologic modifications were associated for isolated CD11B-positive microglia/infiltrated macrophages, with the 3 microglial phenotypes activation: pro-inflammatory, immuno-regulatory and Lgals3 and Mrc1 anti-inflammatory markers up-regulations (Fig. 4e). Likewise, isolated GLAST-positive astrocytes develop different activation phenotypes. Both A1-specific markers (Serping1, Ggta1, H2-D1) and A2-specific markers (Ptx3, Emp1, S100a10, Cd109) were up-regulated (Fig. 4f). These astrocytic activation states were associated to prominent Il6 up-regulation with a no statistically increase of Il1b and Tnf transcripts levels in DFP SE animals compared to control animals (Fig. 4f).

These results indicate important neuroinflammatory activations 24 h after DFP-induced SE. Microglial cells present pro-inflammatory, immuno-regulatory and anti-inflammatory phenotypes. At the same time point, astrocytes present 2 types of activations states: A1 and A2 activation states with joint Il6 mRNA up-regulation.

Microglial and astrocytic activations 3 days after DFP exposure

We next studied microglial and astrocytic activations 3 days after DFP-induced SE. This time point has been widely described by others using IHC techniques and it is thought to be associated to early epileptogenesis [22]. In our model, IHC evaluation of neuroinflammation revealed marked Iba1-positive cell number increase in the 3 hippocampal regions studied (Fig. 5a and 5b). This was associated to an increase of Iba1 immunodensity in the CA3 and DG (Fig. 5a and 5c). Massive increase of GFAP immunodensity was also visible in the 3 hippocampal regions (Fig. 5a and 5d).

Quantitative RT-PCR experiments indicated marked activation of CD11B-positive isolated microglia/infiltrated macrophages. As for the 24 h time point, these cells developed 3 activation states with an increase of pro-inflammatory, immuno-regulatory and anti-inflammatory markers (Fig. 5e). Unlike the other genes, Mrc1 was down-regulated in DFP SE animals compared to control animals (Fig. 5e). In parallel, GLAST-positive astrocytes maintained their A1 and A2-associated activation states. These 2 activations states were however not associated to an increase of Il1b, Tnf or Il6 mRNA in DFP SE animals compared to control animals (Fig. 5f). In all, we observed that cytokines expression seemed more variable in DFP SE animals in astrocytes compared to microglia.

Taken together, these findings show frank microglial and astrocytes activation 3 days after DFP-induced SE. At this time point, the DG is particularly affected by both cell type activations. All described markers are present with an up-regulation of pro-inflammatory, immune-regulatory and anti-inflammatory markers in CD11B-positive cells and A1 and A2-associated reactive markers in GAST-positive astrocytes.

Long lasting seizure activity and neurodegeneration induced by high doses of OPs promote neuroinflammation leading to profound pathological alterations of the brain [5, 7, 17, 20, 23]. We characterize here neuroinflammatory responses at key time points after DFP-induced SE. We confronted classical IHC analysis to RT-qPCR experiments in order to decipher microglial and astrocytic reactive markers expression and extend our understanding of SE-related neuroinflammation. Compared to RT-qPCR on cerebral tissue, RT-qPCR after cell sorting allow to identify the cellular origin of inflammatory genes up-regulation and confers increased sensibility by concentrating a specific cell type. Discussions on the existence of differential M1 and M2 phenotypes in microglial cells is still existing and M1/M2 dichotomy is certainly an oversimplification [24–27]. However diverse opposing impacts of activated microglia on neuronal damage have been demonstrated and modulation of microglial cells polarization towards a M2 phenotype have been described as beneficial [24, 25, 28–30]. Based on differential molecular markers expressions, reactive astrocytes have been also subdivided in A1 and A2 [15]. A1 astrocytes that lose most normal astrocytic functions and gain neurotoxic function are associated to multiple human and experimental models of neurological insult [31–36]. Firstly described after ischemia and postulated to be beneficial as they were associated to up-regulation of many neurotrophic factors, the impact of A2 astrocytes upon neuroinflammatory injury is mostly unclear [15, 16]. It appears that both microglia and astrocytes polarizations are greatly dependent on the type of insult and the delay after the initial insult. Our current understanding of microglia polarization after SE is limited and astrocyte polarization has yet not been studied. Our study reveals sequential activations of microglial and astrocytic phenotypes.

As soon as 1 h post DFP injection, corresponding to 40 min of SE [17], we observe an early pro-inflammatory phenotype in microglia. Previous studies demonstrated that 30–40 min of SE was associated to benzodiazepine-refractoriness in different SE models [9, 19–21, 37–39]. It has been shown that the pro-inflammatory cytokine IL1β could play an important role in benzodiazepine response in SE induced by intrahippocampal injection of KA as IL-1β icv injection prolonged diazepam latency to terminate SE [9]. Furthermore, in this project, SE was responsive to diazepam at 40 min in Il1r1 knock-out mice compared to WT mice [9]. Our results show that, at this very early stage, Il1b mRNA increase is associated to microglia. At the same stage, neurotoxic A1 astrocytic activation is still absent, in accordance with the idea that pro-inflammatory microglia activation precedes pro-inflammatory astrocyte activation [40]. However, at this early time point, we observed an A2-associated reactive phenotype with the up-regulation of Ptgs2, coding for Cox2 protein, and Ptx3 mRNA. Our results further show that this up-regulation of A2-associated markers is concomitant to an Il6 increase. Expression of Il6 protein has been described in astrocytes 12 and 24 h after soman-induced SE in rats, but Il6 presence has not been studied at earlier stages [41]. IL6 is a pleiotropic cytokine, modulating the inflammatory response by exerting both protective and detrimental activities in neuronal tissue. Il6 icv injection has been shown to induce epileptogenesis with the occurrence of cortical seizures detected 3 days after the injection in a dose-dependent manner in C57Bl6 mice [42]. Although A2 astrocytes are thought to be beneficial, A2 astrocytes expressing Cox2 have been proposed as deleterious on oligodendrocyte progenitor cells maturation in a mouse model of neonatal white matter injury [43]. Overall, we described for the first time the early A2 activation phenotype of astrocytes after SE, and its implication in SE maintenance and neuronal degeneration has to be further assessed.

Using the same techniques, while 4 h after DFP injection no modifications were still noticeable by IHC, we observed an up-regulation of pro-inflammatory and immuno-regulatory markers in microglial cells. Interestingly, this time point is associated to a significant increase of Il1rn mRNA, which will persist up to 3 days post-injection, with a \(\tilde\)270 fold increase at 24 h (Table 1). IL1RA protein, encoded by IL1RN, binds the receptor IL1R1 and blocks IL1α and IL1β signaling. IL1β-IL1R1 signaling has been widely implicated in seizures occurrence and epileptogenesis (for review, [8, 44, 45]) and IL1β-IL1R1 signaling blockade has been shown to reduce seizure number, neurodegeneration and epileptogenesis [46–50]. In this regard, soman-induced SE was accompanied with a mild increase of neurodegeneration in Il1r1 and Il1rn knock-out mice but SE duration and severity have not been studied with EEG [51]. We did not observe Il1b mRNA up-regulation in isolated astrocytes at any time points. This is in accordance with the exclusive expression of Il1β at a protein level in activated microglia 24 h after soman-induced SE [41]. Astrocytic expression of Il1β is observed later in time from 4 days [52] in pilocarpine SE model, but discrepancies exist as Il1β has been observed in astrocytes earlier in the pilocarpine model by others [53] or other types of seizures [54–56].

At 24 h after DFP-induced SE, in accordance with the massive neurodegeneration observed in our model [17], both microglial and astrocytic activations were visualized by IHC analysis. In CD11B-positive isolated cells, while pro-inflammatory and immuno-regulatory markers were maintained, anti-inflammatory markers appeared up-regulated. This time point was associated to a \(\tilde\)72 fold increase of the Lgals3 mRNA, this increase persisting at 3 days post-SE (Table 1). Galectin-3 protein, encoded by Lgals3, released by microglia, plays a pivotal role in phagocytosis by binding targeted cells or bacteria [57]. Galectin-3-positive microglia were observed to engulf degenerative neurons in a model of ischemia by middle cerebral artery occlusion [55]. Furthermore, in this model Lgals3 knock-out mice presented increased cell death, suggesting a protective role of Galectin-3 after ischemia [58]. Although, Lgals3 mRNA is strongly up-regulated in microglia after pilocarpine-induced SE, Lgals3 knock-out mice presented a small reduction of cell death in the cortex and no difference in the hippocampus 3 days after pilocarpine injection compared to WT mice, suggesting that, after SE, Galectin-3 protective role is more marginal [59]. Meanwhile, in GLAST-positive isolated cells, while A2-specific markers were maintained, A1-specific markers appeared up-regulated for the first time after DFP exposure. The role of neurotoxic astrogliosis in neurodegeneration has been demonstrated and different mechanisms have been identified, such as, reduced glutamate clearance, adenosine cycle modifications, increased Ca²⁺ signaling and BBB dysfunction [60].

Finally, 3 days after DFP injection, corresponding to early epileptogenesis, previous works have observed strong neuroinflammation in different models of SE [21, 36, 61–63]. In our model, we observed a significant increase in Iba1-positive cell number in all hippocampal regions. This increase was already noticeable at 24 h but increased with time (Table 1). In a SE model induced by icv infusion of KA, Feng and colleagues have observed microglial proliferation and macrophages infiltration at both 24 h and 3 days SE [65]. As the Iba1 marker do not permit to differentiate microglial and infiltrated macrophages, we cannot state if the increases of Iba1-positive cells observed at 24 h and 3 days are due to microglia migration, proliferation or macrophages infiltration. Further work is needed to address this issue in our model. At this time point, in pilocarpine SE model, pro-inflammatory (M1-like) and anti-inflammatory microglial phenotypes (M2a-like) have been observed in the forebrain [64]. However, in SE model induced by intrahippocampal KA, the anti-inflammatory phenotype was not observed at the same time point, showing that models specificities exist [64].

Our work identified sequential microglial and astrocytic phenotypes activations. Given the very early neuroinflammatory modifications observed, targeting these modifications could be a beneficial strategy for the treatment of refractory SE and its cerebral consequences. This type of intervention should probably be provided very early in the course of the SE. Further comprehensive study of A2 reactive astrocytes role in SE is nevertheless needed. An adapted treatment paradigm in order to address specific phases of neuroinflammation could increase therapeutic impact.

AChE

acetylcholinesterase

atropine sulfate

CA1

cornu ammonis 1

CA3

cornu ammonis 3

ChEs

cholinesterases

DFP

Diisopropylfluorophosphate

dentate gyrus

HI-6

asoxime

icv

intracerebroventricular

IHC

immunohistochemistry

intraperitoneal

kainate

OPs

organophosphate compounds

subcutaneous

status epilepticus

SRBD

seizure-related brain damage

wild type

Ethics approval and consent to participate

A statement on ethics for the experiments involving animals is included in the manuscript.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the French Ministry of Armed Forces: Direction Générale de l’Armement (DGA) and Service de Santé des Armées (SSA). Funding sources had no involvement in study design, collection, analysis or interpretation of data, or decision to publish.

Authors’ contributions

C.M, J.E, N.D performed the experiments, the analysis and designed the figures. R.H.A and A.I. helped carry out the experiments. N.D. developed the study design, supervised the project and wrote the manuscript. S.A., N.S.Y, X.B, F.N, G.D contributed to the final manuscript.

Acknowledgments

This work used the imagery platform of IRBA supported by the Service de Santé des Armées. We particularly thank Mr. X. Butigieg for technical support.

Authors’ information

Not applicable

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Time-sequential Neuroinflammatory Response After Organophosphate-induced Status Epilepticus

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Abstract

Figures

Background

Methods

Animals

Drugs

DFP exposure

Immunofluorescence

Neural tissue dissociation and magnetic activated cell sorting

RNA Extraction and RT-qPCR

Statistical analysis

Results

Microglial and astrocytic activations 1 hour after DFP exposure

Microglial and astrocytic activations 4 hours after DFP exposure

Microglial and astrocytic activations 24 hours after DFP exposure

Microglial and astrocytic activations 3 days after DFP exposure

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

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