Acute intoxication with diisopropylfluorophosphate promotes cellular senescence in the adult male rat brain.

Acute intoxication with high levels of organophosphate (OP) cholinesterase inhibitors can cause cholinergic crisis, which is associated with acute, life-threatening parasympathomimetic symptoms, respiratory depression and seizures that can rapidly progress to status epilepticus (SE). Clinical and experimental data demonstrate that individuals who survive these acute neurotoxic effects often develop significant chronic morbidity, including behavioral deficits. The pathogenic mechanism(s) that link acute OP intoxication to chronic neurological deficits remain speculative. Cellular senescence has been linked to behavioral deficits associated with aging and neurodegenerative disease, but whether acute OP intoxication triggers cellular senescence in the brain has not been investigated. Here, we test this hypothesis in a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague-Dawley rats were administered DFP (4 mg/kg, s.c.). Control animals were administered an equal volume (300 µL) of sterile phosphate-buffered saline (s.c.). Both groups were subsequently injected with atropine sulfate (2 mg/kg, i.m.) and 2-pralidoxime (25 mg/kg, i.m.). DFP triggered seizure activity within minutes that rapidly progressed to SE, as determined using behavioral seizure criteria. Brains were collected from animals at 1, 3, and 6 months post-exposure for immunohistochemical analyses of p16, a biomarker of cellular senescence. While there was no immunohistochemical evidence of cellular senescence at 1-month post-exposure, at 3- and 6-months post-exposure, p16 immunoreactivity was significantly increased in the CA3 and dentate gyrus of the hippocampus, amygdala, piriform cortex and thalamus, but not the CA1 region of the hippocampus or the somatosensory cortex. Co-localization of p16 immunoreactivity with cell-specific biomarkers, specifically, NeuN, GFAP, S100ß, IBA1 and CD31, revealed that p16 expression in the brain of DFP animals is neuron-specific. The spatial distribution of p16-immunopositive cells overlapped with expression of senescence associated ß-galactosidase and with degenerating neurons identified by FluoroJade-C (FJC) staining. The co-occurrence of p16 and FJC was positively correlated. This study implicates cellular senescence as a novel pathogenic mechanism underlying the chronic neurological deficits observed in individuals who survive OP-induced cholinergic crisis.

Sex-specific acute and chronic neurotoxicity of acute diisopropylfluorophosphate (DFP)-intoxication in juvenile Sprague-Dawley rats.

Preclinical efforts to improve medical countermeasures against organophosphate (OP) chemical threat agents have largely focused on adult male models. However, age and sex have been shown to influence the neurotoxicity of repeated low-level OP exposure. Therefore, to determine the influence of sex and age on outcomes associated with acute OP intoxication, postnatal day 28 Sprague-Dawley male and female rats were exposed to the OP diisopropylfluorophosphate (DFP; 3.4 mg/kg, s.c.) or an equal volume of vehicle (∼80 µL saline, s.c.) followed by atropine sulfate (0.1 mg/kg, i.m.) and pralidoxime (2-PAM; 25 mg/kg, i.m.). Seizure activity was assessed during the first 4 h post-exposure using behavioral criteria and electroencephalographic (EEG) recordings. At 1 d post-exposure, acetylcholinesterase (AChE) activity was measured in cortical tissue, and at 1, 7, and 28 d post-exposure, brains were collected for neuropathologic analyses. At 1 month post-DFP, animals were analyzed for motor ability, learning and memory, and hippocampal neurogenesis. Acute DFP intoxication triggered more severe seizure behavior in males than females, which was supported by EEG recordings. DFP caused significant neurodegeneration and persistent microglial activation in numerous brain regions of both sexes, but astrogliosis occurred earlier and was more severe in males compared to females. DFP males and females exhibited pronounced memory deficits relative to sex-matched controls. In contrast, acute DFP intoxication altered hippocampal neurogenesis in males, but not females. These findings demonstrate that acute DFP intoxication triggers seizures in juvenile rats of both sexes, but the seizure severity varies by sex. Some, but not all, chronic neurotoxic outcomes also varied by sex. The spatiotemporal patterns of neurological damage suggest that microglial activation may be a more important factor than astrogliosis or altered neurogenesis in the pathogenesis of cognitive deficits in juvenile rats acutely intoxicated with OPs.

Mechanisms of organophosphate neurotoxicity.

The canonical mechanism of organophosphate (OP) neurotoxicity is the inhibition of acetylcholinesterase (AChE). However, multiple lines of evidence suggest that mechanisms in addition to or other than AChE inhibition contribute to the neurotoxic effects associated with acute and chronic OP exposures. Characterizing the role(s) of AChE inhibition versus noncholinergic mechanisms in OP neurotoxicity remains an active area of research with significant diagnostic and therapeutic implications. Here, we review recently published studies that provide mechanistic insights regarding (1) OP-induced status epilepticus, (2) long-term neurologic consequences of acute OP exposures, and (3) neurotoxic effects associated with repeated low-level OP exposures. Key data gaps and challenges are also discussed.

Co-localization of fluorescent signals using deep learning with Manders overlapping coefficient.

Object-based co-localization of fluorescent signals allows the assessment of interactions between two (or more) biological entities using spatial information. It relies on object identification with high accuracy to separate fluorescent signals from the background. Object detectors using convolutional neural networks (CNN) with annotated training samples could facilitate the process by detecting and counting fluorescent-labeled cells from fluorescence photomicrographs. However, datasets containing segmented annotations of colocalized cells are generally not available, and creating a new dataset with delineated masks is label-intensive. Also, the co-localization coefficient is often not used as a component during training with the CNN model. Yet, it may aid with localizing and detecting objects during training and testing. In this work, we propose to address these issues by using a quantification coefficient for co-localization called Manders overlapping coefficient (MOC)1 as a single-layer branch in a CNN. Fully convolutional one-state (FCOS)2 with a Resnet101 backbone served as the network to evaluate the effectiveness of the novel branch to assist with bounding box prediction. Training data were sourced from lab curated fluorescence images of neurons from the rat hippocampus, piriform cortex, somatosensory cortex, and amygdala. Results suggest that using modified FCOS with MOC outperformed the original FCOS model for accuracy in detecting fluorescence signals by 1.1% in mean average precision (mAP). The model could be downloaded from https://github.com/Alphafrey946/Colocalization-MOC.

Allopregnanolone and perampanel as adjuncts to midazolam for treating diisopropylfluorophosphate-induced status epilepticus in rats.

Combinations of midazolam, allopregnanolone, and perampanel were assessed for antiseizure activity in a rat diisopropylfluorophosphate (DFP) status epilepticus model. Animals receiving DFP followed by atropine and pralidoxime exhibited continuous high-amplitude rhythmical electroencephalography (EEG) spike activity and behavioral seizures for more than 5 hours. Treatments were administered intramuscularly 40 min after DFP. Seizures persisted following midazolam (1.8 mg/kg). The combination of midazolam with either allopregnanolone (6 mg/kg) or perampanel (2 mg/kg) terminated EEG and behavioral status epilepticus, but the onset of the perampanel effect was slow. The combination of midazolam, allopregnanolone, and perampanel caused rapid and complete suppression of EEG and behavioral seizures. In the absence of DFP, animals treated with the three-drug combination were sedated but not anesthetized. Animals that received midazolam alone exhibited spontaneous recurrent EEG seizures, whereas those that received the three-drug combination did not, demonstrating antiepileptogenic activity. All combination treatments reduced neurodegeneration as assessed with Fluoro-Jade C staining to a greater extent than midazolam alone, and most reduced astrogliosis as assessed by GFAP immunoreactivity but had mixed effects on markers of microglial activation. We conclude that allopregnanolone, a positive modulator of the GABAA receptor, and perampanel, an AMPA receptor antagonist, are potential adjuncts to midazolam in the treatment of benzodiazepine-refractory organophosphate nerve agent-induced status epilepticus.

Expression of AHI1 Rescues Amyloidogenic Pathology in Alzheimer's Disease Model Cells.

A hallmark of Alzheimer's disease (AD) pathogenesis is the accumulation of extracellular plaques mainly composed of amyloid-ß (Aß) derived from amyloid precursor protein (APP) cleavage. Recent reports suggest that transport of APP in vesicles with huntingtin-associated protein-1 (HAP1) negatively regulates Aß production. In neurons, HAP1 forms a stable complex with Abelson helper integration site-1 (AHI1), in which mutations cause neurodevelopmental and psychiatric disorders. HAP1 and AHI1 interact with tropomyosin receptor kinases (Trks), which are also associated with APP and mediate neurotrophic signaling. In this study, we hypothesize that AHI1 participates in APP trafficking and processing to rescue AD pathology. Indeed, AHI1 was significantly reduced in mouse neuroblastoma N2a cells expressing human Swedish and Indiana APP (designed as AD model cells) and in 3xTg-AD mouse brain. The AD model cells as well as Ahi1-knockdown cells expressing wild-type APP-695 exhibited a significant reduction in viability. In addition, the AD model cells were reduced in neurite outgrowth. APP C-terminal fragment-ß (CTFß) and Aß42 were increased in the AD cell lysates and the culture media, respectively. To investigate the mechanism how AHI1 alters APP activities, we overexpressed human AHI1 in the AD model cells. The results showed that AHI1 interacted with APP physically in mouse brain and transfected N2a cells despite APP genotypes. AHI1 expression facilitated intracellular translocation of APP and inhibited APP amyloidogenic process to reduce the level of APP-CTFß in the total lysates of AD model cells as well as Aß in the culture media. Consequently, AHI1-APP interactions enhanced neurotrophic signaling through Erk activation and led to restored cell survival and differentiation.

Synthesis of mesoporous silica helical fibers using a catanionic-neutral ternary surfactant in a highly dilute silica solution: biomimetic silicification.

Mesoporous silica helical fibers in many different shapes have been synthesized in a highly dilute silicate solution at pH approximately 2.0 by using CnTMAB-SDS-P123 (n = 14-18) ternary surfactant as a template. The mesoporous silica helical fibers possess a well-ordered hexagonal mesostructure, high surface area, and large pore volume. Thus, the microtome sections of the helical fibers demonstrate a concentric mesotructure or two hemiconcentric mesostructures. In addition to triblock copolymer, adding the proper amount of 1-butanol or pentanol can promote the yield of the helical fibers as well. The yield of the surfactant-templated helical fibers is also dependent on the water content, reaction temperature, and pH value of the solution. The mesoporous silica helical fiber can be used as a solid template to prepare mesoporous carbon helical fibers via impregnation of phenol-formaldehyde, pyrolysis, and silica removal.