Alteração na representação de subpopulações de neurônios no epitélio olfatório de camundongos Ric8b cKO / Altered representation of neuronal sub-populations in the olfactory epithelium of Ric8b conditional knock-out mice

O objetivo desse trabalho foi identificar as consequências moleculares e funcionais da falta da proteína Ric8b no epitélio olfatório de camundongos. Para esse fim, comparamos o transcriptoma de epitélio olfatório de camundongos knock-out tecido específico para a proteína RIC8B (Ric8b cKO) com o dos seus irmãos tipo selvagem (WT). Identificamos muitos genes que apresentaram expressão reduzida no epitélio olfatório do camundongo Ric8b cKO, mas também vários genes que apresentaram a sua expressão aumentada. A maioria dos genes com expressão reduzida corresponde a genes normalmente expressos em neurônios olfatórios maduros, como por exemplo os genes de receptores olfatórios, o que é compatível com o fato já conhecido de que os camundongos Ric8b cKO apresentam um menor número desses neurônios. Inesperadamente, apesar de a maioria dos genes de receptores olfatórios ter a sua expressão diminuída no camundongo Ric8b cKO, observamos que um grupo destes genes de receptores teve a sua expressão aumentada. Os camundongos Ric8b cKO apresentaram também genes marcadores de outros tipos celulares que não neurônios canônicos com expressão aumentada no seu epitélio olfatório. Dentre eles, os mais significativamente alterados foram os genes marcadores de neurônios Trpc2+ tipo B (que expressam a guanilato ciclase solúvel Gucy1b2). Sabe-se que este tipo de neurônio é responsável pela sensibilidade a diferentes gases, e concordantemente, observamos que os camundongos Ric8b cKO apresentaram um aumento da sensibilidade a gás carbônico. Como o olfato apresenta um papel importante na regulação de ingestão alimentar, analisamos como os camundongos Ric8b cKO se comportam frente a diferentes dietas. Interessantemente, observamos que esses animais não apresentam preferência por alimento rico em gorduras quando comparado aos seus irmãos tipo selvagem. Nossos resultados sugerem, portanto, que a ausência da proteína RIC8B resulta na alteração de representatividade de neurônios canônicos e não canônicos no epitélio olfatório de camundongos, o que por sua vez leva a alterações funcionais e comportamentais

The objective of this work was to identify the molecular and functional consequences of the lack of the RIC8B protein in the main olfactory epithelium of mice. To this end, we compared the olfactory epithelium transcriptome of Ric8b tissue-specific knock-out mice (Ric8b cKO) with that of their wild-type littermates (WT). We identified many genes with differential expression, many of which were downregulated and also some which were upregulated in the olfactory epithelium of the Ric8b cKO mice. Most of the downregulated genes correspond to genes normally expressed in mature olfactory sensory neurons, such as olfactory receptor genes. This is compatible with the already known fact that the Ric8b cKO mice have less of this kind of neuron. Unexpectedly, even though most of the olfactory receptor genes were downregulated, we observed a subset of these genes that had their expression upregulated in the Ric8b cKO mice. The Ric8b cKO mice also showed upregulation for genes that are markers for cell types other than canonic neurons in their olfactory epithelium. Among these, the most significantly altered were the markers for neurons Trpc2+ type B (that express the soluble guanylate cyclase Gucy1b2). It is known that this kind of neuron is responsible for sensitivity to different gases. Accordingly, we observed that the Ric8b cKO mice presented a higher sensitivity to carbon dioxide. Since olfaction has an important role in food intake, we analyzed how the Ric8b cKO mice behaved with different diets. Interestingly, we observed that the Ric8b cKO mice lack preference for high fat diet when compared to their wild-type littermates. Our results indicate, therefore, that the lack of the RIC8B protein results in altered representativity of canonic and non-canonic neurons in the olfactory epithelium of mice, which then leads to altered function and behavior

Wnt-5a-regulated miR-101b controls COX2 expression in hippocampal neurons

BACKGROUND: Wnt-5a is a member of the WNT family of secreted lipoglycoproteins, whose expression increases during development; moreover, Wnt-5a plays a key role in synaptic structure and function in the adult nervous system. However, the mechanism underlying these effects is still elusive. MicroRNAs (miRNAs) are a family of small non-coding RNAs that control the gene expression of their targets through hybridization with complementary sequences in the 3' UTR, thereby inhibiting the translation of the target proteins. Several evidences indicate that the miRNAs are actively involved in the regulation of neuronal function. RESULTS: In the present study, we examined whether Wnt-5a modulates the levels of miRNAs in hippocampal neurons. Using PCR arrays, we identified a set of miRNAs that respond to Wnt-5a treatment. One of the most affected miRNAs was miR-101b, which targets cyclooxygenase-2 (COX2), an inducible enzyme that converts arachidonic acid to prostanoids, and has been involved in the injury/inflammatory response, and more recently in neuronal plasticity. Consistent with the Wnt-5a regulation of miR-101b, this Wnt ligand regulates COX2 expression in a time-dependent manner in cultured hippocampal neurons. CONCLUSION: The biological processes induced by Wnt-5a in hippocampal neurons, involve the regulation of several miRNAs including miR-101b, which has the capacity to regulate several targets, including COX-2 in the central nervous system

Efeito da atividade física moderada sobre neurônios mioentéricos nitrérgicos do íleo de ratos de meia idade / The effect of moderate physical activity on myoenteric nitrergic neurons in the ileum of middle-aged rats / Efecto de la actividad física moderada en neuronas mientéricas nitrérgicas del íleon de ratas de mediana edad

Poucos são os relatos a respeito do efeito da atividade física sobre o plexo mioentérico ao longo do envelhecimento. Portanto, analisaram-se os neurônios mioentéricos NADPH-diaforase positivos do íleo em ratos que envelheceram praticando atividade física a partir dos seis meses de vida. Dez ratos Wistar foram distribuídos em dois grupos (n = 5/grupo): S (ratos sedentários controles com 12 meses de idade) e T (ratos treinados com 12 meses de idade). Os animais do grupo T realizaram, dos seis aos 12 meses de idade, atividade física regulada conforme resultados obtidos em teste de esforço máximo. Ao final do período experimental, o íleo obtido de cada animal foi processado para a técnica de NADPH-diaforase para evidenciar neurônios mioentéricos em preparados de membrana e quantificar e mensurar a área do pericário desses neurônios. A área do pericário não diferiu (P> 0,05) entre o grupo S (218,49 µm2) e o grupo T (210,59 µm2). A média de neurônios presente em 60 campos microscópicos de preparados de membranas no grupo S (126 neurônios) foi superior (P<0,05) a média encontrada no grupo T (93 neurônios), sugerindo que a atividade física pode inibir ou diminuir a expressão dos neurônios nitrérgicos em ratos de 12 meses de idade que praticaram atividade física desde os seis meses de vida. Os resultados quantitativos sugerem que a falta de atividade física regular propicia aumento na atividade dos neurônios nitrérgicos, contribuindo para o surgimento dos distúrbios da motricidade gastrointestinal que podem ocorrer ao longo do envelhecimento.

There are only a few reports on the effect of physical activity in the myoenteric plexus throughout the aging process. For such, the positive myoenteric NADPH-diaphoresis neurons in the ileum of those rats that aged practicing physical activity from the sixth month were analyzed. Ten Wistar rats were distributed into two groups (n= 5/group): S (12 months-old control sedentary rats) and T (12 months-old trained rats). The animals from the T group practiced controlled physical activity from the age of 6 months to 12 months, according to the maximum effort test results. At the end of the experimental period, the ileum obtained from each animal was processed in the NADPH-diaphoresis technique to highlight myoenteric neurons in membrane preparations, and quantify and measure the perikaryon area from those neurons. The Perikaryon area did not differ (P>0.05) among the S group (218.49 µm2) and the T group (210.59 µm2). The average of neurons in 60 membrane preparations in the S group (126 neurons) was higher (P<0.05) than the average finding in the T group (93 neurons), indicating that the physical activity can inhibit or decrease the nitrergic expression in 12 month-old rats that have practiced physical activity from the age of 6 months. The quantitative results suggest that the lack of regular physical activity provides the elevation of nitrergic neurons, concurring to the emergence of gastrointestinal motility disorders that can occur during the aging process.

Hay pocos informes sobre el efecto de la actividad física sobre el plexo mientérico a lo largo del envejecimiento. Por lo tanto, se analizaron las neuronas mientéricas NADPH-diaforasa positivas del íleon en ratas que envejecieron practicando actividad física a partir de seis meses de vida. Diez ratas Wistar se dividieron en dos grupos (n = 5 / grupo): S (ratas sedentarias de control con 12 meses de edad) y T (ratas entrenadas con 12 meses de edad). Los animales del grupo T realizaron, de los seis a los doce meses de edad, actividad física regulada conforme resultados obtenidos en prueba de esfuerzo máximo. Al final del período experimental, el íleon obtenido de cada animal se procesó por la técnica de NADPH-diaforasa para mostrar las neuronas mientéricas en preparados de membrana, cuantificar y medir el área del pericario de esas neuronas. El área del pericario no difirió (P> 0.05) entre el grupo S (218.49 µm2) y el grupo T (210.59 µm2). El número medio de neuronas presentes en 60 campos microscópicos de preparados de membranas en el grupo S (126 neuronas) fue mayor (P <0,05), promedio encontrado en el grupo T (93 neuronas), lo que sugiere que la actividad física puede inhibir o disminuir la expresión de las neuronas nitrérgicas en ratas de 12 meses de edad que practicaron actividad física desde los seis meses de edad. Los resultados cuantitativos sugieren que la falta de actividad física regular promueve aumento en la actividad de las neuronas nitrérgicas, contribuyendo a la aparición de disturbios de la motricidad gastrointestinal que pueden ocurrir a lo largo del envejecimiento.

Doença de depósito lisossomal induzida pelo consumo de Ipomoea verbascoidea (Convolvulaceae) em caprinos no semiárido de Pernambuco / Lysosomal storage disease induced by the consumption of Ipomoea verbascoidea (Convolvulaceae) in goats at northeastern Brazil

The aim of this paper was to reproduce the poisoning of Ipomoea verbascoidea in goats and describe the epidemiological, clinical and pathological aspects of spontaneous poisoning by this plant in Pernambuco. For this, we studied the epidemiology of the disease in seven municipalities in the semiarid region of the State. Three spontaneously poisoned goats were examined and then euthanized and necropsied (Group I). To reproduce the disease, the dried leaves of I. verbascoidea containing 0.02% swainsonine were supplied at doses of 4g/kg (0.8mg swainsonine/kg) to two groups of three animals. The goats in Group II received daily doses of the plant during 40 days and were euthanized on the 41st day of the experiment. Goats from Group III received daily doses of the plant during 55 days and were euthanized on the 120th day of the experiment. Other three goats constituted the control group (Group IV). In experimental groups, the brain lesions were evaluated by histopathology; additionally the cerebellar lesions were evaluated by morphometry, by measuring the molecular layer thickness, the number of Purkinje cells and the area of the cell bodies of these cells. The main clinical signs and microscopic lesions in goats poisoned were similar to those reported by swainsonine containing plants. In goats of GII and GIII, the first nervous signs were observed between 22th and 29th days; clinically, the disease developed by these animals was similar to the spontaneous cases. The goats of GIII did not recover from the neurologic signs. These results show that the consumption of the plant by 26-28 days after observation of the first clinical signs is enough to cause irreversible damage. By morphometric analysis, the molecular layer of the cerebellum of the goats of Group I and III were thinner than those of goats in the control group, and Purkinje neurons were atrophic. It is suggested that these changes are responsible for the neurological picture observed in goats that stop eating the plant and have sequelae of poisoning.

O objetivo deste trabalho foi reproduzir a intoxicação por Ipomoea verbascoidea em caprinos e descrever os aspectos epidemiológicos, clínicos e histopatológicos da intoxicação espontânea por essa planta no Estado de Pernambuco. Para isso, realizou-se o acompanhamento da epidemiologia da doença em sete municípios do semiárido pernambucano. Três caprinos espontaneamente intoxicados foram examinados e, em seguida eutanasiados e necropsiados (Grupo I). Para reproduzir experimentalmente a doença, as folhas secas de I. verbascoidea contendo 0,02% de swainsonina, foram fornecidas na dose de 4g/kg (0,8mg de swainsonina/kg) a dois grupos de três animais. Os caprinos do Grupo II receberam a planta diariamente por 40 dias e foram eutanasiados no 41º dia de experimento. Os caprinos do Grupo III receberam a planta diariamente por 55 dias e foram eutanasiados no 120º dia de experimento. Outros três caprinos constituíram o grupo controle (Grupo IV). Nos grupos experimentais, as lesões encefálicas foram avaliadas por histopatologia e adicionalmente avaliaram-se as lesões cerebelares por morfometria, mediante mensuração da espessura da camada molecular, do número de neurônios de Purkinje e da área dos corpos celulares dessas células. Os principais sinais clínicos e lesões microscópicas foram semelhantes aos previamente reportados em animais intoxicados por plantas que contem swainsonina. Nos caprinos do GII e GIII, os primeiros sinais clínicos foram observados entre o 22º e 29º dia de experimento; clinicamente a doença desenvolvida por esses animais foi semelhante aos casos espontâneos. Nenhum dos caprinos do GIII se recuperou dos sinais neurológicos. Esse resultado evidencia que o consumo da planta por 26-28 dias após a observação dos primeiros sinais clínicos é suficiente para provocar lesões irreversíveis. Pela análise morfométrica, a camada molecular do cerebelo dos caprinos do Grupo I e III eram mais delgadas que às dos caprinos do grupo controle, e os neurônios de Purkinje estavam atróficos. Sugere-se que essas alterações sejam responsáveis pelo quadro clínico neurológico observado nos caprinos que deixam de ingerir a planta e apresentam seqüelas da intoxicação.

Supplementation with 0.1% and 2% vitamin e in diabetic rats: analysis of myenteric neurons immunostained for myosin-V and nNOS in the jejunum / Suplementação com vitamina E 0,1% e 2% em ratos diabéticos: análise de neurônios mioentéricos imunomarcados para miosina-V e nNOS no jejuno

CONTEXT: Diabetes mellitus is a disease characterized by hyperglycemia that, when allowed to progress long-term untreated, develops vascular and neurological complications, which are responsible for the development of alterations in the enteric nervous system in diabetic patients. In the gastrointestinal tract, diabetes mellitus promotes motor and sensory changes, and in the reflex function of this system, causing gastroparesis, diarrhea, constipation, megacolon, slow gastrointestinal transit, gastric stasis and dilation with decreased or increased peristaltic contractions. Several studies have shown that oxidative stress is the main responsible for the vascular and neurological complications affecting the enteric nervous system of diabetics. OBJECTIVE: The effects of 0.1% and 2% vitamin E on myosin-V- and nNOS-immunoreactive neurons in the jejunum of diabetic rats were investigated. METHODS: Thirty rats were divided into the groups: normoglycemic, normoglycemic treated with 0.1% vitamin E, normoglycemic treated with 2% vitamin E, diabetic, diabetic treated with 0.1% vitamin E, and diabetic treated with 2% vitamin E. The neuronal density and areas of neuron cell bodies were determined. RESULTS: Diabetes (diabetic group) significantly reduced the number of myosin-V-immunoreactive neurons compared with the normoglycemic group. The diabetic treated with 0.1% vitamin E and diabetic treated with 2% vitamin E groups did not exhibit a greater density than the D group (P>0.05). Nitrergic density did not change with diabetes (P>0.05). The areas of myosin-V- and nNOS-immunoreactive neurons significantly increased in the normoglycemic treated with 2% vitamin E and diabetic groups compared with the normoglycemic group. CONCLUSION: Supplementation with 2% vitamin E had a neurotrophic effect only in the area of myosin-V-immunoreactive neurons compared with the diabetic group.

CONTEXTO: O diabetes mellitus (DM) é uma doença caracterizada pela hiperglicemia que a longo prazo, quando não tratada, desenvolve complicações vasculares e neurológicas, responsáveis pelo desenvolvimento das alterações no sistema nervoso entérico de pacientes diabéticos. Em nível gastrointestinal o DM provoca modificações motoras, sensoriais e na função reflexa desse sistema, podendo ocasionar gastroparesia, diarreia, constipação, megacólon, lentidão do trânsito gastrointestinal, estase e dilatação gástrica com diminuição ou aumento de contrações peristálticas. Diversos estudos têm evidenciado que o estresse oxidativo é o principal responsável pelas complicações vasculares e neurológicas que atingem o sistema nervoso entérico de diabéticos. OBJETIVO: O efeito da vitamina E 0,1% e 2 sobre a miosina-V e nNOS imunorreativas em neurônios do jejuno de ratos diabéticos foram investigados. MÉTODOS: Trinta ratos foram divididos em grupos: normoglicêmicos (NU), normoglicêmicos tratados com vitamina E 0,1% (NE1), normoglicêmicos tratados com vitamina E 2% (NE2), diabético (UD), diabéticos tratados com vitamina E 0,1% (DE1), e diabéticos tratados com vitamina E 2% (DE2). A densidade neuronal e áreas de corpos celulares de neurônios foram determinadas. RESULTADOS: Diabetes (UD grupo) reduziu significativamente o número de neurônios miosina-V imunorreativos quando comparado com o grupo UN. Os grupos DE1 e DE2 não exibem uma maior densidade do que o grupo D (P>0,05). Densidade nitrérgicos não se alterou com diabetes (P>0,05). As áreas dos neurônios miosina-V e nNOS imunorreativos aumentaram significativamente nos grupos NE2 e UD comparados com o grupo UN. CONCLUSÃO: A suplementação com vitamina E 2% teve um efeito neurotrófico apenas na área da miosina-V imunorreativos neurônios em comparação com o grupo UD.

Analysis of myosin-V immunoreactive myenteric neurons from arthritic rats / Análise dos neurônios mioentéricos miosina-V imunoreativos de ratos artríticos

CONTEXT: The inflammatory response itself and the consequent oxidative stress are able to promote neurodegeneration. So, it is possible that enteric nervous system is affected by inflammatory diseases threatening quality of life of patients. However, gastrointestinal symptoms of arthritis are usually attributed to anti-inflammatory drugs rather than neural damage. OBJECTIVE: To confirm if the general population of myenteric neurons from the ileum and jejunum of rats is affected by arthritis. METHODS: Twenty Holtzmann rats, 58-day-old male, were used and divided in four groups: control group (C30), arthritic group (Art30), older control group (C60) and older arthritic group (Art60). At 58 days old, the animals in groups Art30 and Art60 received an injection of the complete Freund's adjuvant in order to induce arthritis. The whole-mount preparations of ileum and jejunum were processed for myosin-V immunohistochemistry. Quantitative and morphometric analyses were performed. RESULTS: Groups Art30 and Art60 presented, respectively, a reduction of 2 percent and 6 percent in intestinal area when compared to their control groups. No significant differences were observed in general neuronal density among the four groups (P>0.05). Group C60 presented a reduction of 14.4 percent and 10.9 percent in mean neuronal cell body area when compared to group C30 (P<0.05), for the ileum and jejunum, respectively. The other groups had a similar mean neuronal cell body area (P>0.05). CONCLUSION: Arthritis does not promote quantitative or morphological damages in general myenteric population. However, studies in progress have revealed some significant alterations in myenteric neurons subpopulations (nitrergic and VIP-ergic neurons).

CONTEXTO: A resposta inflamatória e o estresse oxidativo acentuados em decorrência da artrite reumatóide são capazes de promover neurodegeneração. Nessas condições, é possível que o sistema nervoso entérico seja afetado, diminuindo a qualidade de vida dos pacientes. No entanto, os sintomas da artrite no trato gastrointestinal são geralmente associados ao uso de medicamentos anti-inflamatórios do que a um possível dano neural. OBJETIVO: Verificar se a população geral de neurônios mioentéricos do íleo e do jejuno de ratos artríticos é afetada pela artrite. MÉTODOS: Foram utilizados 20 ratos Holtzmann, inicialmente com 58 dias de idade, divididos em 4 grupos: controle com 88 dias (C30); artrítico com 88 dias (Art30); controle com 118 dias (C60) e artrítico com 118 dias (Art60). Os animais dos grupos Art30 e Art60 receberam aos 58 dias de idade o adjuvante completo de Freund para indução da artrite. Os preparados totais de íleo e jejuno foram submetidos a imunoistoquímica para a proteína miosina-V. Realizou-se análises quantitativas e morfométricas dos neurônios. RESULTADOS: Os animais Art30 e Art60 apresentaram, respectivamente, redução de 2 por cento e 6 por cento na área intestinal em relação aos seus controles. Não foram observadas diferenças na densidade neuronal geral entre os quatro grupos (P>0,05). O grupo C60 apresentou redução de 14,4 por cento e 10,9 por cento na área média do corpo celular neuronal em relação ao grupo C30 (P<0,05). Os demais grupos apresentaram área média de corpo celular semelhante (P>0,05). CONCLUSÃO: A artrite não provocou alterações quantitativas ou morfológicas na população mioentérica geral, entretanto, estudos em andamento revelam alterações significativas em subpopulações de neurônios mioentéricos (nitrérgicos e VIP-érgicos).

Preconditioning with oil mixes of high ratio Omega-9: Omega-6 and a low ratio Omega-6:Omega-3 in rats subjected to brain ischemia/reperfusion / Pré-condicionamento com misturas de óleos com Ômega-9: Ômega-6 (alta relação) e Ômega-6:Ômega-3 (baixa relação) em ratos submetidos à isquemia/reperfusão cerebral

PURPOSE: This study aimed to assess the effects of preconditioning with mixtures of oils containing high/low ratio of ω-6/ω-3 and ω-9/ω-6, respectively, in an experimental model of cerebral ischemia-reperfusion (I/R). METHODS: Forty-two Wistar rats were randomly distributed into two groups: control (n=24) and test (n=18). Control group was subdivided in 4 subgroups (n=6): G1: Sham-Water; G2: I/R-Water; G3: Sham-Isolipidic and G4: I/R-Isolipid. The animals received water or a isolipid mixture containing ω-3 oils (8:1 ratio) and ω-9/ω-6 (0.4:1 ratio) by gavage for seven days. Test group included 3 subgroups (n=6) G5: I/R-Mix1, G: 6 I/R-Mix2 and G7: I/R-Mix3. Test group animals received oily mixtures of ω-3 (1.4:1 ratio) and ω-6 (3.4:1 ratio), differing only in source of ω-3: G5 (alpha-linolenic acid); G6 (alpha-linolenic, docosahexaenoic and eicosapentaenoic acids), and G7 (alpha-linolenic and docosahexaenoic acids). On day 7 I/R rats underwent cerebral ischemia with bilateral occlusion of common carotid arteries for 1 hour followed by reperfusion for 3 hours. G1 and G3 animals underwent sham operation. Concluded the experiment, animals were decapitated and their brains sliced for red neurons (RN) count in CA3 area of the hippocampus. Variables were compared using ANOVA-Tukey test. RESULTS: The use of different mix preparations promoted a decrease in red cell count in all three groups (G5/G6/G7), compared with G2/G4, confirming the protective effect of different oil blends, regardless of ω-3 source. CONCLUSION: Pre-conditioning with mixtures of oils containing high ratio ω-6/ω-3 and low ω-9/ω-6 relationship protects brain neurons against I/R injury in an experimental model.

OBJETIVO: Avaliar os efeitos do pré-condicionamento com misturas de óleos contendo relação alta/baixa de ω-6/ω-3 e ω-9/ω-6, respectivamente, em um modelo experimental de isquemia/reperfusão (I/R) cerebral. MÉTODOS: Quarenta e dois ratos foram distribuídos aleatoriamente em dois grupos: controle (n=24) e teste (n=18). Grupo controle foi subdividido em quatro subgrupos (n=6): G1: Sham-Água; G2: I/R-Água; G3: Sham-Isolipídico e G4: I/R-Isolipídico. Os animais receberam água ou uma mistura isolipidica contendo ω-6/ω-3 óleos (8:1) e ω-9/ω-6 (0,4:1) por gavagem, durante sete dias. O grupo teste incluiu três subgrupos (n=6) G5: I/R-Mix1, G: 6 I/R-Mix2 e G7: I/R-Mix3. Animais do grupo teste receberam de misturas de óleos ω-6/ω-3 (1,4:1) e ω-9/ω-6 (3,4:1), diferindo apenas na fonte de -3: G5:alpha-linolênico; G6: ácidos alpha-linolênico, eicosapentaenóico e docosahexaenóico e G7:ácidos alpha-linolênico e docosahexaenóico. No 7º dia os grupos I/R foram submetidos à isquemia cerebral (1h) por oclusão bilateral das artérias carótidas comuns seguida de reperfusão (3h). Ratos G1 e G3 foram submetidos à operação simulada. Concluído o experimento, os animais foram decapitados e seus cérebros fatiados para contagem dos neurônios vermelhos na área CA3 do hipocampo. As variáveis foram comparadas pelo teste de ANOVA-Tukey. RESULTADOS: A utilização de diferentes misturas de óleos promoveu uma diminuição na contagem de células vermelhas nos grupos G5/G6/G7, em comparação com G2/G4, confirmando o efeito protetor das misturas de óleos, independentemente da origem de ω-3. CONCLUSÃO: O pré-condicionamento com misturas de óleos contendo alta proporção de ω-6/ω-3 e baixa proporção de ω-9/ω-6 protege os neurônios cerebrais da lesão de I/R em um modelo experimental.

Chronic stress effects on NPY neuronal population during rat development / Efectos del stress crónico sobre la población neuronal NPY durante el desarrollo de ratas

The aim of this work was to determine the chronical stress effects on the encephalic NPY neurons population during the fetal Central nervous system development. Immunocytochemical techniques were used for this purpose: NPY neurons presented a similar morphology during the gestation days studied but their distribution varied in the anterior, medium and posterior brain. Statistical Highly significant differences in number of NPY positive neurons (p<0.01) among anterior, medium and posterior brain of stressed fetus (SF) were determined depending on the gestation period and the brain area. The NPY neurons were increased in ARC (Arcuate Hypothalamic Nucleus), PH (Posterior Hypothalamic Area) and DM (Dorsomedial Hypothalamic Nucleus) in stressed fetuses (SF) of 17 days, and in ARC of 19 days SF (p< 0.01) were detected in the different brain nucleus. The NPY population increased in PnO (Pontine Reticular Nu, Oral Part) and RITg (Reticulotegmental Nu of the Pons) of 17 days SF, while they were detected in posterior brain at Pyx (Pyramidal Decussation), Rob (Raphe Obscurus Nucleus) and RPA (Raphe Pallidus Nucleus) in SF of 19 days. They also increased in number (p<0.05) in DPGI (Dorsal Paragigantocellular Nu), CGPn (Central Gray of Pons) and PrH (Prepositus Hypoglossal Nucleus) of 17 days SF. Finally, any statistical differences were found among CF and SF in the following nuclei: anterior brain, AH (Anterior Hypothalamic Nucleus), DM (Dorsomedia L Hypothalamic Nucleus) of 17 days; ME (Median Eminence)., VMH (Ventromedial Hypothalamic Nucleus) of 19 days; medium brain in CG (Central Periaqueductal Gray), DR (Dorsal Raphe Nucleus) of 17 days and posterior brain in PnC (Pontine Reticular Nu, Caudal Part), PrH (Prepositus Hypoglossal Nucleus), RMgG (Raphe Magnus Nucleus), IO (Inferior Olive) of 17 days. The increase number of NPY neurons found in the stressed rat fetuses in all periods studied would indicate the participation of the NPY System in...

El propósito del presente estudio fue determinar los efectos del estrés crónico en la población de neuronas NPY encefálicas durante el desarrollo del S.N.C. fetal mediante técnicas inmunocitoquímicas. Se demostró que las neuronas NPY presentan un morfología similar en los días de gestación estudiados, pero su distribución varía en el cerebro anterior, medio y posterior. Se comprobaron diferencias altamente significativas entre el cerebro anterior, medio y posterior (p<0,01) de fetos estresados (FE), variando dicha significación dependiendo del día de la gestación y del área estudiada. En los diferentes núcleos cerebrales del cerebro anterior se detectaron aumentos en ARC (Arcuate Hypothalamic Nucleus), PH (Posterior Hypothalamic Area) de 17 días y DM (Dorsomedia L Hypothalamic Nucleus) y en ARC (Arcuate Hypothalamic Nucleus) de 19días (p<0,01) de F.E. En el cerebro medio se detectaron aumentos en DR (Dorsal Raphe Nucleus) (p<0,01) y PN (Pontine Nucleus) (p<0,05) de 19 F.E. En el cerebro posterior se detectaron aumentos en PnO (Pontine Reticular Nu, Oral Part) y RITg (Reticulotegmental Nu of the Pons) de 17 F. E. y Pyx, (Pyramidal Decussation), Rob (Raphe Obscurus Nucleus) y RPA (Raphe Pallidus Nucleus) de 19 F.E. Asimismo se comprobaron aumentos (p<0,05) en DPGI (Dorsal Paragigantocellular Nu.) de 17 F.E, CGPn (Central Gray of Pons) y PrH (Prepositus Hypoglossal Nucleus), de 19 F.E. Finalmente, no se comprobaron diferencias entre F. C. (fetos controles) y F. E. en los siguientes núcleos del cerebro anterior: AH (Anterior Hypothalamic Nucleus), DM (Dorsomedia L Hypothalamic Nucleus), de 17 días; y EM, (Median Eminence), VMH (Ventromedial Hypothalamic Nucleus) de 19 días. En el cerebro medio CG, (Central Periaqueductal Gray), DR (Dorsal Raphe Nucleus) de 17 días. En el cerebro posterior el PnC, (Pontine Reticular Nu, Caudal Part), PrH (Prepositus Hypoglossal Nucleus), RMgG (Raphe Magnus Nucleus), IO (Inferior Olive) de 17 días del cerebro posterior...

Effect of age on the myosin-V immunoreactive myenteric neurons of rats ileum

Alterations in the gastrointestinal neuromuscular function related to age have been demonstrated in human and animal models. This study analyzes the effects of the aging process on the area of the neuronal cell bodies of the myenteric plexus in the antimesenteric and intermediate regions of the ileal circumference of Wistar, 12 month-old in comparison 3 month-old animals. The ileum was removed and whole-mount preparations immunostained by the antibody anti-myosin-V were processed. The morphometric analyses were performed using a computerized image analysis system, with a subsequent distribution of neurons by size in intervals of 100 micro2. The cellular body morphometry revealed a significant increase in the size of the myosin-V- immunoreactive myenteric neurons from 12 month-old animals when compared with 3 month –old animals. However, significant differences between the regions were not observed; these observations were not age-dependent. The implications of these results in relation to the increase of the body weight, size of the small intestine, general organization of the myenteric plexus, staining method of neurons and the possible factors involved in the regulation and/or control of the volume of neronal cells due to aging, are discussed.

Antioxidant responses of cortex neurons to iron loading

Brain cells have a highly active oxidative metabolism, yet they contain only low to moderate superoxide dismutase and catalase activities. Thus, their antioxidant defenses rely mainly on cellular reduced glutathione levels. In this work, in cortical neurons we characterized viability and changes in reduced and oxidized glutathione levels in response to a protocol of iron accumulation. We found that massive death occurred after 2 days in culture with 10 mM Fe. Surviving cells developed an adaptative response that included increased synthesis of GSH and the maintenance of a glutathione-based reduction potential. These results highlight the fundamental role of glutathione homeostasis in the antioxidant response and provide novel insights into the adaptative mechanisms of neurons subjected to progressive iron loads.

Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 æM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM) amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV). The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

Cleavage of purified neuronal clathrin assembly protein (CALM) by caspase 3 and calpain

The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.

Cleavage of purified neuronal clathrin assembly protein (CALM) by caspase 3 and calpain

The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.

Protéinas y enzimas como posibles mensajeros en la comunicación química interneuronal: parte II / Proteins and enzymes as chemical messengers regulating neuronal transmission: part II

La neurotransmisión química en el cerebro de los mamíferos y en el de las especies no mamíferas, se caracteriza por la liberación neuronal de una amplia gama de moléculas mensajeras, que incluyen neurotransmisores de naturaleza peptídica y no peptídica que poseen la capacidad de facilitar o inhibir la transmisión sináptica mediante la generación de señales eléctricas de tipo excitatorio o inhibitorio. Además de estas sustancias bioactivas, también hay un extenso grupo de macromoléculas de naturaleza protéica que participan en la comunicación química neuronal, generando y regulando diversas respuestas biológicas en el sistema nervioso

Proteínas y enzimas como posibles mensajeros en la comunicación química interneuronal. Parte I / Proteins and enzymes as chemical messangers regulating neuronal transmission. First part

En los últimos 30 años un extenso cúmulo de información ha sido reportada sobre la identificación y caracterización fisiológica y molecular de las proteínas y las enzimas; cuya liberación espontánea e inducida a partir del tejido neuronal y de los tejidos extraneuronales, como las células cromafines de la glándula suprarrenal, han permitido no sólo la subsecuente clonación y aislamiento molecular de estas moléculas protéicas, sino que, además, mediante la aplicación de métodos de hibridación in situ, de la generación de anticuerpos selectivos, conjuntamente con el apoyo de técnicas inmunohistoquímicas, se ha podido detectar el origen, localización y expresión celular de enzimas como la acetilcolinesterasa específica (AChE), y la dopamina-betahidroxilasa (DBH), en diversas regiones del cerebro de distintas especies mamíferas, de donde se ha encontrado su capacidad para ejercer funciones neurotróficas. Asimismo, se han abordado similares planteamientos experimentales para identificar y localizar la expresión celular y subcelular de proteínas como las de la familia de los polipéptidos, representados por las cromograninas y las secretoneurinas, que son polipéptidos, cuya síntesis celular modificaciones postraduccionales, almacenamiento y segregación vesicular, como su exocitosis celular en el tejido neuronal y en los tejidos neuroendocrinos, han sido estudiadas in vivo e in vitro a partir de diferentes preparaciones biológicas, cuyos resultados han sido motivo de múltiples publicaciones. Más aún, estas proteínas parecen ser precursoras de varios péptidos bioactivos, en los que se han observado diversas funciones de tipo autocrino, paracrino, o endocrino, en el tejido neuronal y en los tejidos extraneuronales. Asimismo, una extensa familia de factores tróficos, conocidos como neurotrofinas, incluyendo el Factor de Crecimiento Neuronal (NGF), recientemente se aislaron, identificaron, y caracterizaron molecular y funcionalmente, in vivo e in vitro, con lo que se demostró su capacidad para regular y mantener la supervivencia de diferentes poblaciones de células neuronales en el sistema nervioso central y periférico de múltiples especies de vertebrados e invertebrados, lo que parece mediarse por la activación de diversos subgrupos de receptores membranales, recientemente identificados y localizados en discretas poblaciones neuronales

Age-related decline in multiple unit action potentials of cerebral cortex correlates with the number of lipofuscin-containing neurons.

The present study examined whether there is any obvious correlation between the density of lipofuscin-containing neurons and the spontaneous neuronal action potentials (Multiple Unit Activity, MUA) in the parietal cortex of the aging rat brain. The results showed that MUA counts were decreased with age while the number of lipofuscin-containing neurons was increased. The cortex with the highest percentage of lipofuscin-containing neurons had the lowest MUA counts while the cortex with the lowest percentage of lipofuscin-containing neurons had the highest MUA counts. The inverse correlation between MUA and lipofuscin-containing neuron number was also evident when the population of the lipofuscin-containing neurons was pharmacologically altered in vivo by the administration of anti-lipofuscin drug centrophenoxine. The inverse relationship between MUA and the lipofuscin-containing neuron numbers is consistent with: (i) the correlations of MUA with age-related changes in lipid peroxidation and biochemically measured lipofuscin concentration, and (ii) the oxidative stress-induced impairments of neuronal electrophysiology.

Unilateral enucleation induces an increase of 160 kd neurofilament in lateral geniculate nuclei synapses

Ultrastructural synaptic changes of retinal origin in the pars dorsalis lateral geniculate nuclei (dLGN) after enucleation have been studied in this laboratory, showing a filamentous hypertrophy with maximal expression at 4-6 days post-lesion in monkeys (Pecci Saavedra et al., 1970, 1971). The aim of this work was to elucidate the nature of the newly formed filament in dLGN in post-enucleated rats. Male Wistar rats were fixed with 4 paraformaldehyde plus 0.25 glutaraldehyde in 0.1M phosphate buffer, through the abdominal aorta after 3, 5, and 7 days postenucleation. Sections obtained were incubated with antibodies to the phosphorylated portion of the 160 Kd neurofilaments (1:3000) and anti-GFAP (1:25000). There was an increase in 160 Kd neurofilament staining in axons and degenerating nerve endings in dLGN, as well as a typical astroglial immunostained reaction. Our results show that the newly formed neurofilaments after deafferentation are of the 160 Kd type, commonly present in normal axons.

Myenteric neurons of the mouse small intestine. Morphometry and acetylcholinesterase activity

1. The myenteric plexus of the small intestine of five C57BL/6J male 5-month-old mice was investigated in whole-mount preparations of the muscularis externa by Giemsa staining and by the acetylcholinesterase (AChE) histochemical technique. 2. The neuronal density was 20212 ñ 3038/cm² (mean ñ SEM) in the duodenum, 21948 ñ 1488/cm² in the jejunum, 25048 ñ 2356/cm² in the ilium. The difference in neuronal density between duodenum and ileum was statistically significant (P<0,05). The total serosal surface area of the small intestine was about 30.80 ñ 2.90 cm², and the total number of neurons was estimated at about 690,000. 3. The neuronal cell and nucleus profile areas ranged, respectively, from 23 to 325 µm² and from 6 to 95 µm² in the small intestine of the mice studied. There were no significant differences in any of the 3 regions in terms of average neuronal cell or nucleus profile areas. 4. For the histochemical demonstration of AChE, the "direct coloring" copper ferrocyanide method was used. AChE-positive nerve fibers were distributed in the myenteric plexus which was formed by a primary meshwork of relatively large nerve bundles and a secondary meshwork of finer nerve bundles. Most of the neurons of the plexus displayed AChE activity in the cytoplasm though the neurons presented different reaction intensities. 5. The results of the present study show that the myenteric plexus of the C57BL/6J mouse small intestine contains a large number of neurons which have different sizes and AChE activities

Characterization of concanavalin A-binding neuronal and glial surface glycoproteins from human foetal brain.

Neuronal and glial surface glycoproteins have been isolated from human foetal brains by affinity chromatography on 8 M urea or 6 M guanidine-treated Con A-Sepharose 4B at 4 degrees C and three groups of glycoproteins of molecular mass 65-73 kDa, 52-63 kDa and 43-48 kDa have been identified on SDS/PAGE. These glycoproteins exhibited anomalous behaviour on SDS/PAGE, indicating the existence of a gradation of mutually interconvertible protein-SDS aggregates in dynamic equilibrium with one another. Deglycosylation and deacylation did not alter the SDS/PAGE multiple band pattern. Purified glycoproteins contained 160 +/- 90 micrograms carbohydrate/mg protein, and a sialic acid content of 25 +/- 5 nmole/mg protein. The N-terminals were blocked. The glycoproteins moved preferentially on acid/urea/PAGE. Sepharose 6B gel filtration in the absence of lipid and detergents resolved the glycoproteins into an excluded peak I and a low molecular mass peak II. Peaks I and II were non-interconvertible on Sepharose 6B gel filtration or on reversed phase HPLC in an isopropanol/water/TFA gradient system. Both peaks rendered a single fast moving band of identical mobility on acid/urea/PAGE, suggesting that peak I was possibly a micellar aggregate of the monomeric peak II. The glycoproteins were refractory to digestion by trypsin or pronase and reacted identically towards various lectins.(ABSTRACT TRUNCATED AT 250 WORDS)