RESUMO
A modified method of Ellman's reaction with a continuous flow was used to study the cerebral cholinesterase activity in its biological environment with rat brain slices including the striatum. Comparative studies were performed under various conditions of flow and substrate concentrations (acetylthiocholine bromide) and with or without formaldehyde fixation. We could thus measure either the inhibition rate of cerebral ChE by paraoxon or MPT or the reactivation rate by some oximes in the presence of substrate and after removing excess inhibitor.
Assuntos
Encéfalo/enzimologia , Inibidores da Colinesterase/farmacologia , Oximas/farmacologia , Compostos de Piridínio/farmacologia , Acetilcolinesterase , Animais , Técnicas In Vitro , Cinética , Membranas/enzimologia , Compostos Organofosforados/farmacologia , Ratos , Ratos EndogâmicosRESUMO
A massive and transitory increase in c-fos mRNA and Fos protein occurred in rats intoxicated by a single dose of soman (organophosphate compound and irreversible cholinesterase inhibitor) only in animals that had seizures. Comparison of immunohistochemistry that localizes Fos protein and of in situ hybridization that localizes its mRNA showed that there was an early and explosive expression of mRNA in many cerebral regions followed by transitory immunoreactivity in only some regions (piriform cortex, entorhinal area, hippocampus). The levels of mRNA and c-fos-like immunoreactivity decreased slowly and returned to basal level 24 h after soman administration.
Assuntos
Expressão Gênica/efeitos dos fármacos , Genes fos , Soman/toxicidade , Animais , Autorradiografia , Encéfalo/anatomia & histologia , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Mapeamento Encefálico , Inibidores da Colinesterase/farmacologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
Intoxication with organophosphorus (0P) anticholinesterase agents such as soman triggers irreversible lesions in some cerebral areas. Administration of soman at the LD 50 leads to an increased activity of NADPH-diaphorase (= NO-synthase) in the cerebral endothelial cells from the 6th hour after poisoning. This activity culminates after 24 h, whereas variations in this enzymatic activity are not easily detectable in NADPH-diaphorase positive neurons. Since soman triggers astrocytic oedema leading to a possible decrease in the local cerebral blood flow, it is likely that the induction of endothelial NO-synthase exerts an antagonistic effect, since NO is a vasodilator.
Assuntos
Aminoácido Oxirredutases/biossíntese , Encéfalo/patologia , Circulação Cerebrovascular , Endotélio Vascular/enzimologia , NADPH Desidrogenase/metabolismo , Soman/toxicidade , Acetilcolinesterase/análise , Animais , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Dose Letal Mediana , Masculino , Microcirculação/efeitos dos fármacos , Microcirculação/patologia , NADP/análise , NADP/metabolismo , Óxido Nítrico Sintase , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The cholinesterase equipment of cerebral microvessels was studied in some rodents and carnivores using the Koelle-Friedenwald histochemical method with 3 artificial substrates and specific inhibitors for butyrylcholinesterase or acetylcholinesterase. Our observations reveal a great heterogeneity in cholinesterase types and their distribution in each of the different species studied. Only in the rat, butyrylcholinesterase appears to be a marker for the microvessels provided with a blood-brain barrier.
Assuntos
Encéfalo/enzimologia , Carnívoros/metabolismo , Colinesterases/análise , Endotélio/enzimologia , Roedores/metabolismo , Acetilcolina/metabolismo , Animais , Encéfalo/irrigação sanguínea , Butirilcolinesterase/análise , Butiriltiocolina/metabolismo , Colinesterases/classificação , Histocitoquímica , Especificidade da Espécie , Tiocolina/análogos & derivados , Tiocolina/metabolismoRESUMO
Previous investigations have indicated that the measurement of omega 3 (peripheral-type benzodiazepine) binding site densities could be of widespread applicability in the localization and quantification of neural tissue damage in the central nervous system. In the first step of the present study, the suitability of this approach for the assessment of soman-induced brain damage was validated. Autoradiographic study revealed marked increases of omega 3 site densities in several brain areas of convulsing rats 2 days after soman challenge. These increases were well-correlated with the pattern and the amplitude of neuropathological alterations due to soman and closely related to both glial reaction and macrophage invasion of the lesioned tissues. We then used this marker to assess, in mouse hippocampus, the neuroprotective activity against soman-induced brain damage of NBQX and TCP which are respective antagonists of non-NMDA and NMDA glutamatergic receptors. Injection of NBQX at 20 or 40 mg/kg 5 min prior to soman totally prevented the neuronal damage. Comparatively, TCP had neuroprotective efficacy when administered at 1 mg/kg 5 min prior to soman followed by a reinjection 1 h after. These results demonstrate that both NBQX and TCP afford a satisfactory neuroprotection against soman-induced brain damage. Since it is known that the neuropathology due to soman is closely seizure-related, the neuroprotective activities of NBQX and TCP are discussed in relation with the respective roles of non-NMDA and NMDA receptors in the onset and maintenance of soman-induced seizures.
Assuntos
Encefalopatias/induzido quimicamente , Antagonistas de Aminoácidos Excitatórios , Hipocampo , Receptores de GABA-A/metabolismo , Soman/antagonistas & inibidores , Animais , Autorradiografia , Benzoxazinas , Encéfalo/patologia , Encefalopatias/patologia , Hipocampo/patologia , Isoquinolinas , Ligantes , Masculino , Oxazinas , Fenciclidina/análogos & derivados , Fenciclidina/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Convulsões/patologia , Soman/toxicidadeRESUMO
In rats poisoned with soman, an irreversible organophosphate anticholinesterase, acute changes in blood-brain barrier (BBB) permeability to proteins were investigated, using Evans Blue (EB)-labelled serum albumin and plasmatic gamma-immunoglobulin G (IgG) as indicators. Confirming previously published data, soman produced a conspicuous seizure-related and reversible BBB opening which was greatest after 30 to 60 min of paroxysmal electroencephalographic (EEG) discharges when signs of cerebral hyperactivity (epileptic EEG pattern, hyperoxia) were also at their height. Topographically, the protein leakage was bilateral and restricted to anatomically defined brain structures, some of which being thereafter sites of parenchymal edema and neuronal damage. In these areas (e.g., the thalamus), the edema is probably, at least in part, "vasogenic" in origin, and the possible contribution of the transient BBB opening to the neuronal lesions was questioned. On the other hand, the hippocampus, a region preferentially affected by the soman-induced acute neuropathology, was always free of any protein leakage, suggesting that the edema is unrelated to vascular damage and "cytotoxic" in nature. Finally, no topographic relationship was shown to exist between the increase in cerebrovascular permeability produced by soman and the histochemically-detected inhibition of the parenchymal total cholinesterases (ChE) or endothelial butyrylcholinesterase (BuChE).
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Convulsões/induzido quimicamente , Soman/intoxicação , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Inibidores da Colinesterase/intoxicação , Colinesterases/metabolismo , Eletroencefalografia/efeitos dos fármacos , Masculino , Microscopia de Fluorescência , Oxigênio/metabolismo , Pressão Parcial , Ratos , Ratos Endogâmicos , Convulsões/fisiopatologiaRESUMO
Rats were repeatedly administered with a low dose of diisopropylfluorosphosphate (DFP; 0.2 mg/kg/day, SC, for 9 or 21 days), an irreversible cholinesterase (ChE) inhibitor. Control rats received a daily injection of oil vehicle. Neurochemical changes occurring in the pontomesencephalic tegmentum (PMT), a brain stem region critically involved in behavioral state control, were evaluated at various times of treatment and after DFP withdrawal. First, enzyme assay revealed a profile of ChE inhibition in the whole PMT which looked like that observed in the striatum; both the inhibition and recovery proceeded more slowly than they did in the plasma. Second, quantitative histochemistry indicated that ChE activity in the mesopontine cholinergic nuclei and the pontine reticular formation progressively decreased across the first days of DFP exposure, to reach an asymptotic level of inhibition after 6 days (74-82% inhibition). The inhibition was less pronounced in the locus coeruleus (49%). Third, [3H]QNB autoradiography showed that muscarinic receptor density was unchanged in any of the PMT areas selected. These results are discussed regarding the question of regional variation in susceptibility to anti-ChE agents. To what extent behavioral state alterations occur concomitantly with ChE activity changes is assessed in the companion article.
Assuntos
Inibidores da Colinesterase/farmacologia , Isoflurofato/farmacologia , Ponte/metabolismo , Tegmento Mesencefálico/metabolismo , Animais , Autorradiografia , Colinesterases/metabolismo , Imuno-Histoquímica , Masculino , Antagonistas Muscarínicos , Neostriado/efeitos dos fármacos , Neostriado/enzimologia , Neostriado/metabolismo , Ponte/efeitos dos fármacos , Ponte/enzimologia , Quinuclidinil Benzilato , Ratos , Ratos Wistar , Tegmento Mesencefálico/efeitos dos fármacos , Tegmento Mesencefálico/enzimologiaRESUMO
A series of organophosphorous compounds (OP) was tested using a pharmacohistochemical method applied in vitro on the rat striatum, the central structure which contains the highest levels of acetylcholine and its metabolic enzymes; the OP showed a great variety of action towards the specific cholinesterase (AChE) and non-specific cholinesterase (BuChE). Except for iso-OMPA which is specific for BuChE localized in the microvessels endothelium, all the OP doses used in the present study were more or less potent inhibitors of cholinesterases (ChE). 15 mn after LD 50 doses of OP administered by subcutaneous route, a partial inhibition of the neurophile AChE occurred, revealing some striatal neurons which displayed high residual activity, i.e. the cholinergic interneurons. During the recovery phase following the inhibition of AChE by 1.5 LD 50 doses (the animals being treated with atropine) the AChE reaction product was detected almost simultaneously in some axo-spinous synapses probably non-cholinergic. The partial inhibition and the de novo synthesis of AChE also revealed the presence of small and less reactive non-cholinergic neurons. Among all the OP tested, soman was remarkable for its patchy inhibition of AChE in the striatum. The significance of the alternation of reactive and non-reactive areas is discussed.
Assuntos
Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Colinesterases/metabolismo , Corpo Estriado/enzimologia , Compostos Organofosforados/farmacologia , Animais , Corpo Estriado/citologia , Corpo Estriado/ultraestrutura , Histocitoquímica , Microscopia Eletrônica , Ratos , Relação Estrutura-AtividadeRESUMO
Cats were given systemically the anticholinesterase paraoxon at a dosage (3 mg/kg i.v.) that produced a maximal (over 90%) inhibition of brainstem acetylcholinesterase. All paralyzed and artificially ventilated animals were either unanaesthetized (decerebrated or ventilated with 70% nitrous oxide and 30% oxygen) or anaesthetized (with pentobarbital, alpha-chloralose, or halothane). In unanaesthetized cats, paraoxon produced an immediate rise in arterial blood pressure and did not suppress phrenic nerve respiratory discharges, while in anaesthetized animals it produced an immediate and long-lasting hypotension and a complete arrest of central respiratory activity. It is concluded that acetylcholine accumulation may not suppress respiratory rhythmogenesis and that most anaesthetics may considerably alter the response of cardiorespiratory cholinergic mechanisms to anticholinesterase administration.
Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Músculos Respiratórios/efeitos dos fármacos , Anestesia , Animais , Pressão Sanguínea/efeitos dos fármacos , Gatos , Cloralose , Estado de Descerebração , Eletroencefalografia , Feminino , Halotano , Masculino , Óxido Nitroso , Paraoxon/farmacologia , Pentobarbital , Nervo Frênico/efeitos dos fármacosRESUMO
Organophosphate and carbamate ester insecticides, main causes of pesticide poisoning, inhibit cholinesterase (ChE) enzymes. The aim of this study was to measure and compare baseline values for pseudocholinesterase and acetylcholinesterase (AChE) enzyme activities of different blood fractions in the dog to aid in diagnosis of anticholinesterase poisoning. After collecting blood samples from 23 6-24-mo-old male beagle dogs, Ellman's colorimetric assay was run on plasma, red blood cells (RBC), and whole blood fractions prepared in triplicate. The procedure described in a commercially available kit was applied to plasma and RBC. Hemolyzed whole blood fractions (final dilution 1:8) avoided the time-consuming and laborious separation of plasma and RBC. In addition to the kit substrate acetylthiocholine (ASCh), we used butyrylthiocholine (BSCh) as substrate. Whatever the substrate, ChE activity was lower in RBC than in other blood preparations. It was higher when using ASCh rather than BSCh as substrate (mean IU/L+/-SD): 563+/-144 and 303+/-45 respectively, in contrast to plasma (1640+/-310 and 2510+/-450). Whole blood enzyme activity did not differ significantly according to substrate: ASCh, 1590+/-190; BSCh, 1620+/-250) with a 2 to 3% within-day coefficient of variation. Enzyme activity was significantly lower in dogs <1-y old. This study confirms the low ChE activity in dog RBC compared to other species and other blood fractions. It shows that using whole blood instead of separating RBC from plasma minimizes the variability of ChE activity in the hemoglobin-rich fraction.