RESUMEN
Epileptic seizures and status epilepticus (SE) induced by the poisoning with organophosphorus nerve agents (OP), like soman, are accompanied by neuroinflammation whose role in seizure-related brain damage (SRBD) is not clear. Antagonists of the NMDA glutamate ionotropic receptors are currently among the few compounds able to arrest seizures and provide neuroprotection even during refractory status epilepticus (RSE). Racemic ketamine (KET), in combination with atropine sulfate (AS), was previously shown to counteract seizures and SRBD in soman-poisoned guinea-pigs. In a mouse model of severe soman-induced SE, we assessed the potentials of KET/AS combinations as a treatment for SE/RSE-induced SRBD and neuroinflammation. When starting 30min after soman challenge, a protocol involving six injections of a sub-anesthetic dose of KET (25mg/kg) was evaluated on body weight loss, brain damage, and neuroinflammation whereas during RSE, anesthetic protocols were considered (KET 100mg/kg). After confirming that during RSE, KET injection was to be repeated despite some iatrogenic deaths, we used these proof-of-concept protocols to study the changes in mRNA and related protein contents of some inflammatory cytokines, chemokines and adhesion molecules in cortex and hippocampus 48h post-challenge. In both cases, the KET/AS combinations showed important neuroprotective effects, suppressed neutrophil granulocyte infiltration and partially suppressed glial activation. KET/AS could also reduce the increase in mRNA and related pro-inflammatory proteins provoked by the poisoning. In conclusion, the present study confirms that KET/AS treatment has a strong potential for SE/RSE management following OP poisoning. The mechanisms involved in the reduction of central neuroinflammation remain to be studied.
Asunto(s)
Atropina/farmacología , Sustancias para la Guerra Química/toxicidad , Antagonistas de Aminoácidos Excitadores/farmacología , Ketamina/farmacología , Antagonistas Muscarínicos/farmacología , Soman/toxicidad , Estado Epiléptico/inducido químicamente , Estado Epiléptico/tratamiento farmacológico , Animales , Área Bajo la Curva , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Quimiocina CCL5/genética , Quimiocina CCL5/inmunología , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Masculino , Ratones , Neuroglía/inmunología , Neutrófilos/inmunología , Reacción en Cadena de la Polimerasa , ARN Mensajero/química , ARN Mensajero/genética , Distribución Aleatoria , Estado Epiléptico/inmunologíaRESUMEN
PURPOSE: Neuroinflammation appears as a prominent feature of the mesiotemporal lobe epilepsy syndrome (MTLE) that is observed in human patients and animal models. However, the precise temporal relationship of its development during epileptogenesis remains to be determined. The aim of the present study was to investigate (1) the time course and spatial distribution of neuronal death associated with seizure development, (2) the time course of microglia and astrocyte activation, and (3) the kinetics of induction of mRNAs from neuroinflammatory-related proteins during the emergence of recurrent seizures. METHODS: Experimental MTLE was induced by the unilateral intrahippocampal injection of kainate in C57BL/6 adult mice. Microglial and astrocytic changes in both ipsilateral and contralateral hippocampi were examined by respectively analyzing griffonia simplicifolia (GSA) lectin staining and glial fibrillary acidic protein (GFAP) immunoreactivity. Changes in mRNA levels of selected genes of cytokine and cytokine regulatory proteins (interleukin-1ß, IL-1ß; interleukin-1 receptor antagonist, IL-1Ra; suppressor of cytokine signaling 3, SOCS3) and enzymes of the eicosanoid pathway (group IVA cytosolic phospholipase A2, cPLA(2)-α; cycloxygenase-2, COX-2) were studied by reverse transcription-quantitative real time polymerase chain reaction. KEY FINDINGS: Our data show an immediate cell death occurring in the kainate-injected hippocampus during the initial status epilepticus (SE). A rapid increase of activated lectin-positive cells and GFAP-immunoreactivity was subsequently detected in the ipsilateral hippocampus. In the same structure, Il-1ß, IL-1Ra, and COX-2 mRNA were specifically increased during SE and epileptogenesis with a different time course. Conversely, the expression of SOCS3 mRNA, a surrogate marker of interleukin signaling, was mainly increased in the contralateral hippocampus after SE. SIGNIFICANCE: Our data show that specific neuroinflammatory pathways are activated in a time- and structure-dependent manner with putative distinct roles in epileptogenesis.
Asunto(s)
Citocinas/metabolismo , Epilepsia del Lóbulo Temporal/complicaciones , Regulación de la Expresión Génica/fisiología , Inflamación/etiología , Convulsiones/etiología , Animales , Muerte Celular/efectos de los fármacos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Citocinas/genética , Modelos Animales de Enfermedad , Eicosanoides/genética , Eicosanoides/metabolismo , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/patología , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Inflamación/metabolismo , Ácido Kaínico/toxicidad , Ratones , Ratones Endogámicos C57BL , Lectinas de Plantas , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
The organophosphorus nerve agent soman is an irreversible cholinesterase (ChE) inhibitor that can produce long-lasting seizures and seizure-related brain damage (SRBD) in which acetylcholine and glutamate are involved. Since these neurotransmitters play a key-role in the auditory function, it was hypothesized that a hearing test may be an efficient way for detecting the central effects of soman intoxication. In the present study, distortion product otoacoustic emissions (DPOAEs), a non-invasive audiometric method, were used in rats administered with soman (70 µg/kg). Four hours post-soman, DPOAE intensities were significantly decreased. They returned to baseline one day later. The amplitude of the temporary drop of the DPOAEs was well related to the severity of the intoxication. The greatest change was recorded in the rats that survived long-lasting convulsions, i.e. those that showed the highest ChE inhibition in brain and severe encephalopathy. Furthermore, the administration, immediately after soman, of a three-drug therapy composed of atropine sulfate, HI-6 and avizafone abolished the convulsions, the transient drop of DPOAEs at 4h and the occurrence of SRBD at 28 h without modifying brain ChE inhibition. This showed that DPOAE change was not directly related to soman-induced inhibition of cerebral ChE but rather to its neuropathological consequences. The present findings strongly suggest that DPOAEs represent a promising non-invasive tool to predict SRBD occurrence in nerve agent poisoning and to control the efficacy of a neuroprotective treatment.
Asunto(s)
Lesiones Encefálicas/inducido químicamente , Lesiones Encefálicas/patología , Emisiones Otoacústicas Espontáneas/efectos de los fármacos , Emisiones Otoacústicas Espontáneas/fisiología , Soman/toxicidad , Animales , Lesiones Encefálicas/prevención & control , Cóclea/efectos de los fármacos , Cóclea/fisiología , Predicción , Masculino , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Distribución Aleatoria , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Convulsiones/patología , Convulsiones/prevención & controlAsunto(s)
Epilepsia/inducido químicamente , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Ketamina/uso terapéutico , Organofosfatos/toxicidad , Animales , Atropina/uso terapéutico , Daño Encefálico Crónico/inducido químicamente , Daño Encefálico Crónico/prevención & control , Antagonistas Colinérgicos/uso terapéutico , Combinación de Medicamentos , Epilepsia/prevención & control , Cobayas , Humanos , Fármacos Neuroprotectores/uso terapéuticoRESUMEN
The organophosphorus nerve agent soman is an irreversible cholinesterase (ChE) inhibitor that can produce long-lasting seizures and brain damage in which the neurotransmitters acetylcholine and glutamate are involved. These same neurotransmitters play key-roles in the auditory function. It was then assumed that exploring the hearing function may provide markers of the central events triggered by soman intoxication. In the present study, distortion product otoacoustic emissions (DPOAEs), a non-invasive audiometric method, were used to monitor cochlear functionality in rats administered with a moderate dose of soman (45 microg/kg). DPOAEs were investigated either 4h or 24h post-challenge. In parallel, the effects of soman on whole blood and brain ChE activity and on brain histology were also studied. The first main result is that DPOAE intensities were significantly decreased 4h post-soman and returned to baseline at 24h. The amplitude changes were well related to the severity of symptoms, with the greatest change being recorded in the rats that survived long-lasting convulsions. The second main result is that baseline DPOAEs recorded 8 days before soman appear to predict the severity of symptoms produced by the intoxication. Indeed, the lowest baseline DPOAEs corresponded to the occurrence of long-lasting convulsions and brain damage and to the greatest inhibition in central ChE. These results thus suggest that DPOAEs represent a promising non-invasive tool to assess and predict the central consequences of nerve agent poisoning. Further investigations will be carried out to assess the potential applications and the limits of this non-invasive method.
Asunto(s)
Síndromes de Neurotoxicidad/etiología , Emisiones Otoacústicas Espontáneas/efectos de los fármacos , Soman/toxicidad , Estimulación Acústica , Animales , Audiometría/métodos , Audiometría de Respuesta Evocada/métodos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Química Encefálica/efectos de los fármacos , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/sangre , Inhibidores de la Colinesterasa/toxicidad , Cóclea/efectos de los fármacos , Cóclea/fisiopatología , Inyecciones Subcutáneas , Masculino , Síndromes de Neurotoxicidad/patología , Síndromes de Neurotoxicidad/fisiopatología , Ratas , Ratas Wistar , Índice de Severidad de la Enfermedad , Soman/administración & dosificación , Soman/sangre , Análisis de Supervivencia , Factores de TiempoRESUMEN
Following exposure to the organophosphorus nerve agent soman, the development of long-lasting seizures and build-up of irreversible seizure-related brain damage (SRBD) still represent a therapeutic challenge. A neuro-inflammatory reaction takes place in the brain after poisoning but its characteristics and potential role in SRBD and post-status epilepticus epileptogenesis is not well understood. In the present study we have analyzed by quantitative RT-PCR the time course of changes in mRNA levels of IL-1beta, TNFalpha, IL-6, ICAM-1 and SOCS3 in hippocampus, whole cortex and cerebellum in a mouse model of severe seizures and neuropathy up to 7 days after poisoning. Mice received an injection of the oxime HI-6 (50mg/kg) 5 min prior to the administration of a convulsive dose of soman (172 microg/kg). An important and highly significant increase of the five mRNA levels was recorded in cortex and hippocampus. In the cortex, the activation was generally detected as early as 1h post-intoxication with a peak response recorded between 6 and 24h. In the hippocampus, the gene up-regulation was delayed to 6h post-soman and the peak response observed between 24 and 48 h. After peaking, the response declined (except for ICAM in the hippocampus) but remained elevated, some of them significantly, at day 7. Interestingly, in the cerebellum, some changes were also observed but were several fold smaller. In conclusion, the present study indicates a quick neuro-inflammatory gene response that does not subside over 7 days suggesting a potential role in the neurological consequences of soman-induced status epilepticus.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/genética , Convulsiones/complicaciones , Soman/toxicidad , Animales , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Sustancias para la Guerra Química/toxicidad , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inflamación/etiología , Molécula 1 de Adhesión Intercelular/genética , Interleucina-1beta/genética , Masculino , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Convulsiones/inducido químicamente , Soman/administración & dosificación , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Factores de Tiempo , Factores de Necrosis Tumoral/genéticaRESUMEN
Soman poisoning is known to induce full-blown tonic-clonic seizures, status epilepticus (SE), seizure-related brain damage (SRBD) and lethality. Previous studies in guinea-pigs have shown that racemic ketamine (KET), with atropine sulfate (AS), is very effective in preventing death, stopping seizures and protecting sensitive brain areas when given up to 1h after a supra-lethal challenge of soman. The active ketamine isomer, S(+) ketamine (S-KET), is more potent than the racemic mixture and it also induces less side-effects. To confirm the efficacy of KET and to evaluate the potential of S-KET for delayed medical treatment of soman-induced SE, we studied different S-KET dose regimens using the same paradigm used with KET. Guinea-pigs received pyridostigmine (26 microg/kg, IM) 30min before soman (62 microg/kg, 2 LD(50), IM), followed by therapy consisting of atropine methyl nitrate (AMN) (4 mg/kg, IM) 1min following soman exposure. S-KET, with AS (10mg/kg), was then administered IM at different times after the onset of seizures, starting at 1h post-soman exposure. The protective efficacy of S-KET proved to be comparable to KET against lethality and SRBD, but at doses two to three times lower. As with KET, delaying treatment by 2h post-poisoning greatly reduced efficacy. Conditions that may have led to an increased S-KET brain concentration (increased doses or number of injections, adjunct treatment with the oxime HI-6) did not prove to be beneficial. In summary, these observations confirm that ketamine, either racemic or S-KET, in association with AS and possibly other drugs, could be highly effective in the delayed treatment of severe soman intoxication.
Asunto(s)
Atropina/farmacología , Daño Encefálico Crónico/inducido químicamente , Daño Encefálico Crónico/prevención & control , Inhibidores de la Colinesterasa/toxicidad , Antagonistas de Aminoácidos Excitadores/farmacología , Ketamina/farmacología , Antagonistas Muscarínicos/farmacología , Soman/toxicidad , Estado Epiléptico/inducido químicamente , Estado Epiléptico/prevención & control , Animales , Cobayas , Dosificación Letal Mediana , Masculino , Oximas , Compuestos de Piridinio/farmacología , Estado Epiléptico/mortalidad , EstereoisomerismoRESUMEN
Soman poisoning induces long-term neuropathology characterized by the presence of damaged neurons up to 2 months after exposure in various central brain areas, especially the hippocampal CA1 layer. Rapid depletion of this layer could therefore be expected. Surprisingly, the CA1 layer remained consistently visible, suggesting delayed death of these damaged neurons, potentially accompanied by neuronal regeneration. To address this issue, mice were exposed to a convulsive dose of soman (110 microg/kg followed by 5.0mg/kg of atropine methyl nitrate (MNA) 1 min later) and brains were collected from day 1 to day 90 post-exposure. Damaged and residual healthy neurons were quantified on brain sections using hemalun-phloxin and fluorojade staining or neuronal nuclei antigen (NeuN) immunohistochemistry. On post-soman day 1, a moderate neuronal cell death was noticed in the hippocampal CA1 layer. In this area, an important and steady quantity of damaged neurons (about 48% of the whole pyramidal neurons) was detected from post-soman day 1 to day 30. Thus, throughout this period, damaged neurons seemed to survive, as confirmed by the unmodified depth of the hippocampal CA1 layer. The dramatic disappearance of the damaged neurons occurred only later during the experiment and was almost complete at day 90 after soman exposure. Interestingly, between day 30 and day 90 following poisoning, an increase in the number of residual healthy pyramidal neurons was observed. These different kinetic patterns related to the density of total, damaged and residual healthy neurons after soman poisoning demonstrate that neuronal regeneration is delayed in the hippocampal CA1 layer and is concomitant to the death of damaged neurons.
Asunto(s)
Inhibidores de la Colinesterasa/envenenamiento , Hipocampo/patología , Regeneración Nerviosa/fisiología , Neuronas/patología , Soman/envenenamiento , Animales , Muerte Celular/fisiología , Núcleo Celular/metabolismo , Fluoresceínas , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Masculino , Ratones , Modelos Estadísticos , Compuestos OrgánicosRESUMEN
The organophosphorus compound soman, an irreversible inhibitor of cholinesterases, produces seizure activity and related brain damage. Studies using various biochemical markers of programmed cell death (PCD) suggested that soman-induced cell damage in the brain was apoptotic rather than necrotic. However, it has recently become clear that not all PCD is apoptotic, and the unequivocal demonstration of apoptosis requires ultrastructural examination. Therefore, the present study was undertaken to reinvestigate the damage produced in the brains of mice sacrificed at various times within the first 24 h or at 7 days after a convulsive dose of soman. Classical histology and ultrastructural examination were performed. The immunohistochemical expression of proteins (p53, Bax) involved in PCD, DNA fragmentation (TUNEL method at light and electron microscopy levels) and the glial reaction were also explored. Our study confirms that the severity of lesions depended on the duration of convulsions and shows that cerebral changes were still occurring as late as 7 days after the onset of long-lasting convulsions. Our observations also establish that there was a large variety of ultrastructurally distinct types of cell damage, including hybrid forms between apoptosis and necrosis, but that pure apoptosis was very rare. A prominent expression of p53 and Bax proteins was detected indicating that PCD mechanisms were certainly involved in the morphologically diverse forms of cell death. Since purely apoptotic cells were very rare, these protein expressions were presumably involved either in nonapoptotic cell death mechanisms or in apoptotic mechanisms occurring in parallel with nonapoptotic ones. Moreover, evidence for DNA fragmentation by the TUNEL method was found in apoptotic but also in numerous other morphotypes of cell damage. Therefore, TUNEL-positivity and the expression of PCD-related proteins, in the absence of ultrastructural confirmation, were here shown not to provide proof of apoptosis. In soman poisoning as well as in other cerebral pathologies, premature conclusions on this question can potentially be misleading and might even lead to detrimental therapies.
Asunto(s)
Apoptosis/efectos de los fármacos , Encéfalo , Sustancias para la Guerra Química/toxicidad , Convulsiones/inducido químicamente , Soman/toxicidad , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/ultraestructura , Fragmentación del ADN/efectos de los fármacos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Microscopía Electrónica de Transmisión , Convulsiones/patología , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
To date, only short-term glial reaction has been extensively studied following soman or other warfare neurotoxicant poisoning. In a context of cell therapy by neural progenitor engraftment to repair brain damage, the long-term effect of soman on glial reaction and neural progenitor division was analyzed in the present study. The effect of soman poisoning was estimated in mouse brains at various times ranging from 1 to 90 days post-poisoning. Using immunochemistry and dye staining techniques (hemalun-eosin staining), the number of degenerating neurons, the number of dividing neural progenitors, and microglial, astroglial or oligodendroglial cell activation were studied. Soman poisoning led to rapid and massive (post-soman day 1) death of mature neurons as assessed by hemalun-eosin staining. Following this acute poisoning phase, a weak toxicity effect on mature neurons was still observed for a period of 1 month after poisoning. A massive short-termed microgliosis peaked on day 3 post-poisoning. Delayed astrogliosis was observed from 3 to 90 days after soman poisoning, contributing to glial scar formation. On the other hand, oligodendroglial cells or their precursors were practically unaffected by soman poisoning. Interestingly, neural progenitors located in the subgranular zone of the dentate gyrus (SGZ) or in the subventricular zone (SVZ) of the brain survived soman poisoning. Furthermore, soman poisoning significantly increased neural progenitor proliferation in both SGZ and SVZ brain areas on post-soman day 3 or day 8, respectively. This increased proliferation rate was detected up to 1 month after poisoning.
Asunto(s)
Encéfalo/efectos de los fármacos , Inhibidores de la Colinesterasa/envenenamiento , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Soman/envenenamiento , Células Madre/efectos de los fármacos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Oligodendroglía/patología , Células Madre/metabolismo , Células Madre/patología , Factores de TiempoRESUMEN
Today, organophosphate (OP) nerve agents are still considered as potential threats in both military or terrorism situations. OP agents are potent irreversible inhibitors of central and peripheral acetylcholinesterases. Pretreatment of OP poisoning relies on the subchronic administration of the reversible acetylcholinesterase (AChE) inhibitor pyridostigmine (PYR). Since PYR does not penetrate into the brain, it does not afford protection against seizures and subsequent neuropathology induced by an OP agent such as soman. Comparatively, huperzine (HUP) is a reversible AChE inhibitor that crosses the blood-brain barrier. HUP is presently approved for human use or is in course of clinical trials for the treatment of Alzheimer's disease or myasthenia gravis. HUP is also used as supplementary drug in the USA for correction of memory impairment. Besides, HUP has also been successfully tested for pretreatment of OP poisoning. This review summarizes the therapeutical value of HUP in this field. Moreover, the modes of action of HUP underlying its efficacy against OP agents are described. Efficacy appears mainly related to both the selectivity of HUP for red cell AChE which preserves scavenger capacity of plasma butyrylcholinesterases for OP agents and to the protection conferred by HUP on cerebral AChE. Finally, recent data, showing that HUP seems to be devoid of deleterious effects in healthy subjects, are also presented. Globally, this review reinforces the therapeutical value of HUP for the optimal pretreatment of OP poisoning.
Asunto(s)
Inhibidores de la Colinesterasa/uso terapéutico , Intoxicación por Organofosfatos , Sesquiterpenos/uso terapéutico , Alcaloides , Animales , Sustancias para la Guerra Química/envenenamiento , HumanosRESUMEN
Apoptotic death is known to be an active process requiring the activation of several apoptotic proteins. Depending on the tissue studied and the stimulus used, these processes are distinct. In this work, we studied if there is a putative implication of the p53 and the caspase-3 proteins in kainic acid (KA) and N-methyl-D-aspartic acid (NMDA)-induced apoptosis in organotypic cultures and if there is any relationship between their respective expressions. We found that KA and NMDA both induce apoptosis but only KA-induced apoptosis is p53- and caspase-3-dependent. This demonstrates that KA and NMDA induce apoptosis following different intracellular pathways.