The α4 Nicotinic Acetylcholine Receptor Is Necessary for the Initiation of Organophosphate-Induced Neuronal Hyperexcitability.

Acute intoxication with organophosphorus (OP) cholinesterase inhibitors can produce seizures that rapidly progress to life-threatening status epilepticus. Significant research effort has been focused on investigating the involvement of muscarinic acetylcholine receptors (mAChRs) in OP-induced seizure activity. In contrast, there has been far less attention on nicotinic AChRs (nAChRs) in this context. Here, we address this data gap using a combination of in vitro and in vivo models. Pharmacological antagonism and genetic deletion of α4, but not α7, nAChR subunits prevented or significantly attenuated OP-induced electrical spike activity in acute hippocampal slices and seizure activity in mice, indicating that α4 nAChR activation is necessary for neuronal hyperexcitability triggered by acute OP exposures. These findings not only suggest that therapeutic strategies for inhibiting the α4 nAChR subunit warrant further investigation as prophylactic and immediate treatments for acute OP-induced seizures, but also provide mechanistic insight into the role of the nicotinic cholinergic system in seizure generation.

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.

Strain differences in the extent of brain injury in mice after tetramethylenedisulfotetramine-induced status epilepticus.

Acute intoxication with tetramethylenedisulfotetramine (TETS) can trigger status epilepticus (SE) in humans. Survivors often exhibit long-term neurological effects, including electrographic abnormalities and cognitive deficits, but the pathogenic mechanisms linking the acute toxic effects of TETS to chronic outcomes are not known. Here, we use advanced in vivo imaging techniques to longitudinally monitor the neuropathological consequences of TETS-induced SE in two different mouse strains. Adult male NIH Swiss and C57BL/6J mice were injected with riluzole (10 mg/kg, i.p.), followed 10 min later by an acute dose of TETS (0.2 mg/kg in NIH Swiss; 0.3 mg/kg, i.p. in C57BL/6J) or an equal volume of vehicle (10% DMSO in 0.9% sterile saline). Different TETS doses were administered to trigger comparable seizure behavior between strains. Seizure behavior began within minutes of TETS exposure and rapidly progressed to SE that was terminated after 40 min by administration of midazolam (1.8 mg/kg, i.m.). The brains of vehicle and TETS-exposed mice were imaged using in vivo magnetic resonance (MR) and translocator protein (TSPO) positron emission tomography (PET) at 1, 3, 7, and 14 days post-exposure to monitor brain injury and neuroinflammation, respectively. When the brain scans of TETS mice were compared to those of vehicle controls, subtle and transient neuropathology was observed in both mouse strains, but more extensive and persistent TETS-induced neuropathology was observed in C57BL/6J mice. In addition, one NIH Swiss TETS mouse that did not respond to the midazolam therapy, but remained in SE for more than 2 h, displayed robust neuropathology as determined by in vivo imaging and confirmed by FluoroJade C staining and IBA-1 immunohistochemistry as readouts of neurodegeneration and neuroinflammation, respectively. These findings demonstrate that the extent of injury observed in the mouse brain after TETS-induced SE varied according to strain, dose of TETS and/or the duration of SE. These observations suggest that TETS-intoxicated humans who do not respond to antiseizure medication are at increased risk for brain injury.

Persistent neuropathology and behavioral deficits in a mouse model of status epilepticus induced by acute intoxication with diisopropylfluorophosphate.

Organophosphate (OP) nerve agents and pesticides are a class of neurotoxic compounds that can cause status epilepticus (SE), and death following acute high-dose exposures. While the standard of care for acute OP intoxication (atropine, oxime, and high-dose benzodiazepine) can prevent mortality, survivors of OP poisoning often experience long-term brain damage and cognitive deficits. Preclinical studies of acute OP intoxication have primarily used rat models to identify candidate medical countermeasures. However, the mouse offers the advantage of readily available knockout strains for mechanistic studies of acute and chronic consequences of OP-induced SE. Therefore, the main objective of this study was to determine whether a mouse model of acute diisopropylfluorophosphate (DFP) intoxication would produce acute and chronic neurotoxicity similar to that observed in rat models and humans following acute OP intoxication. Adult male C57BL/6J mice injected with DFP (9.5 mg/kg, s.c.) followed 1 min later with atropine sulfate (0.1 mg/kg, i.m.) and 2-pralidoxime (25 mg/kg, i.m.) developed behavioral and electrographic signs of SE within minutes that continued for at least 4 h. Acetylcholinesterase inhibition persisted for at least 3 d in the blood and 14 d in the brain of DFP mice relative to vehicle (VEH) controls. Immunohistochemical analyses revealed significant neurodegeneration and neuroinflammation in multiple brain regions at 1, 7, and 28 d post-exposure in the brains of DFP mice relative to VEH controls. Deficits in locomotor and home-cage behavior were observed in DFP mice at 28 d post-exposure. These findings demonstrate that this mouse model replicates many of the outcomes observed in rats and humans acutely intoxicated with OPs, suggesting the feasibility of using this model for mechanistic studies and therapeutic screening.

The chemical convulsant diisopropylfluorophosphate (DFP) causes persistent neuropathology in adult male rats independent of seizure activity.

Organophosphate (OP) threat agents can trigger seizures that progress to status epilepticus, resulting in persistent neuropathology and cognitive deficits in humans and preclinical models. However, it remains unclear whether patients who do not show overt seizure behavior develop neurological consequences. Therefore, this study compared two subpopulations of rats with a low versus high seizure response to diisopropylfluorophosphate (DFP) to evaluate whether acute OP intoxication causes persistent neuropathology in non-seizing individuals. Adult male Sprague Dawley rats administered DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im), and pralidoxime (25 mg/kg, im) were monitored for seizure activity for 4 h post-exposure. Animals were separated into groups with low versus high seizure response based on behavioral criteria and electroencephalogram (EEG) recordings. Cholinesterase activity was evaluated by Ellman assay, and neuropathology was evaluated at 1, 2, 4, and 60 days post-exposure by Fluoro-Jade C (FJC) staining and micro-CT imaging. DFP significantly inhibited cholinesterase activity in the cortex, hippocampus, and amygdala to the same extent in low and high responders. FJC staining revealed significant neurodegeneration in DFP low responders albeit this response was delayed, less persistent, and decreased in magnitude compared to DFP high responders. Micro-CT scans at 60 days revealed extensive mineralization that was not significantly different between low versus high DFP responders. These findings highlight the importance of considering non-seizing patients for medical care in the event of acute OP intoxication. They also suggest that OP intoxication may induce neurological damage via seizure-independent mechanisms, which if identified, might provide insight into novel therapeutic targets.

Analysis of inflammation-induced depression of home cage wheel running in rats reveals the difference between opioid antinociception and restoration of function.

Opioids are effective at inhibiting responses to noxious stimuli in rodents, but have limited efficacy and many side effects in chronic pain patients. One reason for this disconnect is that nociception is typically assessed using withdrawal from noxious stimuli in animals, whereas chronic pain patients suffer from abnormal pain that disrupts normal activity. We hypothesized that assessment of home cage wheel running in rats would provide a much more clinically relevant method to assess opioid efficacy to restore normal behavior. Intraplantar injection of Complete Freund's Adjuvant (CFA) into the right hindpaw depressed wheel running and caused mechanical allodynia measured with the von Frey test in both male and female rats. Administration of an ED50 dose of morphine (3.2mg/kg) reversed mechanical allodynia, but did not reverse CFA-induced depression of wheel running. In contrast, administration of a low dose of morphine (1.0mg/kg) restored running for one hour in both sexes, but had no effect on mechanical allodynia. Administration of the atypical opioid buprenorphine had no effect on inflammation-induced depression of wheel running in male or female rats, but attenuated mechanical allodynia in male rats. Administration of buprenorphine and higher doses of morphine depressed wheel running in non-inflamed rats, suggesting that the side effects of opioids interfere with restoration of function. These data indicate that restoration of pain-depressed function requires antinociception in the absence of disruptive side effects. The disruptive side effects of opioids are consistent with the major limitation of opioid use in human pain patients.

Home cage wheel running is an objective and clinically relevant method to assess inflammatory pain in male and female rats.

BACKGROUND: The assessment of nociception in preclinical studies is undergoing a transformation from pain-evoked to pain-depressed tests to more closely mimic the effects of clinical pain. Many inflammatory pain-depressed behaviors (reward seeking, locomotion) have been examined, but these tests are limited because of confounds such as stress and difficulties in quantifying behavior. NEW METHOD: The present study evaluates home cage wheel running as an objective method to assess the magnitude and duration of inflammatory pain in male and female rats. RESULTS: Injection of Complete Freund's Adjuvant (CFA) into the right hindpaw to induce inflammatory pain almost completely inhibited wheel running for 2 days in male and female rats. Wheel running gradually returned to baseline levels within 12 days despite persistent mechanical hypersensitivity (von Frey test). COMPARISON WITH EXISTING METHODS: Continuously monitoring home cage wheel running improves on previous studies examining inflammatory pain-depressed wheel running because it is more sensitive to noxious stimuli, avoids the stress of removing the rat from its cage for testing, and provides a complete analysis of the time course for changes in nociception. CONCLUSIONS: The present data indicate that home cage wheel running is a clinically relevant method to assess inflammatory pain in the rat. The decrease in activity caused by inflammatory pain and subsequent gradual recovery mimics the changes in activity caused by pain in humans. The tendency for pain-depressed wheel running to be greater in female than male rats is consistent with the tendency for women to be at greater risk of chronic pain than men.