RESUMO
The Wistar Audiogenic Rat (WAR) is a well-characterized seizure-prone, inbred rodent strain that, when acutely stimulated with high-intensity sounds, develops brainstem-dependent tonic-clonic seizures that can evolve to limbic-like, myoclonic (forebrain) seizures when the acoustic stimuli are presented chronically (audiogenic kindling). In order to investigate possible mechanisms underlying WAR susceptibility to seizures, we evaluated Na,K-ATPase activity, Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and oxidative stress markers in whole forebrain and whole brainstem samples of naïve WAR, as compared to samples from control Wistar rats. We also evaluated the expression levels of α1 and α3 isoforms of Na,K-ATPase in forebrain samples. We observed increased Na,K-ATPase activity in forebrain samples and increased oxidative stress markers (lipid peroxidation, glutathione peroxidase and superoxide dismutase) in brainstem samples of WAR. The Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and expression levels of α1 and α3 isoforms of Na,K-ATPase were unaltered. In view of previous data showing that the membrane potentials from naïve WAR's neurons are less negative than that from neurons from Wistar rats, we suggest that Na,K-ATPase increased activity might be involved in a compensatory mechanism necessary to maintain WAR's brains normal activity. Additionally, ongoing oxidative stress in the brainstem could bring Na,K-ATPase activity back to normal levels, which may explain why WAR's present increased susceptibility to seizures triggered by high-intensity sound stimulation.
Assuntos
Tronco Encefálico/enzimologia , Estresse Oxidativo/fisiologia , Prosencéfalo/enzimologia , Convulsões , ATPase Trocadora de Sódio-Potássio/metabolismo , Estimulação Acústica/efeitos adversos , Adenosina Trifosfatases/metabolismo , Animais , Tronco Encefálico/patologia , Modelos Animais de Doenças , Glutationa Peroxidase/metabolismo , Excitação Neurológica/fisiologia , Peroxidação de Lipídeos , Neurônios/enzimologia , Prosencéfalo/patologia , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Convulsões/etiologia , Convulsões/metabolismo , Convulsões/patologiaRESUMO
AIMS: The activity of phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (Akt) is enhanced under hypertension. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of PI3K signaling, and its activity is redox-sensitive. In the rostral ventrolateral medulla (RVLM), which is responsible for the maintenance of blood pressure, oxidative stress plays a pivotal role in neurogenic hypertension. The present study evaluated the hypothesis that redox-sensitive inactivation of PTEN results in enhanced PI3K/Akt signaling in RVLM, leading to neurogenic hypertension. RESULTS: Compared to age-matched normotensive Wistar-Kyoto (WKY) rats, PTEN inactivation in the form of oxidation and phosphorylation were greater in RVLM of spontaneously hypertensive rats (SHR). PTEN inactivation was accompanied by augmented PI3K activity and PI3K/Akt signaling, as reflected by the increase in phosphorylation of Akt and mammalian target of rapamycin. Intracisternal infusion of tempol or microinjection into the bilateral RVLM of adenovirus encoding superoxide dismutase significantly antagonized the PTEN inactivation and blunted the enhanced PI3K/Akt signaling in SHR. Gene transfer of PTEN to RVLM in SHR also abrogated the enhanced Akt activation and promoted antihypertension. Silencing PTEN expression in RVLM with small-interfering RNA, on the other hand, augmented PI3K/Akt signaling and promoted long-term pressor response in normotensive WKY rats. INNOVATION: The present study demonstrated for the first time that the redox-sensitive check-and-balance process between PTEN and PI3K/Akt signaling is engaged in the pathogenesis of hypertension. CONCLUSION: We conclude that an aberrant interplay between the redox-sensitive PTEN and PI3k/Akt signaling in RVLM underpins neural mechanism of hypertension.
Assuntos
Tronco Encefálico/enzimologia , Hipertensão/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Tronco Encefálico/fisiopatologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Masculino , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Oxirredução , PTEN Fosfo-Hidrolase/genética , Fosforilação , Extratos Vegetais/genética , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Amyotrophic lateral sclerosis (ALS) is a paralyzing disorder characterized by the progressive degeneration and death of motor neurons and occurs both as a sporadic and familial disease. Mutant SOD1 (mtSOD1) in motor neurons induces vulnerability to the disease through protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities, defective axonal transport- and growth factor signaling, excitotoxicity, and neuro-inflammation.Melittin is a 26 amino acid protein and is one of the components of bee venom which is used in traditional Chinese medicine to inhibit of cancer cell proliferation and is known to have anti-inflammatory and anti-arthritic effects.The purpose of the present study was to determine if melittin could suppress motor neuron loss and protein misfolding in the hSOD1G93A mouse, which is commonly used as a model for inherited ALS. Meltittin was injected at the 'ZuSanLi' (ST36) acupuncture point in the hSOD1G93A animal model. Melittin-treated animals showed a decrease in the number of microglia and in the expression level of phospho-p38 in the spinal cord and brainstem. Interestingly, melittin treatment in symptomatic ALS animals improved motor function and reduced the level of neuron death in the spinal cord when compared to the control group. Furthermore, we found increased of α-synuclein modifications, such as phosphorylation or nitration, in both the brainstem and spinal cord in hSOD1G93A mice. However, melittin treatment reduced α-synuclein misfolding and restored the proteasomal activity in the brainstem and spinal cord of symptomatic hSOD1G93A transgenic mice.Our research suggests a potential functional link between melittin and the inhibition of neuroinflammation in an ALS animal model.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/enzimologia , Meliteno/farmacologia , Meliteno/uso terapêutico , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Comportamento Animal/efeitos dos fármacos , Tronco Encefálico/citologia , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/enzimologia , Tronco Encefálico/patologia , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Dobramento de Proteína/efeitos dos fármacos , Teste de Desempenho do Rota-Rod , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/enzimologia , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Taxa de Sobrevida , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
In this study, we tested the hypothesis that prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and PGHS-2) are expressed throughout the latter half of gestation in ovine fetal brain and pituitary. Hypothalamus, pituitary, hippocampus, brainstem, cortex and cerebellum were collected from fetal sheep at 80, 100, 120, 130, 145days of gestational age (DGA), 1 and 7days postpartum lambs, and from adult ewes (n=4-5 per group). mRNA and protein were isolated from each region, and expression of prostaglandin synthase-1 (PGHS-1) and -2 (PGHS-2) were evaluated using real-time RT-PCR and western blot. PGHS-1 and -2 were detected in every brain region at every age tested. Both enzymes were measured in highest abundance in hippocampus and cerebral cortex, and lowest in brainstem and pituitary. PGHS-1 and -2 mRNA's were upregulated in hypothalamus and pituitary after 100 DGA. The hippocampus exhibited decreases in PGHS-1 and increases in PGHS-2 mRNA after 80 DGA. Brainstem PGHS-1 and -2 and cortex PGHS-2 exhibited robust increases in mRNA postpartum, while cerebellar PGHS-1 and -2 mRNA's were upregulated at 120 DGA. Tissue concentrations of PGE(2) correlated with PGHS-2 mRNA, but not to other variables. We conclude that the regulation of expression of these enzymes is region-specific, suggesting that the activity of these enzymes is likely to be critical for brain development in the late-gestation ovine fetus.
Assuntos
Sistema Nervoso Central/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Hipófise/embriologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Animais , Encéfalo/embriologia , Tronco Encefálico/embriologia , Tronco Encefálico/enzimologia , Sistema Nervoso Central/enzimologia , Cerebelo/embriologia , Cerebelo/enzimologia , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Feminino , Idade Gestacional , Hipotálamo/embriologia , Hipotálamo/enzimologia , Hipófise/enzimologia , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro , OvinosRESUMO
AIMS: To evaluate possible effects of the intracerebroventricular (icv) injection of either O-Tricyclo [5.2.1.0(2,6)] dec-9-yl dithiocarbonate potassium salt (D609), a potent antioxidant and inhibitor of phosphatidylcholine specific phospholipase C (PtdCho-PLC) and acid sphingomyelinase (ASMase), or the spin trap/free radical scavenger N-tert-Butyl-alpha-phenylnitrone (PBN), on mechanical allodynia induced by facial carrageenan injection in mice. METHODS: Balb/c mice received icy injection of D609/PBN plus facial carrageenan injection, and the number of face wash strokes to von Frey hair mechanical stimulation of the maxillary skin was quantified. PtdCho-PLC and ASMase activities were also assayed in the brainstem, thalamus, and somatosensory cortex. RESULTS: Mice that received the icy injection of 10 nmol D609 plus facial carrageenan injection showed significantly fewer face wash strokes evoked by von Frey hair stimulation (indicating reduced mechanical allodynia) at 1 and 3 days post-injection, compared to mice that received icy injection of isotonic saline plus facial carrageenan injection. Mice that received icy injection of 1.13 micromol PBN plus facial carrageenan injection likewise showed significantly fewer face wash strokes after facial carrageenan injection, compared to isotonic saline-injected plus carrageenan-injected controls. D609 injection also resulted in significantly reduced ASMase activity in the brainstem, thalamus, and somatosensory cortex 3 days after injection, compared to controls. CONCLUSION: The icv injections of D609 and PBN were effective in reducing mechanical allodynia after facial carrageenan injection-induced pain. Together, the results point to a possible role of central nervous system sphingolipids and/or free radicals in orofacial pain.
Assuntos
Antioxidantes/uso terapêutico , Encéfalo/enzimologia , Dor Facial/tratamento farmacológico , Sequestradores de Radicais Livres/uso terapêutico , Nervo Maxilar/efeitos dos fármacos , Gânglio Trigeminal/efeitos dos fármacos , Animais , Antioxidantes/administração & dosagem , Encéfalo/efeitos dos fármacos , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/enzimologia , Hidrocarbonetos Aromáticos com Pontes/administração & dosagem , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Carragenina/efeitos adversos , Ventrículos Cerebrais/efeitos dos fármacos , Ventrículos Cerebrais/enzimologia , Óxidos N-Cíclicos/administração & dosagem , Óxidos N-Cíclicos/uso terapêutico , Modelos Animais de Doenças , Dor Facial/induzido quimicamente , Sequestradores de Radicais Livres/administração & dosagem , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Norbornanos , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/enzimologia , Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Estimulação Química , Tálamo/efeitos dos fármacos , Tálamo/enzimologia , Tiocarbamatos , Tionas/administração & dosagem , Tionas/uso terapêutico , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
The distribution of tryptophan hydroxylase (TPH)-containing perikarya and processes in the brainstem and diencephalon of the pigeon (Columba livia) were investigated using single-labeling chromogenic and double-labeling fluorescence immunohistochemical methods for TPH and 5-HT. TPH-immunoreactive (TPH-ir) perikarya were seen extending from the caudal medulla to mid-hypothalamic levels, located in brainstem regions previously described as containing 5-HT-ir somata. Brainstem TPH-ir cell clusters (the midline raphe, and the dorsolateral and ventrolateral serotonergic cell groups) and the circumventricular cerebrospinal fluid-contacting neurons in the taenia choroidea (in the caudal brainstem), recessus infundibuli and paraventricular organ (in the hypothalamus) were shown to co-express 5-HT immunoreactivity. However, heavily labeled TPH-ir cell clusters were observed in the nucleus premamillaris (PMM), in the stratum cellulare internum (SCI), in the nucleus paraventricularis magnocellularis (PVN) and in the medial border of the nucleus dorsomedialis anterior thalami (DMA). Double-labeling experiments indicated that none of these medial hypothalamic TPH-ir cells were immunoreactive to 5-HT. These cells correspond to dopamine- and melatonin-containing neurons previously found in the avian hypothalamus, and appear to be comparable to the mammalian TPH-ir hypothalamic A11-A13 catecholaminergic somata, suggesting that they may be a conserved attribute in the amniote medial hypothalamus.
Assuntos
Tronco Encefálico/enzimologia , Columbidae/metabolismo , Diencéfalo/enzimologia , Neurônios/enzimologia , Serotonina/biossíntese , Triptofano Hidroxilase/metabolismo , Animais , Evolução Biológica , Mapeamento Encefálico , Tronco Encefálico/anatomia & histologia , Columbidae/anatomia & histologia , Diencéfalo/anatomia & histologia , Dopamina/metabolismo , Feminino , Hipotálamo/citologia , Hipotálamo/enzimologia , Imuno-Histoquímica , Masculino , Melatonina/metabolismo , Núcleos da Rafe/citologia , Núcleos da Rafe/enzimologia , Especificidade da Espécie , Transmissão Sináptica/fisiologia , Terceiro Ventrículo/citologia , Terceiro Ventrículo/enzimologiaRESUMO
Ascending sensory information flowing through the visual thalamus is dynamically regulated by a number of modulatory influences. An important part of this ascending modulation is a cholinergic projection from the parabrachial region of the brainstem (PBR). In addition to containing cholinergic fibers, this projection has been identified in some species as the exclusive extrinsic source of fibers containing the neuronal form of nitric oxide synthase (nNOS). In this study, we examined the nitrergic innervation to the adult mouse visual thalamus. Retrograde tract-tracing with fluorescent microspheres was combined with nNOS and choline acetyltransferase (ChAT) immunocytochemistry, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry to identify the source of nitrergic innervation. Double-labeling revealed that only two regions of the mouse brain contained nitrergic neurons that projected to the visual thalamus: the pedunculopontine tegmentum and, to a lesser extent, the lateral dorsal tegmentum (LDT). Though the LDT projected heavily to the mouse visual thalamus, very few of the retrogradely labeled neurons in that region colocalized NADPH-d. These observations suggest that the parabrachial brainstem region is the primary source of nitrergic fibers in the mouse visual thalamus, similar to that found in cat and monkey. Such similarity suggests that the presence of nNOS in presynaptic terminal fields of the visual thalamus is an important conserved property of thalamic physiology and that the mouse is a valid model for studies of nNOS functions in the brain.
Assuntos
Tronco Encefálico/citologia , Vias Neurais/citologia , Óxido Nítrico Sintase/metabolismo , Tálamo/citologia , Animais , Tronco Encefálico/enzimologia , Colina O-Acetiltransferase/metabolismo , Imunofluorescência , Imuno-Histoquímica , Camundongos , NADPH Desidrogenase/metabolismo , Vias Neurais/enzimologia , Tálamo/enzimologiaRESUMO
An Mn for Cu substitution on cellular prion proteins (PrP(c)) in the brain that results in biochemical changes to PrP(c) has been implicated in the pathogenesis of transmissible spongiform encephalopathies. Recent research in the mature bovine does not support this theory. The present study tested this hypothesis by using progeny from gestating cows receiving Cu-deficient diets or Cu-deficient diets coupled with high dietary Mn. Copper-adequate cows (n = 39) were assigned randomly to 1 of 3 treatments: 1) control (adequate in Cu and Mn), 2) Cu deficient (-Cu), or 3) Cu deficient plus high dietary Mn (-Cu+Mn). Cows assigned to treatments -Cu and -Cu+Mn received no supplemental Cu and were supplemented with Mo to further induce Cu deficiency. The -Cu+Mn treatment also received 500 mg of supplemental Mn/kg of dietary DM. Calves were weaned at 180 d and maintained on the same treatments as their respective dams for 260 d. Copper-deficient calves (-Cu and -Cu+Mn) had decreased (P = 0.001) brain (obex) Cu and tended to have increased (P = 0.09) obex Mn relative to control calves. Obex Mn:Cu ratios were substantially increased (P < 0.001) in calves receiving -Cu and -Cu+Mn treatments compared with control calves and were greater (P < 0.001) in -Cu+Mn calves than in -Cu calves. Obex prion protein characteristics, including proteinase K degradability, superoxide dismutase (SOD)-like activity, and glycoform distributions, were largely unaffected. Obex tissue antioxidant capacity was not compromised by perturbations in brain metals, but Cu-deficient calves tended to have decreased (P = 0.06) Cu:Zn SOD activity and increased (P = 0.06) Mn SOD activity. Although obex Cu was decreased because of Cu deficiency and Mn increased because of exposure to high dietary Mn, the obex metal imbalance had minimal effects on PrP(c) functional characteristics in the calves.
Assuntos
Tronco Encefálico/metabolismo , Bovinos/fisiologia , Cobre/deficiência , Cobre/metabolismo , Dieta/veterinária , Príons/fisiologia , Animais , Western Blotting , Tronco Encefálico/enzimologia , Bovinos/metabolismo , Feminino , Fígado/metabolismo , Masculino , Manganês/análise , Manganês/metabolismo , Peptídeo Hidrolases/metabolismo , Gravidez , Príons/metabolismo , Distribuição Aleatória , Superóxido Dismutase/metabolismo , Fatores de TempoRESUMO
Florida manatees have an extensive, well-developed system of vibrissae distributed over their entire bodies and especially concentrated on the face. Although behavioral and anatomical assessments support the manatee's reliance on somatosensation, a systematic analysis of the manatee thalamus and brainstem areas dedicated to tactile input has never been completed. Using histochemical and histological techniques (including stains for myelin, Nissl, cytochrome oxidase, and acetylcholinesterase), we characterized the relative size, extent, and specializations of somatosensory regions of the brainstem and thalamus. The principal somatosensory regions of the brainstem (trigeminal, cuneate, gracile, and Bischoff's nucleus) and the thalamus (ventroposterior nucleus) were disproportionately large relative to nuclei dedicated to other sensory modalities, providing neuroanatomical evidence that supports the manatee's reliance on somatosensation. In fact, areas of the thalamus related to somatosensation (the ventroposterior and posterior nuclei) and audition (the medial geniculate nucleus) appeared to displace the lateral geniculate nucleus dedicated to the subordinate visual modality. Furthermore, it is noteworthy that, although the manatee cortex contains Rindenkerne (barrel-like cortical nuclei located in layer VI), no corresponding cell clusters were located in the brainstem ("barrelettes") or thalamus ("barreloids").
Assuntos
Tronco Encefálico/anatomia & histologia , Tálamo/anatomia & histologia , Tato , Trichechus manatus/anatomia & histologia , Vibrissas/fisiologia , Acetilcolinesterase/análise , Adaptação Fisiológica , Animais , Tronco Encefálico/química , Tronco Encefálico/enzimologia , Tronco Encefálico/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/análise , Corpos Geniculados/anatomia & histologia , Bainha de Mielina/metabolismo , Corpos de Nissl , Tamanho do Órgão , Núcleos Posteriores do Tálamo/anatomia & histologia , Tálamo/química , Tálamo/enzimologia , Tálamo/fisiologia , Trichechus manatus/fisiologia , Núcleos do Trigêmeo/anatomia & histologiaRESUMO
In previous studies, we determined that macrophage migration inhibitory factor (MIF), acting intracellularly via its intrinsic thiol-protein oxidoreductase (TPOR) activity, stimulates basal neuronal delayed-rectifier K(+) current (I(Kv)) and inhibits basal and angiotensin (ANG) II-induced increases in neuronal activity. These findings are the basis for our hypothesis that MIF is a negative regulator of ANG II actions in neurons. MIF has recently been recategorized as a member of the thioredoxin (Trx) superfamily of small proteins. In the present study we have examined whether Trx influences basal and ANG II-modulated I(Kv) in an effort to determine whether the Trx superfamily can exert a general regulatory influence over neuronal activity and the actions of ANG II. Intracellular application of Trx (0.8-80 nM) into rat hypothalamic/brain stem neurons in culture increased neuronal I(Kv), as measured by voltage-clamp recordings. This effect of Trx was abolished in the presence of the TPOR inhibitor PMX 464 (800 nM). Furthermore, the mutant protein recombinant human C32S/C35S-Trx, which lacks TPOR activity, failed to alter neuronal I(Kv). Trx applied at a concentration (0.08 nM) that does not alter basal I(Kv) abolished the inhibition of neuronal I(Kv) produced by ANG II (100 nM). Given our observation that ANG II increases Trx levels in neuronal cultures, it is possible that Trx (like MIF) has a negative regulatory role over basal and ANG II-stimulated neuronal activity via modulation of I(Kv). Moreover, these data suggest that TPOR may be a general mechanism for negatively regulating neuronal activity.
Assuntos
Angiotensina II/metabolismo , Tronco Encefálico/metabolismo , Canais de Potássio de Retificação Tardia/metabolismo , Hipotálamo/metabolismo , Ativação do Canal Iônico , Neurônios/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Tiorredoxinas/metabolismo , Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Benzotiazóis/farmacologia , Tronco Encefálico/citologia , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/enzimologia , Células Cultivadas , Cicloexanonas/farmacologia , Canais de Potássio de Retificação Tardia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hipotálamo/efeitos dos fármacos , Hipotálamo/enzimologia , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Tiorredoxinas/farmacologia , Fatores de Tempo , Regulação para CimaRESUMO
OBJECTIVE: Nitric oxide (NO) is synthesized in the brain through the action of three isoforms of nitric oxide synthase (NOS). The local generation of NO in neurons, glia, and vasculature modulates neuronal activity, as well as regional cerebral blood flow. We propose that, in the fetal brain, cerebral hypoperfusion alters the expression of NOS isoforms, and that estrogen administration modulates the NOS response to hypoperfusion. METHODS: Sixteen chronically catheterized fetal sheep of known gestational age (124 to 128 days' gestation) were subjected to a 10-minute period of brachiocephalic occlusion (BCO) or to sham BCO; half of these fetuses were subjected to subcutaneous implant, which released 17beta-estradiol (E2; 0.25 mg/d) or placebo. Brain tissue was collected for mRNA and protein extraction 1 hour after the start of the BCO or sham BCO. RESULTS: All three isoforms of NOS were identified in fetal brain at both the mRNA and protein levels. BCO increased NOS1 (hippocampus, brainstem), NOS2 (hypothalamus), and NOS3 (hippocampus, cortex) at the protein level. Estradiol alone increased NOS1 (brainstem, cortex), NOS2 (hippocampus, hypothalamus), and NOS3 (brainstem, cerebellum) at the protein level, changes that were not mirrored at the mRNA level. The combination of BCO and estradiol produced smaller changes in NOS1 (brainstem, cortex), NOS2 (hippocampus, hypothalamus), and NOS3 (brainstem) protein levels than those produced by either stimulus alone. CONCLUSIONS: We conclude that the fetal brain expresses all isoforms of NOS, and that NOS expression is altered by both BCO and estradiol, but that the most prevalent effect of estradiol is to reduce specific NOS responses to cerebral hypoperfusion. The present results suggest the possibility that the neuroendocrine responses to estradiol and BCO are modulated by central nervous system (CNS) NO biosynthesis.
Assuntos
Encéfalo/embriologia , Encéfalo/enzimologia , Estradiol/administração & dosagem , Hemodinâmica , Óxido Nítrico Sintase/genética , Ovinos/embriologia , Animais , Tronco Encefálico/enzimologia , Córtex Cerebral/enzimologia , Expressão Gênica/efeitos dos fármacos , Idade Gestacional , Hipocampo/enzimologia , Hipotálamo/enzimologia , Isoenzimas/análise , Isoenzimas/genética , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo I/análise , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/análise , Óxido Nítrico Sintase Tipo III/genética , Hipófise/embriologia , Hipófise/enzimologia , Placebos , RNA Mensageiro/análiseRESUMO
The aim of the study was to explore whether the biphasic time course of the vasospastic response following experimental subarachnoid hemorrhage is associated with any concomitant changes in the amount of cerebral dopamine beta-hydroxylase in the noradrenergic central nervous system. A single-hemorrhage animal model was used. Rabbits were sacrificed from day 1 to day 8 after subarachnoid hemorrhage. Intimal corrugation of the basilar artery and the amount of cerebral dopamine beta-hydroxylase in the hypothalamus and brain stem were measured each day. Vasospastic changes occurred in the biphasic manner following subarachnoid hemorrhage. More profound vasospastic corrugation occurred in the acute phase, followed by a slightly less intense corrugation in the chronic phase (between days 5 and 8 after the subarachnoid hemorrhage). Simultaneously, a clear concomitant biphasic time course developed in the form of an increased amount of dopamine-beta-hydroxylase in the noradrenergic nervous system of the rabbit hypothalamus and brain stem during the acute and chronic phases after the subarachnoid hemorrhage. Statistically significant correlation between basilar artery corrugation and the amount of dopamine beta-hydroxylase was found. These results suggest the possible role of the central sympathetic system in the pathogenesis of vasospasm. At the same time, this study demonstrates the chronological similarity of the vasospastic development after subarachnoid hemorrhage in the animal experimental model with the human time course of vasospasm.
Assuntos
Fibras Adrenérgicas/enzimologia , Artéria Basilar/inervação , Tronco Encefálico/enzimologia , Dopamina beta-Hidroxilase/análise , Hipotálamo/enzimologia , Proteínas do Tecido Nervoso/análise , Hemorragia Subaracnóidea/complicações , Vasoespasmo Intracraniano/fisiopatologia , Doença Aguda , Fibras Adrenérgicas/fisiologia , Animais , Artéria Basilar/patologia , Sangue , Doença Crônica , Cisterna Magna , Modelos Animais de Doenças , Feminino , Injeções , Coelhos , Fatores de Tempo , Túnica Íntima/patologia , Vasoespasmo Intracraniano/enzimologia , Vasoespasmo Intracraniano/etiologiaRESUMO
The distribution of calretinin (CR) in the brainstem and rostral spinal cord of the adult zebrafish was studied by using immunocytochemical techniques. For analysis of some brainstem nuclei and regions, CR distribution was compared with that of complementary markers (choline acetyltransferase, glutamic acid decarboxylase, tyrosine hydroxylase, neuropeptide Y). The results reveal that CR is a marker of various neuronal populations distributed throughout the brainstem, including numerous cells in the optic tectum, torus semicircularis, secondary gustatory nucleus, reticular formation, somatomotor column, gustatory lobes, octavolateral area, and inferior olive, as well as of characteristic tracts of fibers and neuropil. These results indicate that CR may prove useful for characterizing a number of neuronal subpopulations in zebrafish. Comparison of the distribution of CR observed in the brainstem of zebrafish with that reported in an advanced teleost (the gray mullet) revealed a number of similarities, and also some interesting differences. Our results indicate that many brainstem neuronal populations have maintained the CR phenotype in widely divergent teleost lines, so CR studies may prove very useful for comparative analysis.
Assuntos
Tronco Encefálico/metabolismo , Neurônios/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Medula Espinal/metabolismo , Peixe-Zebra/metabolismo , Animais , Tronco Encefálico/citologia , Tronco Encefálico/enzimologia , Calbindina 2 , Colina O-Acetiltransferase/metabolismo , Feminino , Glutamato Descarboxilase/metabolismo , Masculino , Mesencéfalo/citologia , Mesencéfalo/enzimologia , Mesencéfalo/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Neuropeptídeo Y/metabolismo , Rombencéfalo/citologia , Rombencéfalo/enzimologia , Rombencéfalo/metabolismo , Medula Espinal/citologia , Medula Espinal/enzimologia , Distribuição Tecidual , Tirosina 3-Mono-Oxigenase/metabolismo , Peixe-Zebra/anatomia & histologia , Proteínas de Peixe-ZebraRESUMO
Recent studies have reported that l-arginine-derived nitric oxide (NO) in the gracile nucleus modifies the hypotensive responses to electroacupuncture (EA) stimulation of Zusanli (ST 36). The purpose of this study was to examine the influence of EA stimulation of ST 36 on neuronal NO synthase (nNOS) expression in the brainstem nuclei in rats. EA stimulation of ST 36 and a non-acupoint was performed using 3 Hz of stimulation for 10 s every 2 min for a period of 120 min in rats anesthetized with ketamine. Rats in the sham-treated group received surgery and EA needles were placed into the acupoints without performing the stimulation. After 2-h stimulation and sham treatment, animals were perfused with 4% paraformaldehyde. Sections of rat medulla were examined by immunolabeling with a polyclonal antibody directed against nNOS. The brainstem nuclei were also visualized by NADPH-diaphorase histochemistry, a marker of nNOS activity. nNOS expression and NADPH-diaphorase reactivity were quantified by using a microscope with reticule grid to count the number of positive cells over a nucleus. Unilateral EA stimulation of ST 36 in rats caused increases in nNOS immunostained cells in the rostral region of the ipsilateral gracile nucleus, but was not altered in the contralateral gracile nucleus compared with sham-treated rats (P < 0.05, n = 6-7). NADPH-diaphorase-positive cells were also increased in the ipsilateral gracile nucleus of rats with EA stimulation. nNOS immunostaining and NADPH-diaphorase-positive neurons were significantly increased in both ipsilateral and contralateral sides of the medial nucleus tractus solitarius (mNTS) in rats receiving EA ST 36 compared with sham-treated animals (P < 0.05). nNOS immunostaining and NADPH-diaphorase reactivity was neither altered in the gracile nucleus and mNTS of non-acupoint stimulated rats nor other brainstem nuclei in rats with EA ST 36. These results show that nNOS immunoreactivity and NADPH-diaphorase reactivity are consistently increased in the gracile nucleus and the mNTS by EA ST 36. We conclude that EA ST 36 induces nNOS expression in the gracile nucleus and mNTS, and enhanced nNOS-NO in the nuclei may modify central cardiovascular regulation, which contribute to hypotensive effects of acupuncture.
Assuntos
Tronco Encefálico/enzimologia , Eletroacupuntura , Proteínas do Tecido Nervoso/biossíntese , Óxido Nítrico Sintase/biossíntese , Pontos de Acupuntura , Animais , Estimulação Elétrica , Membro Posterior/fisiologia , Imuno-Histoquímica , Masculino , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase Tipo I , Ratos , Ratos Sprague-DawleyRESUMO
Stress is a part of daily life. However, molecular mechanisms underlying the activation of limbic-hypothalamic-pituitary-adrenal (LHPA) axis remains unknown. In this study, we explored whether activation of the mitogen-activated kinase kinase 4 (MKK4)-c-Jun-N-terminal kinase (JNK) signaling pathway may play a role in the activation of the LHPA axis. We found that forced-swim stress induced elevation of activated MKK4 in the hippocampal formation, amygdala, and hypothalamus. Unlike MKK4, a high basal level of JNK activity is present in many brain areas of unstressed mice. Forced-swim stress significantly elevated JNK activity in the hypothalamus and amygdala and, to a lesser extent, in the cortex, CA1 and CA3 regions, and the dentate gyrus. To further investigate the role of MKK4 and JNK in induction of stress responses, we investigated whether a different stress, namely, restraint stress, induced activation of MKK4 or JNK in the brain. We found that restraint stress also induced elevation of activated MKK4 and JNK in the hippocampal formation, amygdala, and hypothalamus. Because MKK4 and JNK were activated within 5 min following stress, we propose that the MKK4-JNK signaling may be an early neural event in the initiation of neuroendocrine, autonomic and behavioral stress responses.
Assuntos
Encéfalo/enzimologia , MAP Quinase Quinase 4 , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Fisiológico/enzimologia , Tonsila do Cerebelo/enzimologia , Animais , Tronco Encefálico/enzimologia , Giro Denteado/enzimologia , Ativação Enzimática/fisiologia , Hipotálamo/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Lobo Parietal/enzimologia , Tálamo/enzimologiaRESUMO
Previous studies have shown that oral exposure of rats to polychlorinated biphenyls (PCBs) results in reduced 5-hydroxytryptamine (5-HT) concentrations in certain brain regions. In the present study, we investigated whether the PCB mixture Aroclor 1254 (0.33 mg/g body weight as a single oral dose) can inhibit the activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis, and reduce 5-HT concentrations in selected brain areas. In two separate experiments, Aroclor 1254 exposure consistently reduced TPH activity in the brainstem (7.2 and 8.7%), frontal cortex (17.4 and 14.8%), and hypothalamus (10.7 and 9.4%) without altering the rats' food intake or growth. Moreover, Aroclor 1254 accumulation in the frontal cortex demonstrated a negative correlation with TPH activity (correlation coefficient -0.82). In addition, 5-HT concentrations decreased in the brainstem and frontal cortex after Aroclor 1254 exposure by 9.1 and 19.7%, respectively. These results suggest that the Aroclor 1254-induced decreases in 5-HT concentrations in certain areas of the rat brain are due to inhibition of TPH activity, similar to our recent observations in Atlantic croaker, and that TPH is one of the targets of PCB neurotoxicity in both fish and mammals.
Assuntos
Encéfalo/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Triptofano Hidroxilase/antagonistas & inibidores , Animais , Encéfalo/enzimologia , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/enzimologia , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/enzimologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/enzimologia , Masculino , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Antagonistas da Serotonina/toxicidadeRESUMO
Neurosteroids are steroids produced within the nervous system. Based on behavioural responses evoked in animals by synthetic steroid injections, several studies suggested neurosteroid involvement in important neurophysiological processes. These observations should be correlated only to neuroactive effects of the injected steroids. Neurosteroids mostly control the CNS activity through allosteric modulation of neurotransmitter receptors within concentration ranges used by neurotransmitters themselves. Therefore, neurosteroid production within pathways controlling a neurophysiological process is necessary to consider neurosteroid involvement in that process. Because of the increasing speculation about pain modulation by neurosteroids based on pharmacological observations, we decided to clarify the situation by investigating neurosteroidogenesis occurrence in sensory pathways, particularly in nociceptive structures. We studied the presence and activity of cytochrome P450side chain cleavage (P450scc) in rat pain pathways. P450scc-immunoreactive cells were localized in dorsal root ganglia (DRG), spinal cord (SC) dorsal horn, nociceptive supraspinal nuclei (SSN) and somatosensory cortex. Incubation of DRG, SSN or SC tissue homogenates with [3H]cholesterol yielded the formation of radioactive metabolites including [3H]pregnenolone of which the synthesis was reduced in presence of aminogluthetimide, a P450scc inhibitor. These first neuroanatomical and neurochemical results demonstrate the occurrence of neurosteroidogenesis in nociceptive pathways and strongly suggest that neurosteroids may control pain mechanisms.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Vias Neurais/enzimologia , Neurônios Aferentes/enzimologia , Animais , Tronco Encefálico/citologia , Tronco Encefálico/enzimologia , Colesterol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/análise , Enzima de Clivagem da Cadeia Lateral do Colesterol/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/enzimologia , Imuno-Histoquímica , Masculino , Vias Neurais/citologia , Neurônios Aferentes/citologia , Especificidade de Órgãos , Pregnenolona/análise , Pregnenolona/biossíntese , Ratos , Ratos Sprague-Dawley , Córtex Somatossensorial/citologia , Córtex Somatossensorial/enzimologia , Medula Espinal/citologia , Medula Espinal/enzimologia , Tálamo/citologia , Tálamo/enzimologia , TrítioRESUMO
OBJECTIVE: To evaluate the antioxidant effect of selenium on Na+, K(+)-ATPase in rat brain in the presence of nitric oxide. METHODS: Male Wistar rats (70 g) were treated as follows: group 1 received 1 microg of i.p. sodium nitroprus-side per kg (SNP), group 2 received 5 microg sodium selenite during 20 days, group 3 received sodium selenite 5 microg + SNP 1 microg and the control group received vehicle 50 microl (0.9% NaCl), same period and route. At the end of treatment, animals were sacrificed and their brain dissected into cortex, hemispheres, cerebellum and brain stem in order to determine lipid peroxidation (TBARS), Na+, K+ ATPase and total ATPase in each section. Blood hemoglobin concentration (Hb) and prostate weight were also assessed. RESULTS: A significant increase of Hb in blood and of proteins in cortex and hemisphere was detected, but TBARS values fell due to the effect of sodium selenite in all examined regions, except for cerebellum. ATPase activity declined in all groups and regions with and without NTP. We conclude that diet supplementary selenium to inhibit NO generation can be a useful treatment in chronic inflammatory diseases.
Assuntos
Antioxidantes/farmacologia , Encéfalo/enzimologia , Óxido Nítrico/fisiologia , Selênio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Química Encefálica , Tronco Encefálico/química , Tronco Encefálico/enzimologia , Cerebelo/química , Cerebelo/enzimologia , Córtex Cerebral/química , Córtex Cerebral/enzimologia , Hemoglobinas/análise , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Proteínas do Tecido Nervoso/análise , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Próstata/anatomia & histologia , Ratos , Ratos Wistar , Selenito de Sódio/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
The sialic acid in the brain is split from sialoglucoconjugates by sialidases (neuraminidases, EC 3.2.1.18), and is postulated to act as an inhibitor of cellular adhesion and to play a role in various membrane functions. Since epilepsy alters cellular interactions and connectivity, it is reasonable to propose that sialidases can be affected by this pathological state or, alternately, by seizures. Therefore, we studied the activity of total, soluble, and membranal sialidases in various brain regions in normal, kindled epileptic and non-epileptic seizing rats. The results showed that in kindled rats, the total activity of the sialidases significantly decreased in cerebral cortex (11.38%) and cerebellum (28.58%), whereas it increased in brainstem (35.51%), hypothalamus (2.88%) and hippocampus (9.37%). The activity of the membranous sialidases in kindled rats followed the same pattern as the total activity, whereas the activity of soluble sialidase was significantly lower than membranous activity. Interestingly, the activity of total and membranal sialidases in non-epileptic seizing rats paralleled that observed in kindled rats. We suggest that the seizure-induced decrease of sialidasic activity may not modify the number of sialic acid molecules bound to gangliosides in cell membranes, as compared to areas of increased activity, that may decrease them. These changes in sialidases' activity may reflect functional disturbances of membrane polysialylated gangliosides related to the functional and anatomical plastic changes associated to seizures. Our data indicate that these changes are related to the presence of seizures rather than to an established epileptic state.
Assuntos
Encéfalo/enzimologia , Epilepsia/enzimologia , Neuraminidase/metabolismo , Animais , Tronco Encefálico/enzimologia , Cerebelo/enzimologia , Córtex Cerebral/metabolismo , Convulsivantes , Antagonistas GABAérgicos , Hipocampo/enzimologia , Hipotálamo/enzimologia , Excitação Neurológica , Masculino , Pentilenotetrazol , Ratos , Ratos Wistar , Convulsões/induzido quimicamenteRESUMO
The in vivo and in vitro effects of methyl parathion, a phosphorothionate insecticide, on cholinergic neurotransmitter systems in the brain of rats were investigated. Three groups of adult female rats received 0, 0.1, or 1.0 mg/kg methyl parathion via dermal exposure for 95 days. Exposure to 0.1 mg/kg methyl parathion produced inhibition of AChE in the caudate-putamen and thalamic nuclei, whereas 1.0 mg/kg resulted in inhibition of AChE in most brain regions. The same doses of methyl parathion had no effect on [(3)H]QNB binding to muscarinic receptors in the brain regions examined. The in vitro study demonstrated that methyl parathion causes preferential inhibition of AChE and [(3)H]QNB binding in specific brain regions. As an inhibitor of AChE, methyl paraoxon was 1,000-fold more potent than was methyl parathion. Similarly, methyl paraoxon showed brain region-specific inhibition of the enzyme. Generally, the brain stem was highly sensitive to organophosphate-induced inhibition of AChE activity and [(3)H]QNB binding. Because central respiratory neurons gather in the brain stem, preferential effects there and in other brain regions may underlie lethal toxicity of methyl parathion and other organophosphates.