RESUMO
Recent experimental evidence suggests combined treatment with midazolam and allopregnanolone is more effective than midazolam alone in terminating seizures triggered by acute organophosphate (OP) intoxication. However, there are concerns that combined midazolam and allopregnanolone increases risk of adverse cardiovascular events. To address this, we used telemetry devices to record cardiovascular responses in adult male Sprague-Dawley rats acutely intoxicated with diisopropylfluorophosphate (DFP). Animals were administered DFP (4 mg/kg, sc), followed immediately by atropine (2 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m.). At 40 min post-exposure, a subset of animals received midazolam (0.65 mg/kg, im); at 50 min, these rats received a second dose of midazolam or allopregnanolone (12 mg/kg, im). DFP significantly increased blood pressure by ~ 80 mmHg and pulse pressure by ~ 34 mmHg that peaked within 12 min. DFP also increased core temperature by ~ 3.5 °C and heart rate by ~ 250 bpm that peaked at ~ 2 h. Heart rate variability (HRV), an index of autonomic function, was reduced by ~ 80%. All acute (within 15 min of exposure) and two-thirds of delayed (hours after exposure) mortalities were associated with non-ventricular cardiac events within 10 min of cardiovascular collapse, suggesting that non-ventricular events should be closely monitored in OP-poisoned patients. Compared to rats that survived DFP intoxication without treatment, midazolam significantly improved recovery of cardiovascular parameters and HRV, an effect enhanced by allopregnanolone. These data demonstrate that midazolam improved recovery of cardiovascular and autonomic function and that the combination of midazolam and allopregnanolone may be a better therapeutic strategy than midazolam alone.
Assuntos
Midazolam , Intoxicação por Organofosfatos , Humanos , Ratos , Masculino , Animais , Ratos Sprague-Dawley , Midazolam/farmacologia , Midazolam/uso terapêutico , Pregnanolona/farmacologia , Isoflurofato/farmacologia , Organofosfatos , Encéfalo , Intoxicação por Organofosfatos/tratamento farmacológicoRESUMO
Acute organophosphate (OP) intoxication can trigger seizures that progress to status epilepticus (SE), and survivors often develop chronic morbidities, including spontaneous recurrent seizures (SRS). The pathogenic mechanisms underlying OP-induced SRS are unknown, but increased BBB permeability is hypothesized to be involved. Previous studies reported BBB leakage following OP-induced SE, but key information regarding time and regional distribution of BBB impairment during the epileptogenic period is missing. To address this data gap, we characterized the spatiotemporal progression of BBB impairment during the first week post-exposure in a rat model of diisopropylfluorophosphate-induced SE, using MRI and albumin immunohistochemistry. Increased BBB permeability, which was detected at 6 h and persisted up to 7 d post-exposure, was most severe and persistent in the piriform cortex and amygdala, moderate but persistent in the thalamus, and less severe and transient in the hippocampus and somatosensory cortex. The extent of BBB leakage was positively correlated with behavioral seizure severity, with the strongest association identified in the piriform cortex and amygdala. These findings provide evidence of the duration, magnitude and spatial breakdown of the BBB during the epileptogenic period following OP-induced SE and support BBB regulation as a viable therapeutic target for preventing SRS following acute OP intoxication.
Assuntos
Barreira Hematoencefálica , Estado Epiléptico , Ratos , Animais , Barreira Hematoencefálica/patologia , Ratos Sprague-Dawley , Organofosfatos/efeitos adversos , Organofosfatos/metabolismo , Estado Epiléptico/metabolismo , Convulsões/metabolismo , Encéfalo/metabolismoRESUMO
Current medical countermeasures for organophosphate (OP)-induced status epilepticus (SE) are not effective in preventing long-term morbidity and there is an urgent need for improved therapies. Rat models of acute intoxication with the OP, diisopropylfluorophosphate (DFP), are increasingly being used to evaluate therapeutic candidates for efficacy in mitigating the long-term neurologic effects associated with OP-induced SE. Many of these therapeutic candidates target neuroinflammation and oxidative stress because of their implication in the pathogenesis of persistent neurologic deficits associated with OP-induced SE. Critical to these efforts is the rigorous characterization of the rat DFP model with respect to outcomes associated with acute OP intoxication in humans, which include long-term electroencephalographic, neurobehavioral, and neuropathologic effects, and their temporal relationship to neuroinflammation and oxidative stress. To address these needs, we examined a range of outcomes at later times post-exposure than have previously been reported for this model. Adult male Sprague-Dawley rats were given pyridostigmine bromide (0.1â¯mg/kg, im) 30â¯min prior to administration of DFP (4â¯mg/kg, sc), which was immediately followed by atropine sulfate (2â¯mg/kg, im) and pralidoxime (25â¯mg/kg, im). This exposure paradigm triggered robust electroencephalographic and behavioral seizures that rapidly progressed to SE lasting several hours in 90% of exposed animals. Animals that survived DFP-induced SE (~70%) exhibited spontaneous recurrent seizures and hyperreactive responses to tactile stimuli over the first 2â¯months post-exposure. Performance in the elevated plus maze, open field, and Pavlovian fear conditioning tests indicated that acute DFP intoxication reduced anxiety-like behavior and impaired learning and memory at 1 and 2â¯months post-exposure in the absence of effects on general locomotor behavior. Immunohistochemical analyses revealed significantly increased expression of biomarkers of reactive astrogliosis, microglial activation and oxidative stress in multiple brain regions at 1 and 2â¯months post-DFP, although there was significant spatiotemporal heterogeneity across these endpoints. Collectively, these data largely support the relevance of the rat model of acute DFP intoxication as a model for acute OP intoxication in the human, and support the hypothesis that neuroinflammation and/or oxidative stress represent potential therapeutic targets for mitigating the long-term neurologic sequelae of acute OP intoxication.
Assuntos
Encéfalo , Modelos Animais de Doenças , Isoflurofato/toxicidade , Síndromes Neurotóxicas , Estresse Oxidativo/efeitos dos fármacos , Animais , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/patologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Masculino , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Intoxicação por Organofosfatos/metabolismo , Intoxicação por Organofosfatos/patologia , Ratos , Ratos Sprague-Dawley , Estado Epiléptico/induzido quimicamenteRESUMO
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.
Assuntos
Ondas Encefálicas/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Convulsivantes/toxicidade , Isoflurofato/toxicidade , Degeneração Neural , Síndromes Neurotóxicas/etiologia , Convulsões/induzido quimicamente , Acetilcolinesterase/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Proteínas Ligadas por GPI/metabolismo , Masculino , Síndromes Neurotóxicas/diagnóstico por imagem , Síndromes Neurotóxicas/enzimologia , Síndromes Neurotóxicas/fisiopatologia , Ratos Sprague-Dawley , Convulsões/diagnóstico por imagem , Convulsões/enzimologia , Convulsões/fisiopatologia , Fatores de Tempo , Microtomografia por Raio-XRESUMO
Acute intoxication with picrotoxin or the rodenticide tetramethylenedisulfotetramine (TETS) can cause seizures that rapidly progress to status epilepticus and death. Both compounds inhibit γ-aminobutyric acid type-A (GABAA) receptors with similar potency. However, TETS is approximately 100 × more lethal than picrotoxin. Here, we directly compared the toxicokinetics of the two compounds following intraperitoneal administration in mice. Using LC/MS analysis we found that picrotoxinin, the active component of picrotoxin, hydrolyses quickly into picrotoxic acid, has a short in vivo half-life, and is moderately brain penetrant (brain/plasma ratio 0.3). TETS, in contrast, is not metabolized by liver microsomes and persists in the body following intoxication. Using both GC/MS and a TETS-selective immunoassay we found that mice administered TETS at the LD50 of 0.2 mg/kg in the presence of rescue medications exhibited serum levels that remained constant around 1.6 µM for 48 h before falling slowly over the next 10 days. TETS showed a similar persistence in tissues. Whole-cell patch-clamp demonstrated that brain and serum extracts prepared from mice at 2 and 14 days after TETS administration significantly blocked heterologously expressed α2ß3γ2 GABAA-receptors confirming that TETS remains pharmacodynamically active in vivo. This observed persistence may contribute to the long-lasting and recurrent seizures observed following human exposures. We suggest that countermeasures to neutralize TETS or accelerate its elimination should be explored for this highly dangerous threat agent.
Assuntos
Encéfalo/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/toxicidade , Convulsivantes/toxicidade , Antagonistas GABAérgicos/toxicidade , Picrotoxina/análogos & derivados , Convulsões/induzido quimicamente , Animais , Biotransformação , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Convulsivantes/farmacocinética , Antagonistas GABAérgicos/farmacocinética , Dose Letal Mediana , Masculino , Camundongos , Picrotoxina/farmacocinética , Picrotoxina/toxicidade , Receptores de GABA-A/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Sesterterpenos , Distribuição Tecidual , ToxicocinéticaRESUMO
Organophosphates (OPs), a class of phosphorus-containing chemicals that act by disrupting cholinergic transmission, include both toxic and fast-acting chemical warfare agents as well as less toxic but more easily accessible OP pesticides. The classical atropine/2-PAM antidote fails to protect against long-term symptoms following acute intoxication with OPs at levels that trigger status epilepticus. Acute OP intoxication also causes a robust neuroinflammatory response, which is implicated in the pathogenesis of long-term effects. In this study, we characterized the profiles of lipid mediators, important players in neuroinflammation, in the rat model of acute DFP intoxication. The profiles of lipid mediators were monitored in three different regions of the brain (cortex, hippocampus, and cerebellum) at 0, 1, 3, 7, 14, and 28â¯days post-exposure. The distribution pattern of lipid mediators was distinct in the three brain regions. In the cerebellum, the profile is dominated by LOX metabolites, while the lipid mediator profiles in cortex and hippocampus are dominated by COX metabolites followed by LOX and CYP 450 metabolites. Following acute DFP intoxication, most of the pro-inflammatory lipid mediators (e.g., PGD2 and PGE2) increased rapidly from day 1, while the concentrations of some anti-inflammatory lipid mediators (e.g. 14,15 EpETrE) decreased after DFP intoxication but recovered by day 14 post-exposure. The lipidomics results suggest two potential treatment targets: blocking the formation of prostaglandins by inhibiting COX and stabilizing the anti-inflammatory lipid mediators containing epoxides by inhibiting the enzyme soluble epoxide hydrolase (sEH).
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Inibidores da Colinesterase/toxicidade , Isoflurofato/toxicidade , Lipidômica/métodos , Organofosfatos/toxicidade , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Acute intoxication with organophosphorus (OP) cholinesterase inhibitors can trigger convulsions that progress to life-threatening status epilepticus. Survivors face long-term morbidity including mild-to-severe decline in memory. It is posited that neuroinflammation plays a key role in the pathogenesis of OP-induced neuropsychiatric deficits. Rigorous testing of this hypothesis requires preclinical models that recapitulate relevant phenotypic outcomes. Here, we describe a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) that exhibits persistent neuroinflammation and cognitive impairment. METHODS: Neuroinflammation, neurodegeneration, and cognitive function were compared in adult male Sprague Dawley rats injected with an acutely toxic dose of DFP vs. vehicle controls at multiple time points up to 36 days post-exposure. Neuroinflammation was quantified using immunohistochemical biomarkers of microglia (ionized calcium-binding adapter molecule 1, IBA1) and activated astrocytes (glial fibrillary acidic protein, GFAP) and positron emission tomography (PET) imaging of [11C]-(R)-PK11195, a ligand for the 18-kDa mitochondrial membrane translocator protein (TSPO). FluoroJade-B staining was used to assess neurodegeneration; Pavlovian conditioning, to assess cognitive function. RESULTS: Animals exhibited moderate-to-severe seizures within minutes of DFP injection that continued for up to 6 h post-injection. As indicated by IBA1 and GFAP immunoreactivity and by PET imaging of TSPO, acute DFP intoxication triggered neuroinflammation in the hippocampus and cortex during the first 3 days that peaked at 7 days and persisted to 21 days post-exposure in most animals. Neurodegeneration was detected in multiple brain regions from 1 to 14 days post-exposure. All DFP-intoxicated animals exhibited significant deficits in contextual fear conditioning at 9 and 20 days post-exposure compared to vehicle controls. Whole-brain TSPO labeling positively correlated with seizure severity score, but did not correlate with performance in the contextual fear-conditioning task. CONCLUSIONS: We describe a preclinical model in which acute DFP intoxication causes seizures, persistent neuroinflammation, neurodegeneration, and memory impairment. The extent of the neuroinflammatory response is influenced by seizure severity. However, the observation that a subset of animals with moderate seizures and minimal TSPO labeling exhibited cognitive deficits comparable to those of animals with severe seizures and significant TSPO labeling suggests that DFP may impair learning and memory circuitry via mechanisms independent of seizures or neuroinflammation.
Assuntos
Inibidores da Colinesterase/toxicidade , Disfunção Cognitiva/induzido quimicamente , Encefalite/induzido quimicamente , Isoflurofato/toxicidade , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Disfunção Cognitiva/diagnóstico por imagem , Condicionamento Clássico/efeitos dos fármacos , Encefalite/diagnóstico por imagem , Comportamento Exploratório/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Imageamento por Ressonância Magnética , Masculino , Proteínas dos Microfilamentos/metabolismo , Tomografia por Emissão de Pósitrons , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Análise de Regressão , Fatores de TempoRESUMO
We previously demonstrated that antigen sensitization increases vulnerability to airway hyperreactivity induced by the organophosphorus pesticide (OP) parathion. Sensitization also changes the mechanism of parathion-induced airway hyperreactivity to one that is dependent on IL-5. To determine whether this effect can be generalized to other OPs, and to other classes of pesticides, we measured airway responsiveness to vagal stimulation or intravenous acetylcholine in nonsensitized and ovalbumin-sensitized guinea pigs 24 hours after a single subcutaneous injection of the OPs diazinon or chlorpyrifos, or the pyrethroid permethrin. Sensitization exacerbated the effects of chlorpyrifos on bronchoconstriction in response to vagal stimulation or intravenous acetylcholine. Pretreatment with function-blocking IL-5 antibody prevented chlorpyrifos-induced airway hyperreactivity in sensitized, but not in nonsensitized, guinea pigs. In sensitized guinea pigs, blocking IL-5 decreased eosinophil activation, as measured by decreased eosinophil major basic protein in the trachea. In contrast, sensitization did not alter diazinon-induced airway hyperreactivity, and permethrin did not cause airway hyperreactivity in either nonsensitized or sensitized guinea pigs. None of the pesticides affected inflammatory cells in the bronchoalveolar lavage fluid or blood. We have previously shown that three different OPs cause airway hyperreactivity via loss of neuronal M2 muscarinic receptor function. Similar to parathion, but unlike diazinon, the mechanism of chlorpyrifos-induced airway hyperreactivity is changed by sensitization. Thus, OP-induced airway hyperreactivity is dependent on sensitization status and on the OP used, which may influence therapeutic approaches.
Assuntos
Asma/imunologia , Hiper-Reatividade Brônquica/imunologia , Broncoconstrição/efeitos dos fármacos , Imunização , Inseticidas/farmacologia , Ovalbumina/farmacologia , Acetilcolina/farmacologia , Animais , Anticorpos Neutralizantes/farmacologia , Asma/induzido quimicamente , Asma/genética , Asma/patologia , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar/química , Broncoconstrição/imunologia , Clorpirifos/farmacologia , Diazinon/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Cobaias , Injeções Intravenosas , Injeções Subcutâneas , Interleucina-5/antagonistas & inibidores , Interleucina-5/genética , Interleucina-5/imunologia , Permetrina/farmacologia , Traqueia/efeitos dos fármacos , Traqueia/imunologia , Traqueia/patologia , Nervo Vago/efeitos dos fármacos , Nervo Vago/imunologiaRESUMO
BACKGROUND: Neuregulin-1 (NRG-1) has been shown to act as a neuroprotectant in animal models of nerve agent intoxication and other acute brain injuries. We recently demonstrated that NRG-1 blocked delayed neuronal death in rats intoxicated with the organophosphate (OP) neurotoxin diisopropylflurophosphate (DFP). It has been proposed that inflammatory mediators are involved in the pathogenesis of OP neurotoxin-mediated brain damage. METHODS: We examined the influence of NRG-1 on inflammatory responses in the rat brain following DFP intoxication. Microglial activation was determined by immunohistchemistry using anti-CD11b and anti-ED1 antibodies. Gene expression profiling was performed with brain tissues using Affymetrix gene arrays and analyzed using the Ingenuity Pathway Analysis software. Cytokine mRNA levels following DFP and NRG-1 treatment was validated by real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: DFP administration resulted in microglial activation in multiple brain regions, and this response was suppressed by treatment with NRG-1. Using microarray gene expression profiling, we observed that DFP increased mRNA levels of approximately 1,300 genes in the hippocampus 24 h after administration. NRG-1 treatment suppressed by 50% or more a small fraction of DFP-induced genes, which were primarily associated with inflammatory responses. Real-time RT-PCR confirmed that the mRNAs for pro-inflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were significantly increased following DFP exposure and that NRG-1 significantly attenuated this transcriptional response. In contrast, tumor necrosis factor α (TNFα) transcript levels were unchanged in both DFP and DFP + NRG-1 treated brains relative to controls. CONCLUSION: Neuroprotection by NRG-1 against OP neurotoxicity is associated with the suppression of pro-inflammatory responses in brain microglia. These findings provide new insight regarding the molecular mechanisms involved in the neuroprotective role of NRG-1 in acute brain injuries.
Assuntos
Inibidores da Colinesterase/toxicidade , Inibidores da Colinesterase/uso terapêutico , Encefalite/induzido quimicamente , Isoflurofato/toxicidade , Neuregulina-1/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Animais , Encéfalo/patologia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intra-Arteriais , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro , Ratos , Ratos Sprague-DawleyRESUMO
Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABAAR) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABAAR positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15mg/kg, ip). Administration of a high dose of diazepam (5mg/kg, ip) immediately following the second clonic seizure (approximately 20min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABAAR antagonists. The sEH inhibitor TUPS (1mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5mg/kg, ip) and TUPS (1mg/kg, ip, starting 1h after diazepam and repeated every 24h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication.
Assuntos
Anti-Inflamatórios/administração & dosagem , Anticonvulsivantes/administração & dosagem , Encéfalo/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes , Diazepam/administração & dosagem , Encefalite/prevenção & controle , Inibidores Enzimáticos/administração & dosagem , Epóxido Hidrolases/antagonistas & inibidores , Moduladores GABAérgicos/administração & dosagem , Compostos de Fenilureia/administração & dosagem , Piperidinas/administração & dosagem , Convulsões/prevenção & controle , Animais , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Ondas Encefálicas/efeitos dos fármacos , Modelos Animais de Doenças , Esquema de Medicação , Quimioterapia Combinada , Eletroencefalografia , Encefalite/induzido quimicamente , Encefalite/enzimologia , Encefalite/fisiopatologia , Epóxido Hidrolases/metabolismo , Masculino , Camundongos , Convulsões/induzido quimicamente , Convulsões/enzimologia , Convulsões/fisiopatologia , Fatores de TempoRESUMO
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.
RESUMO
Acute poisoning with organophosphorus cholinesterase inhibitors (OPs), such as OP nerve agents and pesticides, can cause life threatening cholinergic crisis and status epilepticus (SE). Survivors often experience significant morbidity, including brain injury, acquired epilepsy, and cognitive deficits. Current medical countermeasures for acute OP poisoning include a benzodiazepine to mitigate seizures. Diazepam was long the benzodiazepine included in autoinjectors used to treat OP-induced seizures, but it is now being replaced in many guidelines by midazolam, which terminates seizures more quickly, particularly when administered intramuscularly. While a direct correlation between seizure duration and the extent of brain injury has been widely reported, there are limited data comparing the neuroprotective efficacy of diazepam versus midazolam following acute OP intoxication. To address this data gap, we used non-invasive imaging techniques to longitudinally quantify neuropathology in a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) with and without post-exposure intervention with diazepam or midazolam. Magnetic resonance imaging (MRI) was used to monitor neuropathology and brain atrophy, while positron emission tomography (PET) with a radiotracer targeting translocator protein (TSPO) was utilized to assess neuroinflammation. Animals were scanned at 3, 7, 28, 65, 91, and 168 days post-DFP and imaging metrics were quantitated for the hippocampus, amygdala, piriform cortex, thalamus, cerebral cortex and lateral ventricles. In the DFP-intoxicated rat, neuroinflammation persisted for the duration of the study coincident with progressive atrophy and ongoing tissue remodeling. Benzodiazepines attenuated neuropathology in a region-dependent manner, but neither benzodiazepine was effective in attenuating long-term neuroinflammation as detected by TSPO PET. Diffusion MRI and TSPO PET metrics were highly correlated with seizure severity, and early MRI and PET metrics were positively correlated with long-term brain atrophy. Collectively, these results suggest that anti-seizure therapy alone is insufficient to prevent long-lasting neuroinflammation and tissue remodeling.
Assuntos
Lesões Encefálicas , Estado Epiléptico , Ratos , Animais , Diazepam/farmacologia , Midazolam/farmacologia , Midazolam/uso terapêutico , Isoflurofato/farmacologia , Organofosfatos , Doenças Neuroinflamatórias , Neuroproteção , Ratos Sprague-Dawley , Encéfalo/metabolismo , Benzodiazepinas/farmacologia , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/diagnóstico por imagem , Estado Epiléptico/tratamento farmacológico , Tomografia por Emissão de Pósitrons , Proteínas de Transporte/metabolismo , Imageamento por Ressonância Magnética , Lesões Encefálicas/metabolismo , Atrofia/patologiaRESUMO
Acute intoxication with organophosphate (OP) cholinesterase inhibitors poses a significant public health risk. While currently approved medical countermeasures can improve survival rates, they often fail to prevent chronic neurological damage. Therefore, there is need to develop effective therapies and quantitative metrics for assessing OP-induced brain injury and its rescue by these therapies. In this study we used a rat model of acute intoxication with the OP, diisopropylfluorophosphate (DFP), to test the hypothesis that T2 measures obtained from brain magnetic resonance imaging (MRI) scans provide quantitative metrics of brain injury and therapeutic efficacy. Adult male Sprague Dawley rats were imaged on a 7T MRI scanner at 3, 7 and 28 days post-exposure to DFP or vehicle (VEH) with or without treatment with the standard of care antiseizure drug, midazolam (MDZ); a novel antiseizure medication, allopregnanolone (ALLO); or combination therapy with MDZ and ALLO (DUO). Our results show that mean T2 values in DFP-exposed animals were: (1) higher than VEH in all volumes of interest (VOIs) at day 3; (2) decreased with time; and (3) decreased in the thalamus at day 28. Treatment with ALLO or DUO, but not MDZ alone, significantly decreased mean T2 values relative to untreated DFP animals in the piriform cortex at day 3. On day 28, the DUO group showed the most favorable T2 characteristics. This study supports the utility of T2 mapping for longitudinally monitoring brain injury and highlights the therapeutic potential of ALLO as an adjunct therapy to mitigate chronic morbidity associated with acute OP intoxication.
Assuntos
Lesões Encefálicas , Intoxicação por Organofosfatos , Ratos , Masculino , Animais , Ratos Sprague-Dawley , Isoflurofato/toxicidade , Organofosfatos , Inibidores da Colinesterase/farmacologia , Intoxicação por Organofosfatos/tratamento farmacológico , Intoxicação por Organofosfatos/patologia , Lesões Encefálicas/induzido quimicamente , Encéfalo , Midazolam/farmacologiaRESUMO
Organophosphorus pesticides (OPs) are implicated in human asthma. We previously demonstrated that, at concentrations that do not inhibit acetylcholinesterase activity, the OP parathion causes airway hyperreactivity in guinea pigs as a result of functional loss of inhibitory M2 muscarinic receptors on parasympathetic nerves. Because macrophages are associated with asthma, we investigated whether macrophages mediate parathion-induced M2 receptor dysfunction and airway hyperreactivity. Airway physiology was measured in guinea pigs 24 h after a subcutaneous injection of parathion. Pretreatment with liposome-encapsulated clodronate induced alveolar macrophage apoptosis and prevented parathion-induced airway hyperreactivity in response to electrical stimulation of the vagus nerves. As determined by qPCR, TNF-α and IL-1ß mRNA levels were increased in alveolar macrophages isolated from parathion-treated guinea pigs. Parathion treatment of alveolar macrophages ex vivo did not significantly increase IL-1ß and TNF-α mRNA but did significantly increase TNF-α protein release. Consistent with these data, pretreatment with the TNF-α inhibitor etanercept but not the IL-1ß receptor inhibitor anakinra prevented parathion-induced airway hyperreactivity and protected M2 receptor function. These data suggest a novel mechanism of OP-induced airway hyperreactivity in which low-level parathion activates macrophages to release TNF-α-causing M2 receptor dysfunction and airway hyperreactivity. These observations have important implications regarding therapeutic approaches for treating respiratory disease associated with OP exposures.
Assuntos
Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/imunologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Paration/toxicidade , Fator de Necrose Tumoral alfa/imunologia , Animais , Apoptose/efeitos dos fármacos , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/patologia , Ácido Clodrônico/farmacologia , Etanercepte , Feminino , Cobaias , Humanos , Imunoglobulina G/farmacologia , Inseticidas/toxicidade , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/antagonistas & inibidores , Receptores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genéticaRESUMO
The neurosteroid allopregnanolone (ALLO) is under investigation as a treatment for benzodiazepine-refractory status epilepticus (SE). Here, we assess the cardiopulmonary safety of intravenous ALLO by itself and after a clinically recommended dose of midazolam (MDZ) in two healthy adult beagles. Each dog received ALLO (1 mg/kg, IV), and after a washout period of 2 weeks, each dog was dosed with MDZ (0.2 mg/kg, IV) followed 10 minutes later by ALLO. Behavioral state, vital signs, arterial blood gases, blood chemistries, and plasma ALLO concentrations were monitored for up to 6 hours after dosing. The dogs appeared sleepy but were fully responsive after both treatments. No depression of mean arterial pressure or respiratory rate was noted. Blood gas measurements failed to show evidence of drug-induced acute respiratory acidosis. Estimated maximum plasma ALLO concentrations were in the range of 1500 to 3000 ng/ml. The results indicate that intravenous ALLO can be used safely to treat benzodiazepine-refractory SE, even when administered shortly after a benzodiazepine.
Assuntos
Midazolam , Estado Epiléptico , Cães , Animais , Midazolam/uso terapêutico , Pregnanolona/uso terapêutico , Estado Epiléptico/tratamento farmacológico , Administração IntravenosaRESUMO
Tetramethylenedisulfotetramine (tetramine; TETS) is a potent convulsant poison that is considered to be a chemical threat agent. To provide a basis for the investigation of antidotes for TETS-induced seizures, we characterized the convulsant activity of TETS in mice and rats when administered by the intraperitoneal, intravenous, oral, and intraventricular routes as a single acute dose and with repeated sublethal doses. In mice, parenteral and oral TETS caused immobility, myoclonic body jerks, clonic seizures of the forelimbs and/or hindlimbs, tonic seizures, and death. The CD50 values for clonic and tonic seizures after oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5-100 µg) in rats also caused clonic-tonic seizures and death. In mice, repeated sublethal doses of TETS at intervals of 2, 24, and 48 h failed to result in the development of persistent enhanced seizure responsivity ("kindling") as was observed with repeated pentylenetetrazol treatment. In mice, sublethal doses of TETS that produced clonic seizures did not cause observable structural brain damage as assessed with routine histology and Fluoro-Jade B staining 7 days after treatment. However, 1 to 3 days after a single convulsant dose of TETS the expression of glial fibrillary acidic protein, an astrocyte marker, and ionized calcium binding adaptor molecule 1, a microglia marker, were markedly increased in cortex and hippocampus. Although TETS doses that are compatible with survival are not associated with overt evidence of cellular injury or neurodegeneration, there is transient reactive astrocytosis and microglial activation, indicating that brain inflammatory responses are provoked.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/toxicidade , Convulsivantes/toxicidade , Convulsões/induzido quimicamente , Convulsões/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Extremidades , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/induzido quimicamente , Gliose/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Pentilenotetrazol/farmacologia , Picrotoxina/efeitos adversos , Ratos , Ratos Sprague-DawleyRESUMO
Current medical countermeasures against organophosphate (OP) nerve agents are effective in reducing mortality, but do not sufficiently protect the CNS from delayed brain damage and persistent neurological symptoms. In this study, we examined the efficacy of neuregulin-1 (NRG-1) in protecting against delayed neuronal cell death following acute intoxication with the OP diisopropylflurophosphate (DFP). Adult male Sprague-Dawley rats were pretreated with pyridostigmine (0.1 mg/kg BW, i.m.) and atropine methylnitrate (20 mg/kg BW, i.m.) prior to DFP (9 mg/kg BW, i.p.) intoxication to increase survival and reduce peripheral signs of cholinergic toxicity but not prevent DFP-induced seizures or delayed neuronal injury. Pretreatment with NRG-1 did not protect against seizures in rats exposed to DFP. However, neuronal injury was significantly reduced in most brain regions by pretreatment with NRG-1 isoforms NRG-EGF (3.2 µg/kg BW, i.a) or NRG-GGF2 (48 µg/kg BW, i.a.) as determined by FluroJade-B labeling in multiple brain regions at 24 h post-DFP injection. NRG-1 also blocked apoptosis and oxidative stress-mediated protein damage in the brains of DFP-intoxicated rats. Administration of NRG-1 at 1h after DFP injection similarly provided significant neuroprotection against delayed neuronal injury. These findings identify NRG-1 as a promising adjuvant therapy to current medical countermeasures for enhancing neuroprotection against acute OP intoxication.
Assuntos
Encéfalo/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Isoflurofato/toxicidade , Neuregulina-1/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Convulsões/prevenção & controle , Animais , Atropina/farmacologia , Encéfalo/citologia , Encéfalo/metabolismo , Imuno-Histoquímica , Masculino , Neurônios/metabolismo , Isoformas de Proteínas , Brometo de Piridostigmina/farmacologia , Ratos , Ratos Sprague-Dawley , Convulsões/metabolismoRESUMO
It has recently been reported that soluble epoxide hydrolase (sEH), the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), is expressed in axons of cortical neurons; however, the functional relevance of axonal sEH localization is unknown. Immunocytochemical analyses demonstrate predominant axonal localization of sEH in primary cultures of not only cortical but also sympathetic and sensory neurons. Morphometric analyses of cultured sensory neurons indicate that exposure to a regioisomeric mixture of EETs (0.01-1.0 µM) causes a concentration-dependent increase in axon outgrowth. This axon promoting activity is not a generalized property of all regioisomers of EETs as axonal growth is enhanced in sensory neurons exposed to 14,15-EET but not 8,9- or 11,12-EET. 14,15-EET also promotes axon outgrowth in cultured cortical neurons. Co-exposure to EETs and either of two structurally diverse pharmacological inhibitors of sEH potentiates the axon-enhancing activity of EETs in sensory and cortical neurons. Mass spectrometry indicates that sEH inhibition significantly increases EETs and significantly decreases dihydroxyeicosatrienoic acid metabolites in neuronal cell cultures. These data indicate that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration.
Assuntos
Axônios/fisiologia , Córtex Cerebral/fisiologia , Ácidos Eicosanoicos/farmacologia , Células Receptoras Sensoriais/fisiologia , Animais , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Epóxido Hidrolases/metabolismo , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Imuno-Histoquímica , Neurônios/efeitos dos fármacos , Gravidez , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
Organophosphate (OP) neurotoxins cause acute cholinergic toxicity and seizures resulting in delayed brain damage and persistent neurological symptoms. Testing novel strategies for protecting against delayed effects of acute OP intoxication has been hampered by the lack of appropriate animal models. In this study, we characterize the spatiotemporal pattern of cellular injury after acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague-Dawley rats received pyridostigmine (0.1 mg/kg, im) and atropine methylnitrate (20mg/kg, im) prior to DFP (9 mg/kg, ip) administration. All DFP-treated animals exhibited moderate to severe seizures within minutes after DFP injection but survived up to 72 h. AChE activity was significantly depressed in the cortex, hippocampus, subcortical brain tissue and cerebellum at 1h post-DFP injection and this inhibition persisted for up to 72 h. Analysis of neuronal injury by Fluoro-Jade B (FJB) labeling revealed delayed neuronal cell death in the hippocampus, cortex, amygdala and thalamus, but not the cerebellum, starting at 4h and persisting until 72 h after DFP treatment, although temporal profiles varied between brain regions. At 24h post-DFP injection, the pattern of FJB labeling corresponded to TUNEL staining in most brain regions, and FJB-positive cells displayed reduced NeuN immunoreactivity but were not immunopositive for astrocytic (GFAP), oligodendroglial (O4) or macrophage/microglial (ED1) markers, demonstrating that DFP causes a region-specific delayed neuronal injury mediated in part by apoptosis. These findings indicate the feasibility of this model for testing neuroprotective strategies, and provide insight regarding therapeutic windows for effective pharmacological intervention following acute OP intoxication.
Assuntos
Encéfalo/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Isoflurofato/toxicidade , Neurônios/efeitos dos fármacos , Animais , Encéfalo/patologia , Masculino , Modelos Animais , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Fatores de TempoRESUMO
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.