RESUMEN
We evaluated the efficacy of aerosolized acetylcholinesterase (AChE) reactivator oxime MMB-4 in combination with the anticholinergic atropine sulfate for protection against respiratory toxicity and lung injury following microinstillation inhalation exposure to nerve agent soman (GD) in guinea pigs. Anesthetized animals were exposed to GD (841 mg/m(3), 1.2 LCt(50)) and treated with endotracheally aerosolized MMB-4 (50 µmol/kg) plus atropine sulfate (0.25 mg/kg) at 30 sec post-exposure. Treatment with MMB-4 plus atropine increased survival to 100% compared to 38% in animals exposed to GD. Decreases in the pulse rate and blood O(2) saturation following exposure to GD returned to normal levels in the treatment group. The body-weight loss and lung edema was significantly reduced in the treatment group. Similarly, bronchoalveolar cell death was significantly reduced in the treatment group while GD-induced increase in total cell count was decreased consistently but was not significant. GD-induced increase in bronchoalveolar protein was diminished after treatment with MMB-4 plus atropine. Bronchoalveolar lavage AChE and BChE activity were significantly increased in animals treated with MMB-4 plus atropine at 24 h. Lung and diaphragm tissue also showed a significant increase in AChE activity in the treatment group. Treatment with MMB-4 plus atropine sulfate normalized various respiratory dynamics parameters including respiratory frequency, tidal volume, peak inspiratory and expiratory flow, time of inspiration and expiration, enhanced pause and pause post-exposure to GD. Collectively, these results suggest that aerosolization of MMB-4 plus atropine increased survival, decreased respiratory toxicity and lung injury following GD inhalation exposure.
Asunto(s)
Atropina/administración & dosificación , Inhibidores de la Colinesterasa/toxicidad , Reactivadores de la Colinesterasa/administración & dosificación , Oximas/administración & dosificación , Sustancias Protectoras/administración & dosificación , Soman/toxicidad , Acetilcolinesterasa/metabolismo , Administración por Inhalación , Aerosoles , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Butirilcolinesterasa/metabolismo , Sustancias para la Guerra Química/toxicidad , Combinación de Medicamentos , Cobayas , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/patología , Pulmón/fisiopatología , MasculinoRESUMEN
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the blood and tissues of animals that are treated with a number of endotracheally aerosolized therapeutics for protection against inhalation toxicity to sarin. Therapeutics included, aerosolized atropine methyl bromide (AMB), scopolamine or combination of AMB with salbutamol, sphingosine 1-phosphate, keratinocyte growth factor, adenosine A1 receptor antisense oligonucleotide (EPI2010), 2,3-diacetyloxybenzoic acid (2,3 DABA), oxycyte, and survanta. Guinea pigs exposed to 677.4 mg/m(3) or 846.5 mg/m(3) (1.2 LCt(50)) sarin for 4 min using a microinstillation inhalation exposure technique and treated 1 min later with the aerosolized therapeutics. Treatment with all therapeutics significantly increased the survival rate with no convulsions throughout the 24 h study period. Blood AChE activity determined using acetylthiocholine as substrate showed 20% activity remaining in sarin-exposed animals compare to controls. In aerosolized AMB and scopolamine-treated animals the remaining AChE activity was significantly higher (45-60%) compared to sarin-exposed animals (p < 0.05). Similarly, treatment with all the combination therapeutics resulted in significant increase in blood AChE activity in comparison to sarin-exposed animals although the increases varied between treatments (p < 0.05). BChE activity was increased after treatment with aerosolized therapeutics but was lesser in magnitude compared to AChE activity changes. Various tissues showed elevated AChE activity after therapeutic treatment of sarin-exposed animals. Increased AChE and BChE activities in animals treated with nasal therapeutics suggest that enhanced breathing and reduced respiratory toxicity/lung injury possibly contribute to rapid normalization of chemical warfare nerve agent inhibited cholinesterases.
Asunto(s)
Acetilcolinesterasa/metabolismo , Broncodilatadores/uso terapéutico , Inhibidores de la Colinesterasa/toxicidad , Antagonistas Muscarínicos/uso terapéutico , Sarín/toxicidad , Acetilcolinesterasa/sangre , Animales , Antídotos/uso terapéutico , Butirilcolinesterasa/sangre , Butirilcolinesterasa/metabolismo , Sustancias para la Guerra Química/toxicidad , Cobayas , Pulmón/efectos de los fármacos , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/enzimología , Masculino , Terapia RespiratoriaRESUMEN
The chemical warfare nerve agent (CWNA) soman irreversibly inhibits acetylcholinesterase (AChE) causing seizure, neuropathology and neurobehavioral deficits. Pyridostigmine bromide (PB), the currently approved pretreatment for soman, is a reversible AChE inhibitor that does not cross the blood-brain barrier (BBB) to protect against central nervous system damage. [-]-Huperzine A, a natural reversible AChE inhibitor, rapidly passes through the BBB and has numerous neuroprotective properties that are beneficial for protection against soman. However, [-]-Huperzine A is toxic at higher doses due to potent AChE inhibition which limits the utilization of its neuroprotective properties. [+]-Huperzine A, a synthetic stereoisomer of [-]-Huperzine A and a weak inhibitor of AChE, is non-toxic. In this study, we evaluated the efficacy of [+]-Huperzine A for protection against soman toxicity in guinea pigs. Pretreatments with [+]-Huperzine A, i.m., significantly increased the survival rate in a dose-dependent manner against 1.2× LD(50) soman exposures. Behavioral signs of soman toxicity were significantly reduced in 20 and 40 mg/kg [+]-Huperzine A treated animals at 4 and 24 h compared to vehicle and PB controls. Electroencephalogram (EEG) power spectral analysis showed that [+]-Huperzine A significantly reduces soman-induced seizure compared to PB. [+]-Huperzine A (40 mg/kg) preserved higher blood and brain AChE activity compared to PB in soman exposed animals. These data suggest that [+]-Huperzine A protects against soman toxicity stronger than PB and warrant further development as a potent medical countermeasure against CWNA poisoning.
Asunto(s)
Alcaloides/uso terapéutico , Inhibidores de la Colinesterasa/uso terapéutico , Sesquiterpenos/uso terapéutico , Soman/toxicidad , Acetilcolinesterasa/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Temperatura Corporal/efectos de los fármacos , Sustancias para la Guerra Química/toxicidad , Cobayas , Masculino , Fármacos Neuroprotectores/administración & dosificación , Bromuro de Piridostigmina/metabolismo , Bromuro de Piridostigmina/uso terapéutico , Convulsiones/inducido químicamente , Convulsiones/prevención & control , EstereoisomerismoRESUMEN
The efficacy of endotracheal aerosolization of atropine sulfate for protection against soman (GD)-induced respiratory toxicity was investigated using microinstillation technique in guinea pigs. GD (841 mg/m(3), 1.3 LCt(50) or 1121 mg/m(3), 1.7 LCt(50)) was aerosolized endotracheally to anesthetized male guinea pigs that were treated with atropine sulfate (5.0 mg/kg) 30 s postexposure by endotracheal microinstillation. Animals exposed to 841 mg/m(3) and 1121 mg/m(3)GD resulted in 31 and 13% while treatment with atropine sulfate resulted in 100 and 50% survival, respectively. Cholinergic symptoms and increased body weight loss were reduced in atropine-treated animals compared to GD controls. Diminished pulse rate and blood O(2) saturation in GD-exposed animals returned to normal levels after atropine treatment. Increased cell death, total cell count and protein in the bronchoalveolar fluid (BALF) in GD-exposed animals returned to normal levels following atropine treatment. GD exposure increased glutathione and superoxide dismutase levels in BALF and that were reduced in animals treated with atropine. Respiratory parameters measured by whole-body barometric plethysmography revealed that treatment with atropine sulfate resulted in normalization of respiratory frequency, tidal volume, time of expiration, time of inspiration, end expiratory pause, pseudo lung resistance (Penh) and pause at 4 and 24 h post 841 mg/m(3) GD exposure. Lung histopathology showed that atropine treatment reduced bronchial epithelial subepithelial inflammation and multifocal alveolar septal edema. These results suggest that endotracheal aerosolization of atropine sulfate protects against respiratory toxicity and lung injury induced by microinstillation inhalation exposure to lethal doses of GD.
Asunto(s)
Atropina/farmacología , Exposición por Inhalación/efectos adversos , Enfermedades Respiratorias/inducido químicamente , Enfermedades Respiratorias/prevención & control , Soman/toxicidad , Tráquea/metabolismo , Acetilcolinesterasa/sangre , Acetilcolinesterasa/metabolismo , Administración por Inhalación , Animales , Peso Corporal/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/química , Butirilcolinesterasa/sangre , Butirilcolinesterasa/metabolismo , Sustancias para la Guerra Química/toxicidad , Cobayas , Frecuencia Cardíaca , Dosificación Letal Mediana , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Pletismografía Total , Sarín/toxicidad , Volumen de Ventilación Pulmonar/efectos de los fármacosRESUMEN
The protective efficacy of the antimuscarinic agent scopolamine was evaluated against soman (o-pinacolyl methylphosphonofluoridate [GD])-induced respiratory toxicity in guinea pigs. Anesthetized animals were exposed to GD (841 mg/m(3)) by microinstillation inhalation exposure and treated 30 seconds later with endotracheally aerosolized scopolamine (0.25 mg/kg) and allowed to recover for 24 hours. Treatment with scopolamine significantly increased survival and reduced clinical signs of toxicity and body weight loss in GD-exposed animals. Analysis of bronchoalveolar lavage (BAL) fluid showed normalization of GD-induced increased cell death, total cell count, and protein following scopolamine treatment. The BAL fluid acetylcholinesterase and butyrylcholinesterase levels were also increased by scopolamine treatment. Respiratory dynamics parameters were normalized at 4 and 24 hours post-GD exposure in scopolamine-treated animals. Lung histology showed that scopolamine treatment reduced bronchial epithelial and subepithelial inflammation and multifocal alveolar septal edema. These results suggest that aerosolized scopolamine considerably protects against GD-induced respiratory toxicity.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Pulmón/efectos de los fármacos , Antagonistas Muscarínicos/farmacología , Sustancias Protectoras/farmacología , Escopolamina/farmacología , Soman/toxicidad , Acetilcolinesterasa/sangre , Acetilcolinesterasa/metabolismo , Aerosoles , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Butirilcolinesterasa/sangre , Butirilcolinesterasa/metabolismo , Recuento de Células , Cobayas , Pulmón/metabolismo , Pulmón/patología , Masculino , Pruebas de Función Respiratoria , Tráquea/efectos de los fármacos , Tráquea/patologíaRESUMEN
Barometric whole-body plethysmography (WBP) was used to examine pulmonary functions at 4 and 24 hours postexposure to soman (GD) in guinea pigs without therapeutics to improve survival. Endotracheal aerosolization by microinstillation was used to administer GD (280, 561, and 841 mg/m(3)) or saline to anesthetized guinea pigs. Significant increases in respiratory frequency (RF), tidal volume (TV), and minute volume (MV) were observed with 841 mg/m(3) GD at 4 hours and that were reduced at 24 hours postexposure. A dose-dependent increase in peak inspiration flow and peak expiration flow was present at 4-hour post-GD exposure that was reduced at 24 hours. Time of inspiration and expiration were decreased in all doses of GD exposure at 4 and 24 hours, with significant inhibition at 841 mg/m(3). End-expiratory pause (EEP) increased at 280 and 561 mg/m(3), but decreased in animals exposed 841 mg/m(3) at 24 hours postexposure. Pseudo-lung resistance (Penh) and pause followed similar patterns and increased at 4 hours, but decreased at 24 hours postexposure to 841 mg/m(3) of GD compared to control. These studies indicate GD exposure induces dose-dependent changes in pulmonary function that are significant at 841 mg/m(3) at 4 hours and remains 24 hours postexposure. Furthermore, at 4 hours, GD induces bronchoconstriction possibly due to copious airway secretion and ongoing lung injury in addition to cholinergic effects, while at 24 hours GD induces bronchodilation a possible consequence of initial compensatory mechanisms.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Pulmón/efectos de los fármacos , Pruebas de Función Respiratoria , Soman/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Cobayas , Pulmón/fisiología , PletismografíaRESUMEN
Respiratory toxicity and lung injury following inhalation exposure to chemical warfare nerve agent soman was examined in guinea pigs without therapeutics to improve survival. A microinstillation inhalation exposure technique that aerosolizes the agent in the trachea was used to administer soman to anesthetized age and weight matched male guinea pigs. Animals were exposed to 280, 561, 841, and 1121 mg/m(3) concentrations of soman for 4 min. Survival data showed that all saline controls and animals exposed to 280 and 561 mg/m(3) soman survived, while animals exposed to 841, and 1121 mg/m(3) resulted in 38% and 13% survival, respectively. The microinstillation inhalation exposure LCt(50) for soman determined by probit analysis was 827.2mg/m(3). A majority of the animals that died at 1121 mg/m(3) developed seizures and died within 15-30 min post-exposure. There was a dose-dependent decrease in pulse rate and blood oxygen saturation of animals exposed to soman at 5-6.5 min post-exposure. Body weight loss increased with the dose of soman exposure. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase and butyrylcholinesterase activity was inhibited dose-dependently in soman treated groups at 24h. BAL cells showed a dose-dependent increase in cell death and total cell counts following soman exposure. Edema by wet/dry weight ratio of the accessory lung lobe and trachea was increased slightly in soman exposed animals. An increase in total bronchoalveolar lavage fluid protein was observed in soman exposed animals at all doses. Differential cell counts of BAL and blood showed an increase in total lymphocyte counts and percentage of neutrophils. These results indicate that microinstillation inhalation exposure to soman causes respiratory toxicity and acute lung injury in guinea pigs.
Asunto(s)
Lesión Pulmonar Aguda/inducido químicamente , Sustancias para la Guerra Química/toxicidad , Exposición por Inhalación/efectos adversos , Soman/toxicidad , Acetilcolinesterasa/sangre , Animales , Líquido del Lavado Bronquioalveolar/química , Butirilcolinesterasa/sangre , Relación Dosis-Respuesta a Droga , Cobayas , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Oxígeno/sangre , Pérdida de Peso/efectos de los fármacosRESUMEN
The toxicity of organophosphorous (OP) nerve agents is attributed to their irreversible inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine (ACh) and is followed by the release of excitatory amino acids (EAA). EAAs sustain seizure activity and induce neuropathology due to over-stimulation of N-methyl-d-aspartate (NMDA) receptors. Huperzine A (Hup A), a blood-brain barrier permeable selective reversible inhibitor of AChE, has been shown to reduce EAA-induced cell death by interfering with glutamate receptor-gated ion channels in primary neuronal cultures. Although [-]-Hup A, the natural isomer, inhibits AChE approximately 38-fold more potently than [+]-Hup A, both [-]- and [+]-Hup A block the NMDA channel similarly. Here, we evaluated the protective efficacy of [+]-Hup A for NMDA-induced seizure in a rat model. Rats implanted with radiotelemetry probes to record electroencephalography (EEG), electrocardiography (ECG), body temperature, and physical activity were administered various doses of [+]-Hup A (intramuscularly) and treated with 20 microg/kg NMDA (intracerebroventricular) 20-30 min later. For post-exposure, rats were treated with [+]-Hup A (3 mg/kg, intramuscularly) 1 min after NMDA (20 microg/kg). Our data showed that pre- and post-exposure, [+]-Hup A (3 mg/kg) protects animals against NMDA-induced seizures. Also, NMDA-administered animals showed increased survival following [+]-Hup A treatment. [+]-Hup A has no visible effect on EEG, heart-rate, body temperature, or physical activity, indicating a reduced risk of side effects, toxicity, or associated pathology. Our results suggest that [+]-Hup A protects against seizure and status epilepticus (SE) by blocking NMDA-induced excitotoxicity in vivo. We propose that [+]-Hup A, or a unique combination of [+]- and [-]-Hup A, may prove to be effective for pre- and post-exposure treatment of lethal doses of OP-induced neurotoxicity.
Asunto(s)
N-Metilaspartato/toxicidad , Convulsiones/prevención & control , Sesquiterpenos/uso terapéutico , Estado Epiléptico/prevención & control , Acetilcolinesterasa/sangre , Alcaloides , Animales , Temperatura Corporal , Modelos Animales de Enfermedad , Electroencefalografía , Frecuencia Cardíaca , Masculino , Actividad Motora , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Convulsiones/fisiopatología , Estado Epiléptico/inducido químicamente , Estado Epiléptico/fisiopatologíaRESUMEN
Chemokines and their receptors are of great interest within the milieu of immune responses elicited in the central nervous system in response to trauma. Chemokine (C-C motif)) ligand 2 (CCL2), which is also known as monocyte chemotactic protein-1, has been implicated in the pathogenesis of traumatic brain injury (TBI), brain ischemia, Alzheimer's disease, and other neurodegenerative diseases. In this study, we investigated the time course of CCL2 accumulation in cerebrospinal fluid (CSF) after exposures to single and repeated blast overpressures of varied intensities along with the neuropathological changes and motor deficits resulting from these blast conditions. Significantly increased concentrations of CCL2 in CSF were evident by 1 h of blast exposure and persisted over 24 h with peak levels measured at 6 h post-injury. The increased levels of CCL2 in CSF corresponded with both the number and intensities of blast overpressure and were also commensurate with the extent of neuromotor impairment and neuropathological abnormalities resulting from these exposures. CCL2 levels in CSF and plasma were tightly correlated with levels of CCL2 messenger RNA in cerebellum, the brain region most consistently neuropathologically disrupted by blast. In view of the roles of CCL2 that have been implicated in multiple neurodegenerative disorders, it is likely that the sustained high levels of CCL2 and the increased expression of its main receptor, CCR2, in the brain after blast may similarly contribute to neurodegenerative processes after blast exposure. In addition, the markedly elevated concentration of CCL2 in CSF might be a candidate early-response biomarker for diagnosis and prognosis of blast-induced TBI.
Asunto(s)
Traumatismos por Explosión/líquido cefalorraquídeo , Lesiones Traumáticas del Encéfalo/líquido cefalorraquídeo , Quimiocina CCL2/líquido cefalorraquídeo , Animales , Biomarcadores/líquido cefalorraquídeo , Traumatismos por Explosión/sangre , Lesiones Traumáticas del Encéfalo/sangre , Quimiocina CCL2/sangre , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Dephosphorylation of phosphorylated Tau (pTau) protein, which is essential for the preservation of neuronal microtubule assemblies and for protection against trauma-induced tauopathy and chronic traumatic encephalopathy (CTE), is primarily achieved in brain by tissue non-specific alkaline phosphatase (TNAP). Paired helical filaments (PHFs) and Tau isolated from Alzheimer's disease (AD) patients' brains have been shown to form microtubule assemblies with tubulin only after treatment with TNAP or protein phosphatase-2A, 2B and -1, suggesting that Tau protein in the PHFs of neurons in AD brain is hyperphosphorylated, which prevents microtubule assembly. Using blast or weight drop models of traumatic brain injury (TBI) in rats, we observed pTau accumulation in the brain as early as 6h post-injury and further accumulation which varied regionally by 24h post-injury. The pTau accumulation was accompanied by reduced TNAP expression and activity in these brain regions and a significantly decreased plasma total alkaline phosphatase activity after the weight drop. These results reveal that both blast- and impact acceleration-induced head injuries cause an acute decrease in the level/activity of TNAP in the brain, which potentially contributes to trauma-induced accumulation of pTau and the resultant tauopathy. The regional changes in the level/activity of TNAP or accumulation of pTau after these injuries did not correlate with the accumulation of amyloid precursor protein, suggesting that the basic mechanism underlying tauopathy in TBI might be distinct from that associated with AD.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Lesiones Encefálicas/metabolismo , Encéfalo/enzimología , Proteínas tau/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Masculino , Fosforilación , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Repeated blast exposures commonly induce traumatic brain injury (TBI) characterized by diffuse axonal injury (DAI). We hypothesized that degradation of cytoskeletal proteins in the brain can lead to DAI, and evaluated α-II spectrin degradation in the pathophysiology of blast-induced TBI using the tightly-coupled three repetitive blast exposure mice model with a 1-30 min window in between exposures. Degradation of α-II spectrin and the expression profiles of caspase-3 and calpain-2, the major enzymes involved in the degradation were analyzed in the frontal cortex and cerebellum using Western blotting with specific antibodies. DAI at different brain regions was evaluated by neuropathology with silver staining. Repeated blast exposures resulted in significant increases in the α-II spectrin degradation products in the frontal cortex and cerebellum compared to sham controls. Expression of active caspase-3, which degrades α-II spectrin, showed significant increase in the frontal cortex after blast exposure at all the time points studied, while cerebellum showed an acute increase which was normalized over time. The expression of another α-II spectrin degrading enzyme, calpain-2, showed a rapid increase in the frontal cortex after blast exposure and it was significantly higher in the cerebellum at later time points. Neuropathological analysis showed significant levels of DAI at the frontal cortex and cerebellum at multiple time points after repeated blast injury. In summary, repeated blast exposure results in specific degradation of α-II spectrin in the brain along with differential expression of caspase-3/calpain-2 suggesting cytoskeletal breakdown as a possible contributor of DAI after repeated blast exposure.
Asunto(s)
Traumatismos por Explosión/metabolismo , Lesiones Encefálicas/metabolismo , Encéfalo/metabolismo , Espectrina/metabolismo , Animales , Axones/patología , Traumatismos por Explosión/patología , Encéfalo/patología , Lesiones Encefálicas/patología , Calpaína/metabolismo , Caspasa 3/metabolismo , Proteínas del Citoesqueleto/metabolismo , RatonesRESUMEN
The pathophysiology of blast-induced traumatic brain injury (TBI) and subsequent behavioral deficits are not well understood. Unraveling the mechanisms of injury is critical to derive effective countermeasures against this form of neurotrauma. Preservation of the integrity of cellular DNA is crucial for the function and survival of cells. We evaluated the effect of repeated blast exposures on the integrity of brain DNA and tested the utility of cell-free DNA (CFD) in plasma as a biomarker for the diagnosis and prognosis of blast-induced polytrauma. The results revealed time-dependent breakdown in cellular DNA in different brain regions, with the maximum damage at 24 h post-blast exposures. CFD levels in plasma showed a significant transient increase, which was largely independent of the timing and severity of brain DNA damage; maximum levels were recorded at 2 h after repeated blast exposure and returned to baseline at 24 h. A positive correlation was observed between the righting reflex time and CFD level in plasma at 2 h after blast exposure. Brain DNA damage subsequent to repeated blast was associated with decreased mitochondrial membrane potential, increased release of cytochrome C, and up-regulation of caspase-3, all of which are indicative of cellular apoptosis. Shock-wave-induced DNA damage and initiation of mitochondrial-driven cellular apoptosis in the brain after repeated blast exposures indicate that therapeutic strategies directed toward inhibition of DNA damage or instigation of DNA repair may be effective countermeasures.
Asunto(s)
Traumatismos por Explosión/metabolismo , Lesiones Encefálicas/metabolismo , Encéfalo/metabolismo , Fragmentación del ADN , Mitocondrias/metabolismo , Animales , Apoptosis/fisiología , Biomarcadores/metabolismo , Traumatismos por Explosión/fisiopatología , Encéfalo/fisiopatología , Lesiones Encefálicas/fisiopatología , Caspasa 3/metabolismo , Explosiones , Masculino , Ratones , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
The neuropathologic mechanisms after exposure to lethal doses of nerve agent are complex and involve multiple biochemical pathways. Effective treatment requires drugs that can simultaneously protect by reversible binding to the acetylcholinesterase (AChE) and blocking cascades of seizure related brain damage, inflammation, neuronal degeneration as well as promoting induction of neuroregeneration. [-]-Huperzine A ([-]-Hup A), is a naturally occurring potent reversible AChE inhibitor that penetrates the blood-brain barrier. It also has several neuroprotective effects including modification of beta-amyloid peptide, reduction of oxidative stress, anti-inflammatory, anti-apoptotic and induction and regulation of nerve growth factor. Toxicities at higher doses restrict the neuroporotective ability of [-]-Hup A for treatment. The synthetic stereoisomer, [+]-Hup A, is less toxic due to poor AChE inhibition and is suitable for both pre-/post-exposure treatments of nerve agent toxicity. [+]-Hup A block the N-methyl-D-aspartate (NMDA)-induced seizure in rats, reduce excitatory amino acid induced neurotoxicity and also prevent soman induced toxicity with minimum performance decrement. Unique combinations of two stereo-isomers of Hup A may provide an excellent pre/post-treatment drug for the nerve agent induced seizure/status epilepticus. We investigated a combination of [+]-Hup A with a small dose of [-]-Hup A ([+] and [-]-Hup A) against soman toxicity. Our data showed that pretreatment with a combination [+] and [-]-Hup A significantly increased the survival rate and reduced behavioral abnormalities after exposure to 1.2 × LD(50) soman compared to [+]-Hup A in guinea pigs. In addition, [+] and [-]-Hup A pretreatment inhibited the development of high power of EEG better than [+]-Hup A pretreatment alone. These data suggest that a combination of [+] and [-]-Hup A offers better protection than [+]-Hup A and serves as a potent medical countermeasure against lethal dose nerve agent toxicity in guinea pigs.
Asunto(s)
Alcaloides/administración & dosificación , Inhibidores de la Colinesterasa/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , Sesquiterpenos/administración & dosificación , Soman/antagonistas & inhibidores , Soman/toxicidad , Acetilcolinesterasa/metabolismo , Alcaloides/química , Animales , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/química , Cobayas , Masculino , Fármacos Neuroprotectores/química , Ratas , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Sesquiterpenos/química , EstereoisomerismoRESUMEN
Blast-induced traumatic brain injury is complex and involves multiple factors including systemic pathophysiological factors in addition to direct brain injuries. We hypothesize that systemic activation of platelets/leukocytes plays a major role in the development and exacerbation of brain injury after blast exposure. A mouse model of repeated blast exposure that results in significant neuropathology, neurobehavioral changes and regional specific alterations in various biomolecules in the brain was used for the proposed study. Activation of platelets was evaluated by flow cytometry and serotonin content was analyzed by ELISA. Expression of myeloperoxidase was analyzed by Western blotting. Histopathology of the brain was used to assess blast-induced cerebral vasoconstriction. The data showed an increase in the activation of platelets at 4h after repeated blast exposures, indicating changes in platelet phenotype in blast neurotrauma. Platelet serotonin concentration showed a significant decrease at 4h after blast with a concurrent increase in the plasma serotonin levels, confirming the early onset of platelet activation after repeated blast exposures. Blood, plasma and brain myeloperoxidase enzyme activity and expression was increased in repeated blast exposed mice at multiple time points. Histopathological analysis of the brains of blast exposed mice showed constriction of blood vessels compared to the respective controls, a phenomenon similar to the reported cerebral vasoconstriction in blast affected victims. These results suggest that repeated blast exposure leads to acute activation of platelets/leukocytes which can augment the pathological effects of brain injury. Platelet/leukocyte targeted therapies can be evaluated as potential acute treatment strategies to mitigate blast-induced neurotrauma.
Asunto(s)
Traumatismos por Explosión/metabolismo , Lesiones Encefálicas/metabolismo , Encéfalo/metabolismo , Animales , Traumatismos por Explosión/fisiopatología , Plaquetas/metabolismo , Encéfalo/irrigación sanguínea , Lesiones Encefálicas/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Peroxidasa/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Serotonina/metabolismo , VasoconstricciónRESUMEN
Glial fibrillary acidic protein (GFAP), a protein enriched in astrocytes, and Tau, a protein abundant in neuronal microtubules, are being widely studied as biomarkers of brain injury, and persistent severity-dependent increases in brain and blood have been reported. Studies on the acute changes of these proteins after blast exposure are limited. Using a mouse model of closely-coupled repeated blast exposures, we have evaluated acute changes in the levels of GFAP and total Tau by Western blotting. Brain levels of GFAP and Tau proteins decreased significantly at 6 h and increased considerably at 24 h after repeated blast exposures. Plasma samples showed a similar initial decrease and later increase over this timeframe. This biphasic pattern points to possible absorption or sequestration of these proteins from plasma immediately after repeated blast exposures. Liver and spleen tissue showed significant increases in the levels of GFAP and Tau protein at 6 and 24 h post-blast exposures whereas semi-quantitative RT-PCR analysis of liver showed no significant changes in the levels of GFAP or Tau mRNAs. These results suggest that blast exposure causes transient changes in cell membrane integrity in multiple organs leading to abnormal migration of proteins from the tissues to the plasma and vice versa. This transient changes in cell membrane permeability and subsequent bidirectional movement of molecules may contribute to the pathophysiology of TBI and polytrauma after blast exposure.
Asunto(s)
Traumatismos por Explosión/metabolismo , Lesiones Encefálicas/metabolismo , Permeabilidad de la Membrana Celular , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas tau/metabolismo , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Traumatismos por Explosión/sangre , Traumatismos por Explosión/complicaciones , Lesiones Encefálicas/sangre , Lesiones Encefálicas/complicaciones , Proteína Ácida Fibrilar de la Glía/sangre , Hígado/metabolismo , Masculino , Ratones , Bazo/metabolismo , Proteínas tau/sangreRESUMEN
Use of improvised explosive devices has significantly increased the incidence of traumatic brain injury (TBI) and associated neuropsychiatric deficits in the recent wars in Iraq and Afghanistan. Acute deleterious effects of single and repeated blast exposure can lead to long-term neurobiological effects and neuropsychiatric deficits. Using in vitro and in vivo shock tube models of blast-induced TBI, we studied changes in mitochondrial energy metabolism after blast exposure. Single and repeated blast exposures in vitro resulted in significant decreases in neuronal adenosine triphosphate (ATP) levels at 6 h post-blast that returned towards normal levels by 24 h. Similar changes in ATP also were observed in the cerebral cortices of mice subjected to single and repeated blast exposures. In neurons, mitochondrial glutamate oxaloacetate transaminase (GOT2) plays a critical role in metabolism and energy production. Proteomic analysis of brain cortices showed a significant decrease in GOT2 levels 6 h after repeated blast exposures, which was further confirmed by Western blotting. Western blot analysis of GOT2 and pyruvate dehydrogenase in the cortex showed direct correlation only between GOT2 and ATP levels. Activity of GOT2 in the isolated cortical mitochondria also showed significant decrease at 6 h supporting the results of proteomic and Western blot analyses. Knowing the significant role of GOT2 in the neuronal mitochondrial energy metabolism, it is quite likely that the down regulation of GOT2 after blast exposure is playing a significant role in mitochondrial dysfunction after blast exposure.
Asunto(s)
Aspartato Aminotransferasas/metabolismo , Traumatismos por Explosión/enzimología , Traumatismos por Explosión/patología , Mitocondrias/enzimología , Mitocondrias/fisiología , Enfermedades Mitocondriales/patología , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Línea Celular , Corteza Cerebral/enzimología , Corteza Cerebral/lesiones , Corteza Cerebral/metabolismo , Ciclo del Ácido Cítrico , Electroforesis en Gel de Poliacrilamida , Metabolismo Energético , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Proteómica , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Cholinergic activity has been recognized as a major regulatory component of stress responses after traumatic brain injury (TBI). Centrally acting acetylcholinesterase (AChE) inhibitors are also being considered as potential therapeutic candidates against TBI mediated cognitive impairments. We have evaluated the expression of molecules involved in cholinergic and inflammatory pathways in various regions of brain after repeated blast exposures in mice. Isoflurane anesthetized C57BL/6J mice were restrained and placed in a prone position transverse to the direction of the shockwaves and exposed to three 20.6 psi blast overpressures with 1-30 min intervals. Brains were collected at the 6h time point after the last blast exposure and subjected to cDNA microarray and microRNA analysis. cDNA microarray analysis showed significant changes in the expression of cholinergic (muscarinic and nicotinic) and gammaaminobutyric acid and glutamate receptors in the midbrain region along with significant changes in multiple genes involved in inflammatory pathways in various regions of the brain. MicroRNA analysis of cerebellum revealed differential expression of miR-132 and 183, which are linked to cholinergic anti-inflammatory signaling, after blast exposure. Changes in the expression of myeloperoxidase in the cerebellum were confirmed by Western blotting. These results indicate that early pathologic progression of blast TBI involves dysregulation of cholinergic and inflammatory pathways related genes. Acute changes in molecules involved in the modulation of cholinergic and inflammatory pathways after blast TBI can cause long-term central and peripheral pathophysiological changes.
Asunto(s)
Acetilcolina/metabolismo , Traumatismos por Explosión/metabolismo , Lesiones Encefálicas/metabolismo , Mediadores de Inflamación/metabolismo , Acetilcolinesterasa/metabolismo , Animales , Traumatismos por Explosión/genética , Encéfalo/metabolismo , Lesiones Encefálicas/genética , Cerebelo/lesiones , Cerebelo/metabolismo , Progresión de la Enfermedad , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Transducción de Señal , Distribución TisularRESUMEN
The mechanisms of central auditory processing involved in auditory/vestibular injuries and subsequent tinnitus and hearing loss in Active Duty servicemembers exposed to blast are not currently known. We analyzed the expression of hearing-related genes in different regions of the brain 6 h after repeated blast exposures in mice. Preliminary data showed that the expression of the deafness-related genes otoferlin and otoancorin was significantly changed in the hippocampus after blast exposures. Differential expression of cadherin and protocadherin genes, which are involved in hearing impairment, was observed in the hippocampus, cerebellum, frontal cortex, and midbrain after repeated blasts. A series of calcium-signaling genes that are known to be involved in auditory signal processing were also found to be significantly altered after repeated blast exposures. The hippocampus and midbrain showed significant increase in the gene expression of hearing loss-related antioxidant enzymes. Histopathology of the auditory cortex showed more significant injury in the inner layer compared to the outer layer. In summary, mice exposed to repeated blasts showed injury to the auditory cortex and significant alterations in multiple genes in the brain known to be involved in age- or noise-induced hearing impairment.
Asunto(s)
Corteza Auditiva/fisiopatología , Enfermedades Auditivas Centrales/genética , Traumatismos por Explosión/fisiopatología , Proteínas Ligadas a GPI/metabolismo , Pérdida Auditiva Provocada por Ruido/genética , Hipocampo/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Enfermedades Auditivas Centrales/metabolismo , Lesiones Encefálicas/fisiopatología , Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Explosiones , Proteínas Ligadas a GPI/genética , Pérdida Auditiva Provocada por Ruido/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
The chemical warfare nerve agent, soman irreversibly inhibits acetylcholinesterase (AChE) leading to hypercholinergy and seizures which trigger glutamate toxicity and status epilepticus ultimately resulting in neuropathology and neurobehavioral deficits. The standard emergency treatment comprising of anticholinergic, AChE reactivator and anticonvulsant does not completely protect against soman toxicity. We have evaluated imidazenil, a new anticonvulsant imidazo benzodiazepine with high affinity and intrinsic efficacy at α5-, α2-, and α3- but low intrinsic efficacy at α1-containing GABA(A) receptors and is devoid of cardiorespiratory depression, sedative/hypnoitc and amnestic actions and does not elicit tolerance and dependence liabilities unlike diazepam, for protection against soman toxicity. Guinea pigs implanted with bipotential radiotelemetry probes for recording EEG and ECG were administered with 26 µg/kg pyridostigmine bromide 30 min prior to 2× LD(50) soman exposure and 1 min later treated with a combination of 2mg/kg atropine sulfate and 25mg/kg 2-pralidoxime and various doses of imidazenil. Intramuscular administration of imidazenil, dose-dependently protected against 2× LD(50) of soman toxicity up to 1mg/kg. Further increase in the dose of imidazenil to 2.5mg/kg was less effective than 1mg/kg probably due to non-specific actions at sites other than GABA(A) receptors. Compared to vehicle group, 1mg/kg imidazenil treatment showed optimal increase in survival rate, reduction in behavioral manifestations and high power of EEG spectrum as well as neuronal necrosis. These data suggest that imidazenil is an effective anticonvulsant for medical countermeasure against soman-induced toxicity.
Asunto(s)
Benzodiazepinas/uso terapéutico , Sustancias para la Guerra Química/toxicidad , Imidazoles/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Síndromes de Neurotoxicidad/tratamiento farmacológico , Síndromes de Neurotoxicidad/etiología , Soman/toxicidad , Acetilcolinesterasa/sangre , Acetilcolinesterasa/metabolismo , Análisis de Varianza , Animales , Atropina/uso terapéutico , Peso Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Reactivadores de la Colinesterasa/uso terapéutico , Modelos Animales de Enfermedad , Esquema de Medicación , Electrocardiografía/métodos , Electroencefalografía/métodos , Cobayas , Dosificación Letal Mediana , Masculino , Antagonistas Muscarínicos/uso terapéutico , Compuestos de Pralidoxima/uso terapéutico , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Telemetría , Factores de TiempoRESUMEN
Acetylcholinesterase (AChE) which catalyzes the hydrolysis of the neurotransmitter acetylcholine has been recognized as one of the major regulators of stress responses after traumatic brain injury (TBI). Repeated blast exposure induces TBI (blast TBI) with a variable neuropathology at different brain regions. Since AChE inhibitors are being used as a line of treatment for TBI, we sought to determine the time course of AChE activity in the blood and different brain regions after repeated blast exposures using modified Ellman assay. Our data showed that repeated blast exposures significantly reduced AChE activity in the whole-blood and erythrocytes by 3-6h, while plasma AChE activity was significantly increased by 3h post-blast. In the brain, significant increase in AChE activity was observed at 6h in the frontal cortex, while hind cortex and hippocampus showed a significant decrease at 6h post-blast, which returned to normal levels by 7 days. AChE activity in the cerebellum and mid brain showed a decrease at 6h, followed by significant increase at 3 days and that was decreased significantly at 14 days post-blast. Medulla region showed decreased AChE activity at 24h post-blast, which was significantly increased at 14 days. These results suggest that there are brain regional and time-related changes in AChE activity after tightly coupled repeated blast exposures in mice. In summary, acute and chronic regional specific changes in the AChE activity after repeated blast exposures warrant systematic evaluation of the possibility of AChE inhibitor therapeutics against blast TBI.