Shifts in the spatiotemporal profile of inflammatory phenotypes of innate immune cells in the rat brain following acute intoxication with the organophosphate diisopropylfluorophosphate

Abstract

Acute intoxication with cholinesterase inhibiting organophosphates (OP) can produce life-threatening cholinergic crisis and status epilepticus (SE). Survivors often develop long-term neurological consequences, including spontaneous recurrent seizures (SRS) and impaired cognition. Numerous studies implicate OP-induced neuroinflammation as a pathogenic mechanism contributing to these chronic sequelae; however, little is known about the inflammatory phenotype of innate immune cells in the brain following acute OP intoxication. Thus, the aim of this study was to characterize the natural history of microglial and astrocytic inflammatory phenotypes following acute intoxication with the OP, diisopropylfluorophosphate (DFP). Adult male and female Sprague-Dawley rats were administered a single dose of DFP (4 mg/kg, sc) followed by standard medical countermeasures. Within minutes, animals developed benzodiazepine-resistant SE as determined by monitoring seizures using a modified Racine scale. At 1, 3, 7, 14, and 28 d post-exposure (DPE), neuroinflammation was assessed using translocator protein (TSPO) positron emission tomography (PET) and magnetic resonance imaging (MRI). In both sexes, we observed consistently elevated radiotracer uptake across all examined brain regions and time points. A separate group of animals was euthanized at these same time points to collect tissues for immunohistochemical analyses. Colocalization of IBA-1, a marker for microglia, with iNOS or Arg1 was used to identify pro- and anti-inflammatory microglia, respectively; colocalization of GFAP, a marker for astrocytes, with C3 or S100A10, pro- and anti-inflammatory astrocytes, respectively. We observed shifts in the inflammatory profiles of microglia and astrocyte populations during the first month post-intoxication, largely in hyperintense inflammatory lesions in the piriform cortex and amygdala regions. In these areas, iNOS⁺ proinflammatory microglial cell density peaked at 3 and 7 DPE, while anti-inflammatory Arg1⁺ microglia cell density peaked at 14 DPE. Pro- and anti-inflammatory astrocytes emerged within 7 DPE, and roughly equal ratios of C3⁺ pro-inflammatory and S100A10⁺ anti-inflammatory astrocytes persisted at 28 DPE. In summary, microglia and astrocytes adopted mixed inflammatory phenotypes post-OP intoxication, which evolved over one month post exposure. These activated cell populations were most prominent in the piriform and amygdala areas and were more abundant in males compared to females. The temporal relationship between microglial and astrocytic responses suggests that initial microglial activity may influence delayed, persistent astrocytic responses. Further, our findings identify putative windows for inhibition of OP-induced neuroinflammatory responses in both sexes to evaluate the therapeutic benefit of anti-inflammation in this context.

Keywords: Astrocytes; Microglia; Nerve agent; Pesticide; Seizures; Status epilepticus.

Fig. 1

a Adult male and female Sprague–Dawley rats were administered DFP followed one min later by atropine sulfate (AS) and 2-pralidoxime (2-PAM). Control animals were administered vehicle (Veh) in place of DFP. At 40 and 50 min post-DFP intoxication, animals received midazolam (MDZ). Behavioral seizures were monitored over the first 4 h post-intoxication. At 1, 3, 7, 14, and 28 d post exposure (DPE), animals were either subjected to translocator protein (TSPO) positron emission tomography (PET) and magnetic resonance imaging (MRI) or euthanized and brains harvested for histological analyses. b Modified Racine scale used to evaluate behavioral seizures. Only those animals with an average seizure score of ≥ 2.5 prior to MDZ intervention were included for the remainder of the study (5/42 males and 5/42 females were excluded). c, d Behavioral seizure scores in males (c) and females (d) after administration of DFP or Veh. Vertical dotted lines indicate the timing of MDZ treatment. Data are presented as mean ± SD (n = 20–21 Veh and 37 DFP animals per sex)

Fig. 2

a Longitudinal [¹⁸F]DPA-714 PET SUVR_Q1 maps. Data are overlaid on corresponding T₂-weighted images from the same animals. The [¹⁸F]DPA-714 uptake observed in the Veh animal at 1 DPE (left column, also Supplementary Fig. 1) is largely due to non-specific binding. By comparison, male and female DFP animals (right columns) display significant radiotracer uptake in the amygdala and piriform cortex (left arrow), hippocampus (down arrow), and thalamus (up-left arrow). In all scans there is minor signal penetration from outside the brain along the skull and jaw bones due to [¹⁸F] binding in bone following defluorination of the radiotracer. b, c Regional [¹⁸F]DPA-714 uptake data quantified by standard uptake value (SUV) analysis normalized to the SUV derived from the first quartile of a cerebellar reference region (SUVR_Q1). Geometric mean ratio (GMR, dot) of the mean SUVR_Q1 with 95% confidence intervals (CIs, bars) in (b) male (n = 3–4 Veh; 7–9 DFP) and (c) female (n = 3–5 Veh, 6–9 DFP). CIs that do not include 1 (the gray horizontal line) and are shaded blue indicate a significant difference between SUVR_Q1 in DFP vs. Veh after FDR correction

Fig. 3

a Representative photomicrographs of piriform cortex-amygdala immunostained for IBA1 (red) to identify microglia and counterstained with DAPI (blue) to identify cell nuclei. b, c Geometric mean ratio (GMR) (dot) of the mean density of IBA1⁺ cells in various brain regions of animals intoxicated with DFP relative to Veh at 1, 3, 7, 14, and 28 DPE with 95% confidence intervals (bars) in males (b) and females (c). The y-axis is shown as a log-scale. Confidence intervals that do not include 1 (the gray horizontal line) and are shaded blue indicate a significant difference in the density of IBA1⁺ nuclei between DFP and Veh after FDR correction

Fig. 4

a Representative photomicrographs of piriform cortex-amygdala immunostained for IBA1 (red) to identify microglia and iNOS (green) to identify pro-inflammatory cells, then counterstained with DAPI (blue) to identify cell nuclei. Solid boxes identify the field in the lower magnification image that is shown at higher magnification. b, c (GMR) (dot) of the mean density of iNOS⁺ IBA1⁺ cells in various brain regions of animals intoxicated with DFP relative to Veh at 1, 3, 7, 14, and 28 DPE with 95% CI (bars) in males (b) and females (c). The y-axis is shown as a log-scale. CI that does not include 1 (the gray horizontal line) and is shaded blue indicates a significant difference in the density of iNOS⁺ IBA1⁺ nuclei between DFP and Veh after FDR correction

Fig. 5

a Representative photomicrographs of piriform cortex-amygdala immunostained for IBA1 (red) to identify microglia and Arg1 (green) to identify anti-inflammatory cells, then counter-stained with DAPI (blue) to identify cell nuclei as observed at lower and higher magnification. Solid boxes identify the field in the lower magnification image that is shown at higher magnification. b, c GMR (dot) of the mean density of Arg1⁺ IBA1⁺ nuclei in in various brain regions of animals intoxicated with DFP relative to Veh at 1, 3, 7, 14, and 28 DPE with 95% CI (bars) in males (b) and females (c). b In males, differences in Arg1⁺ IBA1⁺ nuclei density did not vary with DPE so overall difference by brain region is displayed. The y-axis is shown as a log-scale. A CI that does not include 1 (the gray horizontal line) and is shaded blue indicates a significant difference in the density of Arg1⁺ IBA1⁺ nuclei between DFP and Veh after FDR correction

Fig. 6

a Representative photomicrographs of piriform cortex-amygdala immunostained for GFAP (green) to identify astrocytes and counterstained with DAPI (blue) to identify cell nuclei. b, c GMR (dot) of the mean density of GFAP⁺ nuclei in in various brain regions of animals intoxicated with DFP relative to Veh at 1, 3, 7, 14 and 28 DPE with 95% CI (bars) in males (b) and females (c). b In males, differences in GFAP⁺ nuclei density did not vary across DPE, so overall difference by brain region is displayed. The y-axis is shown as a log-scale. A CI that does not include 1 (the gray horizontal line) and is shaded blue indicates a significant difference in the density of GFAP⁺ nuclei between DFP and Veh after FDR correction

Fig. 7

a Representative photomicrographs of piriform cortex-amygdala immunostained for GFAP (green) to identify astrocytes, C3 (red) to identify pro-inflammatory cells, and counterstained with DAPI (blue) to identify cell nuclei shown at lower and higher magnifications. Solid boxes identify the field in the lower magnification image that is shown at higher magnification. b, c Geometric mean ratio (GMR) (dot) of the mean C3⁺ GFAP⁺ nuclei density in various brain regions of animals intoxicated with DFP relative to Veh at 1, 3, 7, 14, and 28 DPE with 95% confidence intervals (bars) in males (b) and females (c). b In males, differences in C3⁺ GFAP⁺ nuclei density did not vary DPE so overall difference by brain region is displayed. The y-axis is shown as a log-scale. Confidence intervals that do not include 1 (the gray horizontal line) and are shaded blue indicate a significant difference in the density of C3⁺ GFAP⁺ nuclei between DFP and Veh after FDR correction

Fig. 8

a Representative photomicrographs of piriform cortex-amygdala immunostained for GFAP (green) to identify astrocytes and S100A10 (red) to identify anti-inflammatory cells, then counterstained with DAPI (blue) to identify cell nuclei. Solid boxes identify the field in the lower magnification image that is shown at higher magnification. b, c GMR (dot) of the mean density of S100A10⁺ GFAP⁺ nuclei in various brain regions of animals intoxicated with DFP relative to Veh at 1, 3, 7, 14, and 28 DPE with 95% CI (bars) in males (b) and females (c). b In males, differences in S100A10⁺ GFAP⁺ nuclei density did not vary across DPE, so overall difference by brain region is displayed. The y-axis is shown as a log-scale. A CI that does not include 1 (the gray horizontal line) and is shaded blue indicates a significant difference in the density of S100A10⁺ GFAP⁺ nuclei between DFP and Veh after FDR correction

Fig. 9

Simplified temporal schematic representing the density of (a) microglial and (b) astrocytic pro- and anti-inflammatory cells, red and blue, respectively, in the amygdala and piriform cortex of male rats over 28 days following acute DFP intoxication