RESUMO
Nitric oxide (NO) is a gaseous messenger that plays a role in neurotransmission, long term potentiation, depression and cerebral blood flow. Increases in intracellular calcium levels activate the enzyme NOS, and the NO released then diffuse to adjacent cells and activate guanylate cyclase. NO mediates the increase in cerebral blood flow during seizure activity. Therefore, the present study was aimed to investigate the change of NOS and calcium binding proteins in the rat cerebral cortex following seizure. Rats were injected with kainate (KA) and killed at 6 hours, 1, 3, 5 and 10 days after seizure. Expressional change of nNOS, calbindin D28k and parvalbumin was assessed by histochemistry, immunohistochemistry and microdensitometry in the rat brain. The intensity of the NADPH -d staining in rat cortical neurons showed a marked susceptibility to KA administration. At 6 hours and 3 days after seizure, the optical density of the NADPH -d staining was increased relative to the signal in saline treated control rats. At 5 and 10 days after seizure, the optical density of NADPH -d staining was not significantly different in most cortical regions compared to controls. In the hippocampus, the optical density of NADPH -d staining was highest at 5 days after seizure. The optical densities of calbindin D28k and parvalbumin positive neurons were various in the cerebral cortex, hippocampus and caudatoputamen during postseizure period. These results indicate that the calcium binding proteins investigated here are not essential for determining the activation of nNOS/NADPH -d positive neurons in the cerebral cortex and striatum.
Assuntos
Animais , Ratos , Encéfalo , Calbindina 1 , Calbindinas , Cálcio , Proteínas de Ligação ao Cálcio , Proteínas de Transporte , Córtex Cerebral , Depressão , Guanilato Ciclase , Hipocampo , Imuno-Histoquímica , Ácido Caínico , Potenciação de Longa Duração , NADP , Neurônios , Óxido Nítrico , Convulsões , Transmissão SinápticaRESUMO
We have investigated the neural cell damage and the change in the expression of NOS in the rat hippocampus, one of the brain structures most vulnerable to seizures. Rats were injected with kainic acid (KA) and sacrificed 6 h, 1 d, 3 d and 6 d after KA administration. The neural cell damage and the expression pattern of NOS was studied using silver impregnation, NADPH-diaphorase (NADPH-d) histochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Silver impregnation revealed that kainic acid caused pyramical cell damage which was most severe in the CA1/CA2 subfield and hilus and to a lesser degree in the CA3 region. The optical densities of NADPH-d-positive neurons in the CA1, CA3 and dentate gyrus (DG) regions of the hippocampus were shown to have increased in samples obtained 1 d and 3 d after injection of KA. The number of NADPH-d-positive neurons in the CA1 and CA3 regions of the hippocampus was shown to have decreased in samples obtained 3 d and 6 d after injection of KA. However, the number of NADPH-d-positive neurons in the DG region did not change significantly. The increase in the levels of nNOS, iNOS and eNOS mRNA reached maximal values in samples obtained 1 d after KA treatment. Our findings indicate that the KA-induced seizures induce neural cell damage, increase NOS activity and upregulate the expression of NOS mRNA, which suggests the possibility of a functional role of NOS in bringing about changes in the cells in the hippocampus following seizures.
Assuntos
Animais , Ratos , Encéfalo , Giro Denteado , Hipocampo , Ácido Caínico , Neurônios , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase , Óxido Nítrico , RNA Mensageiro , Convulsões , PrataRESUMO
Administration of kainic acid (KA) results in induction of epileptiform activity and motor seizures. Nitric oxide (NO) mediates the increase in cerebral blood flow during seizure activity. However, the production site of NO has not been clearly defined. Recent studies showed that constitutive nitric oxide synthase may be induced under certain conditions. Therefore, this study was aimed to investigate the change in endothelial nitric oxide synthase (eNOS), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) since these are involved in cerebral blood flow. Rats were treated with KA and killed at 6 hours, 1, 3, 6 and 12 days after seizure. Expressional changes were assessed by immunohisto-chemistry and RT-PCR. eNOS was detected in the blood vessels of the cerebral cortex of the control group, but was not detected in neurons. eNOS-positive neurons were induced in the cerebral cortex at 1 and 3 days after seizure and found in specific cortical areas, such as primary motor cortex, secondary motor cortex, primary somatosensory cortex, secondary somatosensory cortex, insular cortex, ectorhinal cortex, parietal association cortex, temporal association cortex, auditory cortex and visual cortex. The levels of eNOS mRNA increased at 1 and 3 days after seizure compared to controls. The staining intensity of eNOS-positive microvessels was elevated in samples obtained 1, 3, and 6 days after seizure compared to the control group. However, NPY- and VIP-positive neurons, and glial fibrillary acidic protein-positive astrocytes were not induced in the cerebral cortex after seizure. Therefore, specific neuroactive substances may be induced in the cerebral cortex after seizure. Nitric oxide, a free radical synthesized in the brain by NOS, is a messenger molecule that mediates vascular dilatation and neural transmission. Therefore, neurons showing induced eNOS-positivity and upregulated eNOS-positive microvessels may affect the cerebral blood flow and neuronal activity in the cerebral cortex after seizure.
Assuntos
Animais , Ratos , Astrócitos , Córtex Auditivo , Vasos Sanguíneos , Encéfalo , Córtex Cerebral , Dilatação , Ácido Caínico , Microvasos , Córtex Motor , Neurônios , Neuropeptídeo Y , Óxido Nítrico , Óxido Nítrico Sintase , Óxido Nítrico Sintase Tipo III , Rabeprazol , RNA Mensageiro , Convulsões , Córtex Somatossensorial , Transmissão Sináptica , Peptídeo Intestinal Vasoativo , Córtex VisualRESUMO
Nitric oxide is synthesized by cells containing the nitric oxide synthase (NOS), and NADPH-diaphorase (NADPH-d) is a selective histochemical marker for the NOS in the brain. The influence of feeding rats only half the amount of their normal daily intake of a purified diet on NOS was measured in the cerebral cortex by immunohistochemistry and NADPH-d histochemistry. iNOS was not detected in the cerebral cortex of control group. iNOS-positive neurons were induced in the cerebral cortex at 1 week after food restriction and found in specific cortical areas, such as primary motor cortex, secondary motor cortex, primary somatosensory cortex, secondary somatosensory cortex, parietal association cortex, auditory cortex, visual cortex, temporal association cortex and retrosplenial cortex. At 2 weeks after food restriction, iNOS-positive neurons were not found in all cortical areas. At 4 weeks after food restriction, iNOS-positive neurons were found in ectorhinal cortex and perirhinal cortex. In samples obtained 3 days after food restriction, the staining intensity of NADPH-d-positive neurons was decreased in most cortrical regions compared to the control group. At 1 week after food restriction, the staining intensity of NADPH-d was significantly increased in isocortical regions compared to the control group. At 9 weeks after food restriction, the staining intensity of NADPH-d was significantly decreased in all cortical regions. NO, a free radical synthesized in the brain by NOS, is a messenger molecule that mediates vascular dilatation and neural transmission. Therefore, neurons showing induced iNOS-positivity and upregulated NADPH-d-positive neurons may affect the neuronal activity in the cerebral cortex after food restriction.
Assuntos
Animais , Ratos , Córtex Auditivo , Encéfalo , Córtex Cerebral , Dieta , Dilatação , Imuno-Histoquímica , Córtex Motor , Neurônios , Óxido Nítrico Sintase , Óxido Nítrico , Rabeprazol , Córtex Somatossensorial , Transmissão Sináptica , Córtex VisualRESUMO
Kainic acid (KA) is a frequently used excitotoxin in experimental epilepsy research. The excitatory effect of KA leads to generalized convulsions when KA is administered systematically at convulsant doses. Nitric oxide (NO) is a gaseous messenger that plays a role in neurotransmission. NO is formed by NO synthase (NOS) from arginine. Purification and molecular cloning led to identification of at least three NOS isoforms designated as neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). In the central nervous system, NO seems to be involved in plasticity and cytotoxicity. Therefore, the present study has investigated the congruence of mRNA induction and protein expression of nNOS, eNOS, iNOS and neuropeptide Y (NPY) following KA-induced seizure activity. The patterns of NOS and NPY were studied by NADPH-diaphorase histochemistry, immunohistochemistry and RT-PCR in the rat brain. NADPH-d displayed a region-specific induction pattern. Regions of NADPH-d induction were the motor cortex and perirhinal cortex of KA treated group. Whereas NADPH-d neurons were not induced in auditory cortex, visual cortex, cingulate cortex, insular cortex, retrosplenial cortex and ectorhinal cortex of KA treated group. NPY neurons were not induced in all cortical areas of KA treated group. Subsequent to cortical neuronal induction, NADPH-d activity was increased in constitutive NADPH-d neurons of 1 and 3 days group of KA treatment. 1 and 3 days following KA administration, increased levels of nNOS, eNOS and iNOS mRNA were seen in the cerebral cortex. However, the level of NPY mRNA was decreased in 6 and 12 days after seizure. These findings demonstrate that mRNAs encoding for NOS isoforms are translated into the respective proteins following excitotoxic seizure.
Assuntos
Animais , Ratos , Arginina , Córtex Auditivo , Encéfalo , Sistema Nervoso Central , Córtex Cerebral , Clonagem Molecular , Epilepsia , Giro do Cíngulo , Imuno-Histoquímica , Ácido Caínico , Córtex Motor , Neurônios , Neuropeptídeo Y , Neuropeptídeos , Neurotoxinas , Óxido Nítrico Sintase , Óxido Nítrico , Plásticos , Isoformas de Proteínas , RNA Mensageiro , Convulsões , Transmissão Sináptica , Córtex VisualRESUMO
Nitric oxide (NO), a free radical that has been postulated to act as a neurotransmitter, neuromodulator, or second messenger molecule in nervous system, is synthesized from L-arginine by nitric oxide synthase (NOS). The NADPH-diaphorase (NADPH-d) histochemical techenique provides a simple and robust method to stain the selected populations of NOS neurons in the brain. This study was aimed to clarify the distribution of NOS neurons in the brain stem of rats. To verify the distribution of NOS neurons in the brain stem, the neurons were stained by the NOS immunohis-tochemistry and NADPH-d histochemistry. Image analyzer-assisted densitometry and cell counting method have been used to quantitatively characterize groups of neuronal cells. Double labeling of NOS immunohistochemistry and NADPH-d histochemistry showed the coexistence of NOS and NADPH-d in same neurons. Neuronal cell bodies exhibiting NOS/NADPH-d staining were found in particular nuclei throughout the brain stem. The number of NOS/NADPH-d neurons were variable in brain stem nuclei. The NADPH-d neurons exhibited different intensities of reaction product. Some groups, including paradorsal raphe nucleus, laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus were extremely heavily stained. Other neurons such as those in the interpeduncular nucleus, central gray, substantia nigra lateralis, nucleus solitarius and raphe obscurus nucleus were moderately stained, while other neurons such supragenual nucleus, lateral paragigantocellular nucleus and prepositus hypoglossal nucleus were weakly stained. The present study describes the many locations within the brain stem in which NADPH-d occurs. Since NADPH-d activity colocalizes with NOS, the results indicate the likely involvement of nitric oxide in the neuronal functions of many brain stem nuclei of rats.
Assuntos
Animais , Ratos , Arginina , Tronco Encefálico , Encéfalo , Contagem de Células , Densitometria , Imuno-Histoquímica , Sistema Nervoso , Neurônios , Neurotransmissores , Óxido Nítrico Sintase , Óxido Nítrico , Núcleo Tegmental Pedunculopontino , Núcleos da Rafe , Sistemas do Segundo Mensageiro , Núcleo Solitário , Substância NegraRESUMO
Nitric oxide has been considered to be an important modulator of the epileptic seizure response. Previous studies have mainly focused on the nitric oxide synthase (NOS) expressed in glial cells and vascular endothelial cells in the brain following seizures, while less data have been available reading the change of neuronal NOS (nNOS) produced in neurons. Polypeptide growth factors play a central role in a variety of environmentally induce structural changes in the cortex and hippocampus of adult brain. neuregulin is widely expressed in the central and peripheral nerve cells and Schwann cells, glia, oligodendrocytes and muscle cells, to control cellular proliferation, differentiation and migration. erbB family are the receptors of the neuregulin and consist of erbB2, erbB3 and erbB4. We have, therefore, investigated the change in the expression of nNOS and erbB4 in the rat hippocampus, one of the brain structures most vulnerable to seizures. Rats were injected with kainic acid (KA) and sacrificed 6 h, 1 d, 3 d and 6 d after KA administration. The expression pattern of nNOS and erbB4 was studied using reverse transcription-polymerase chain reaction analysis, NADPH-diaphorase (NADPH-d) histochemistry and immunohistochemistry. The increase in the level of nNOS reached maximal values in samples obtained 1 d after KA treatment. The optical densities of NADPH-d-positive neurons in the CA1 and dentate gyrus (DG) regions of the hippocampus were shown to have increased in samples obtained 1 d and 3 d after injection of KA. The number of NADPH-d-positive neurons in the CA1 regions of the hippocampus was shown to have decreased in samples obtained 3 d and 6 d after injection of KA. However, the number of NADPH-d-positive neurons in the DG region did not change significantly. We show that erbB4 immunoreactivity is increased in hippocampus, reaching maximal levels 3 d after KA treatment, some NOS neurons contain erbB4 protein. We propose that the survival of NOS neuron in the hippocampus after injection of KA is associated with expression of erbB4, neuregulin receptor.
Assuntos
Adulto , Animais , Humanos , Ratos , Encéfalo , Proliferação de Células , Giro Denteado , Células Endoteliais , Epilepsia , Hipocampo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Ácido Caínico , Células Musculares , Neuroglia , Neurônios , Óxido Nítrico Sintase , Óxido Nítrico , Oligodendroglia , Nervos Periféricos , Células de Schwann , ConvulsõesRESUMO
Administration of kainate (KA) results in the induction of epileptiform activity and limbic motor seizures. Nitric oxide (NO) is a gaseous messenger that plays a role in neural transmission, long term potentiation, depression and cerebral blood flow. NO is formed by NO synthase (NOS) from arginine. NO mediates the increase in cerebral blood flow during seizure activity. However, the production site of NO has not been clearly defined. Recent report showed that constitutive NOS may be induced under certain conditions. Therefore, the present study was aimed to investigate the change of NOS and calbindin D28k in the rat cerebral cortex following seizure. Rats were injected with KA and killed at 6 hours, 1, 3, 6 and 12 days after seizure. Expressional change of nNOS and calbindin D28k was assessed by histochemistry, immunohistochemistry and RT-PCR in the rat brain. Induced NADPH-d positive neurons were observed in the cerebral cortex of 1, 3, 6 and 12 days after seizure and found in specific cortical areas, such as motor cortex, somatosensory cortex, auditory cortex, visual cortex, ectorhinal cortex and perirhinal cortex. The level of nNOS mRNA increased at 1, 3, 6 and 12 days after seizure compared with control group. Induced calbindin D28k positive neurons were observed in motor cortex and somatosensory cortex 1 and 3 days after seizure. The level of calbindin D28k mRNA in the cerebral cortex was slightly decreased at 1 day after seizure. Therefore, in this study, the induced NADPH-d, calbindin D28k positive neurons and upregulated NADPH-d positive neurons may influence the cerebral blood flow and neuronal activity in the cerebral cortex during post-seizure period.