RESUMO
The omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) is enriched in neural membranes of the CNS, and recent studies have shown a role of DHA metabolism by 15-lipoxygenase-1 (Alox15) in prefrontal cortex resolvin D1 formation, hippocampo-prefrontal cortical long-term-potentiation, spatial working memory, and anti-nociception/anxiety. In this study, we elucidated epigenetic regulation of Alox15 via histone modifications in neuron-like cells. Treatment of undifferentiated SH-SY5Y human neuroblastoma cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate significantly increased Alox15 mRNA expression. Moreover, Alox15 expression was markedly upregulated by Class I HDAC inhibitors, MS-275 and depsipeptide. Co-treatment of undifferentiated SH-SY5Y cells with the p300 histone acetyltransferase (HAT) inhibitor C646 and TSA or sodium butyrate showed that p300 HAT inhibition modulated TSA or sodium butyrate-induced Alox15 upregulation. Differentiation of SH-SY5Y cells with retinoic acid resulted in increased neurite outgrowth and Alox15 mRNA expression, while co-treatment with the p300 HAT inhibitor C646 and retinoic acid modulated the increases, indicating a role of p300 HAT in differentiation-associated Alox15 upregulation. Increasing Alox15 expression was found in primary murine cortical neurons during development from 3 to 10 days-in-vitro, reaching high levels of expression by 10 days-in-vitro-when Alox15 was not further upregulated by HDAC inhibition. Together, results indicate regulation of Alox15 mRNA expression in neuroblastoma cells by histone modifications, and increasing Alox15 expression in differentiating neurons. It is possible that one of the environmental influences on the immature brain that can affect cognition and memory, may take the form of epigenetic effects on Alox15 and metabolites of DHA.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Histonas/metabolismo , Neuroblastoma/metabolismo , Acetilação/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/metabolismo , Epigênese Genética/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Células-Tronco Neurais/metabolismo , Neurônios/metabolismoRESUMO
Arachidonic acid and docosahexaenoic acid (DHA) released by the action of phospholipases A2 (PLA2) on membrane phospholipids may be metabolized by lipoxygenases to the anti-inflammatory mediators lipoxin A4 (LXA4) and resolvin D1 (RvD1), and these can bind to a common receptor, formyl-peptide receptor 2 (FPR2). The contribution of this receptor to axonal or dendritic outgrowth is unknown. The present study was carried out to elucidate the distribution of FPR2 in the rat CNS and its role in outgrowth of neuronal processes. FPR2 mRNA expression was greatest in the brainstem, followed by the spinal cord, thalamus/hypothalamus, cerebral neocortex, hippocampus, cerebellum and striatum. The brainstem and spinal cord also contained high levels of FPR2 protein. The cerebral neocortex was moderately immunolabelled for FPR2, with staining mostly present as puncta in the neuropil. Dentate granule neurons and their axons (mossy fibres) in the hippocampus were very densely labelled. The cerebellar cortex was lightly stained, but the deep cerebellar nuclei, inferior olivary nucleus, vestibular nuclei, spinal trigeminal nucleus and dorsal horn of the spinal cord were densely labelled. Electron microscopy of the prefrontal cortex showed FPR2 immunolabel mostly in immature axon terminals or 'pre-terminals', that did not form synapses with dendrites. Treatment of primary hippocampal neurons with the FPR2 inhibitors, PBP10 or WRW4, resulted in reduced lengths of axons and dendrites. The CNS distribution of FPR2 suggests important functions in learning and memory, balance and nociception. This might be due to an effect of FPR2 in mediating arachidonic acid/LXA4 or DHA/RvD1-induced axonal or dendritic outgrowth.
Assuntos
Axônios/metabolismo , Encéfalo/metabolismo , Dendritos/metabolismo , Receptores de Lipoxinas/biossíntese , Medula Espinal/metabolismo , Animais , Axônios/química , Axônios/ultraestrutura , Encéfalo/ultraestrutura , Química Encefálica/fisiologia , Sobrevivência Celular/fisiologia , Sistema Nervoso Central/química , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/ultraestrutura , Dendritos/química , Dendritos/ultraestrutura , Masculino , Ratos , Ratos Wistar , Receptores de Lipoxinas/análise , Medula Espinal/química , Medula Espinal/ultraestruturaRESUMO
Silver nanoparticles (AgNPs) are among the most commonly used nanomaterials, but thus far, little is known about ways to mitigate against potential toxic effects of exposure. In this study, we examined the potential effects of AgNPs on mitochondrial function and cellular ATP levels, and whether these could be prevented by treatment with docosahexaenoic acid (DHA) and L-carnitine (LC). Acute exposure of AgNPs for 1 h to SH-SY5Y cells resulted in decreased mitochondrial membrane potential, and decreased ATP and ADP levels, indicating mitochondrial damage and reduced production of ATP. Incubation of cells with DHA partially reduced, while treatment with LC and DHA completely abolished the AgNP induced decreases in ATP and ADP levels. This could be due to a LC-facilitated entry of DHA to mitochondria, for repair of damaged phospholipids. It is postulated that DHA and LC may be useful for treatment of accidental environmental exposure to AgNPs.
Assuntos
Trifosfato de Adenosina/metabolismo , Carnitina/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Difosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfolipídeos/metabolismoRESUMO
Anatomy is an important component in the vertical integration of basic science and clinical practice. Two common pedagogies are cadaveric dissection and examination of prosected specimens. Comparative studies mostly evaluate their immediate effectiveness. A randomized controlled trial design was employed to compare both the immediate and long-term effectiveness of dissection and prosection. Eighty third-year medical students undergoing their surgical rotation from the Yong Loo Lin School of Medicine were randomized into two groups: dissection and prosection. Each participated in a one-day hands-on course following a similar outline that demonstrated surgical anatomy in the context of its clinical relevance. A pre-course test was conducted to establish baseline knowledge. A post-course test was conducted immediately after and at a one-year interval to evaluate learner outcome and knowledge retention. A post-course survey was conducted to assess participant perception. Thirty-nine and thirty-eight participants for the dissection and prosection groups, respectively, were included for analysis. There was no significant difference between mean pre-course test scores between the dissection and prosection groups [12.6 (3.47) vs. 12.7 (3.16), P > 0.05]. Both the mean immediate [27.9 (4.30) vs. 24.9 (4.25), P < 0.05] and 1 year [23.9 (4.15) vs. 19.9 (4.05), P < 0.05] post-course test scores were significantly higher in the dissection group. However, when adjusted for course duration [dissection group took longer than prosection group (mean 411 vs. 265 min)], these findings were negated. There is no conclusive evidence of either pedagogy being superior in teaching surgical anatomy. Based on learner surveys, dissection provides a greater learner experience.
Assuntos
Anatomia , Educação de Graduação em Medicina , Estudantes de Medicina , Humanos , Anatomia/educação , Dissecação/educação , Mãos , Extremidade Superior/anatomia & histologia , Cadáver , Ensino , CurrículoRESUMO
Energy requiring apoptosis and presumably unregulated necrosis are considered conceptually and morphologically distinct forms of cell death which have been initially identified as two exclusive pathways. However, several apoptotic characteristics have been observed in the necrotic core lesion in ischemia which led to the controversial theory that cell death advances via a number of hybrid pathways among a continuum between the two processes. ATP availability has been shown to influence the decision between apoptosis and necrosis. The aims of our study are 1) to determine if combined inhibitors administration of pan-caspase inhibitor Carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) and non-selective poly (ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide (3-AB) can further reduce infarct volume compared to single modality of either inhibitor following ischemic insult, 2) to ascertain the pharmacological intervention up to 24 hour post-middle cerebral artery occlusion (MCAo), and 3) to correlate intracellular ATP level with infarct volume. Single modality treatment was optimised at 3 mg/kg z-VAD-fmk and 30 mg/kg 3-AB with infarct volume measured at 24.13%+/-3.89% and 26.98%+/-2.22% respectively, while untreated control group was determined at 45.97%+/-1.86%. Combined inhibitors treatment rendered further reduction in infarct volume, measuring 7.228%+/-1.988%, 21.02%+/-1.06%, 24.40%+/-2.12% at 30 min, 6 h, 24 h post-ischemia respectively. In conclusion, the combined inhibitors administration of both z-VAD-fmk and 3-AB show further increased in infarct volume reduction with our ischemic model up to the 24 hour post-MCAo. However, in our in vivo study, no correlation between intracellular ATP level and infarct size was established.
Assuntos
Clorometilcetonas de Aminoácidos/administração & dosagem , Benzamidas/administração & dosagem , Caspases/metabolismo , Inibidores Enzimáticos/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspases/efeitos dos fármacos , Infarto Cerebral/metabolismo , Infarto Cerebral/patologia , Infarto Cerebral/prevenção & controle , Quimioterapia Combinada , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Masculino , Necrose/enzimologia , Necrose/prevenção & controle , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Resultado do TratamentoRESUMO
Heat shock proteins (HSPs) are evolutionarily conserved molecules and play important roles in fundamental cellular processes. They serve as molecular chaperones and hence provide a protective function in ensuring cell survival and repair of cellular damage after a stressful stimulus. This paper summarizes the current knowledge about the different roles of HSPs in aging and disease, focusing on the neurodegenerative disorders of Alzheimer's marks disease, Parkinson's disease, Huntington's marks disease, and prion disease.
Assuntos
Proteínas de Choque Térmico/fisiologia , Doenças Neurodegenerativas/fisiopatologia , Envelhecimento/fisiologia , Animais , HumanosRESUMO
Docosahexaenoic acid (DHA) is enriched in membrane phospholipids of the central nervous system (CNS) and has a role in aging and neuropsychiatric disorders. DHA is metabolized by the enzyme Alox15 to 17S-hydroxy-DHA, which is then converted to 7S-hydroperoxy,17S-hydroxy-DHA by a 5-lipoxygenase, and thence via epoxy intermediates to the anti-inflammatory molecule, resolvin D1 (RvD1 or 7S,8R,17S-trihydroxy-docosa-Z,9E,11E,13Z,15E,19Z-hexaenoic acid). In this study, we investigated the distribution and function of Alox15 in the CNS. RT-PCR of the CNS showed that the prefrontal cortex exhibits the highest Alox15 mRNA expression level, followed by the parietal association cortex and secondary auditory cortex, olfactory bulb, motor and somatosensory cortices, and the hippocampus. Western blot analysis was consistent with RT-PCR data, in that the prefrontal cortex, cerebral cortex, hippocampus, and olfactory bulb had high Alox15 protein expression. Immunohistochemistry showed moderate staining in the olfactory bulb, cerebral cortex, septum, striatum, cerebellar cortex, cochlear nuclei, spinal trigeminal nucleus, and dorsal horn of the spinal cord. Immuno-electron microscopy showed localization of Alox15 in dendrites, in the prefrontal cortex. Liquid chromatography mass spectrometry analysis showed significant decrease in resolvin D1 levels in the prefrontal cortex after inhibition or antisense knockdown of Alox15. Alox15 inhibition or antisense knockdown in the prefrontal cortex also blocked long-term potentiation of the hippocampo-prefrontal cortex pathway and increased errors in alternation, in the T-maze test. They indicate that Alox15 processing of DHA contributes to production of resolvin D1 and LTP at hippocampo-prefrontal cortical synapses and associated spatial working memory performance. Together, results provide evidence for a key role of anti-inflammatory molecules generated by Alox15 and DHA, such as resolvin D1, in memory. They suggest that neuroinflammatory brain disorders and chronic neurodegeneration may 'drain' anti-inflammatory molecules that are necessary for normal neuronal signaling, and compromise cognition.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Memória de Curto Prazo/fisiologia , Córtex Pré-Frontal/metabolismo , Memória Espacial/fisiologia , Animais , Encéfalo/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
Endothelial dysfunction is a major feature of vascular diseases. A practical, minimally invasive method to effectively "probe" gene transcription for an individual patient's endothelium has potential to "customize" assessment for an individual at risk of vascular disease as well as pathophysiologic insight in an in vivo human, clinical context. Published literature lacks a methodology to identify endothelial differential gene expression in individuals with vascular disease. We describe a methodology to do so. The aim of this study was to specifically utilize (a) cutaneous microvascular biopsy, (b) laser capture microdissection, (c) cDNA amplification, (d) suppression subtractive hybridization, (e) high-throughput sequencing techniques, (f) real-time polymerase chain reaction (PCR), and (g) in combination of these methods, to profile differential gene expression in the context of cardiovascular and cerebrovascular disease. Endothelial cells were obtained by laser capture microdissection from a patient and a healthy sibling's microvascular biopsy tissues. Endothelial RNA was extracted, reverse transcribed, and amplified to ds cDNA. Suppression subtractive hybridization was used to establish an endothelial differential gene expression library. Real-time PCR confirmed SERP1, caspase 8, IGFBP7, S100A4, F85, and F147 up-regulation between 1.4- and 3.47-fold. The authors have successfully established a methodology to profile endothelial differential gene expression and identified six differentially expressed genes. This minimally invasive novel method has potential wide application in the customized assessment of many patients suffering vascular diseases.
Assuntos
Células Endoteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Adulto , Animais , Vasos Sanguíneos/ultraestrutura , Mapeamento Cromossômico , DNA Complementar , Bases de Dados de Ácidos Nucleicos , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Células Endoteliais/ultraestrutura , Biblioteca Gênica , Humanos , Lasers , Masculino , Camundongos , Camundongos SCID , Hibridização de Ácido Nucleico , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cysteine is known to cause neuronal cell death and has been reported to be elevated in brain ischemia, but it has not been studied in clinical stroke. In this study, we correlated plasma levels of cyst(e)ine with long-term clinical outcome at 3 months in acute stroke. Patients were classified into 3 groups at 3 months as follows: good outcome (Rankin 0-1, n = 11), poor outcome (Rankin 2-5, n = 20), and dead (n = 5). Their plasma cyst(e)ine levels within 24 hours of stroke onset were 61 +/- 12, 67 +/- 9, and 82 +/- 14 micromol/L (standard deviation), respectively. The correlation between early plasma cyst(e)ine levels and long-term clinical outcome assessed at 3 months is significant with p < 0.001. None of the other 4 amino acids studied showed any significant correlation. Cyst(e)ine was also significantly elevated in patients who had early stroke deterioration (p < 0.02). Dose-dependent administration of cysteine increased the infarct volume by approximately 30% in a rat stroke model. This effect of cysteine was abolished by aminooxyacetic acid, an inhibitor of the enzyme cystathionine beta-synthase that converts cysteine to hydrogen sulfide (H2S), indicating that this novel neuromodulator may be acting as a mediator of ischemic brain damage. Raised plasma cyst(e)ine in patients with stroke may reflect increased production of H2S in the brain and thus predispose to poor outcome in clinical stroke. Inhibition of H2S formation may therefore be a novel approach in acute stroke therapy.
Assuntos
Cisteína/sangue , Cistina/sangue , Sulfeto de Hidrogênio/metabolismo , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminoácidos/sangue , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cisteína/administração & dosagem , Cistina/administração & dosagem , Modelos Animais de Doenças , Feminino , Humanos , Infarto da Artéria Cerebral Média , Masculino , Pessoa de Meia-Idade , Prognóstico , Ratos , Ratos Wistar , Acidente Vascular Cerebral/patologiaRESUMO
Exercise has been shown to influence learning and memory. Most studies were performed with a voluntary running paradigm (e.g. running wheel) in mice. However, such effects of exercise on learning and memory are less well demonstrated using a forced running paradigm (e.g. treadmill). The present study was designed to examine the effects of 12 weeks of forced treadmill running on learning and memory performance in rats. We have previously shown that forced running resulted in qualitative and quantitative changes in the cholinergic neurons of the horizontal diagonal band of Broca (HDB) in the septum. This study was conducted in order to determine whether or not these changes occur simultaneously with enhanced learning and memory. The one-day version of the Morris water maze (MWM) test [Frick, K.M., Stillner, E.T., Berger-Sweeney, J., 2000. Mice are not little rats: species differences in a one-day water maze task. NeuroReport 11, 3461-3465] was used to test spatial learning and memory after the exercise period. Our data showed that runners displayed better spatial learning and memory when compared to nonrunners. This was evidently shown by a reduction in the time required for spatial acquisition (p<0.05) and superior probe trial performance (p<0.05). A shorter distance swam by the runners also suggested improved learning over the nonrunners (p<0.05). In an attempt to revalidate our earlier quantitative results, we used design-based stereology (DBS) to estimate the number of cholinergic neuronal profile population in the medial septum and diagonal band (MSDB). We confirmed that forced running increased the cholinergic neuronal profile subpopulation in the HDB (Coefficient of Error<0.2). Taken together, these results indicate that forced exercise could influence learning and memory with a concomitant increase in the number of cholinergic neurons in the HDB.
Assuntos
Memória/fisiologia , Condicionamento Físico Animal , Comportamento Espacial/fisiologia , Acetilcolinesterase/metabolismo , Análise de Variância , Animais , Comportamento Animal , Contagem de Células/métodos , Teste de Esforço/métodos , Imuno-Histoquímica/métodos , Masculino , Aprendizagem em Labirinto/fisiologia , Neurônios/metabolismo , Ratos , Ratos Wistar , Tempo de Reação/fisiologia , Núcleos Septais/citologia , Fatores de TempoRESUMO
Group IVA cytosolic phospholipase A2 (cPLA2 or PLA2G4A) is a key enzyme that contributes to inflammation via the generation of arachidonic acid and eicosanoids. While much is known about regulation of cPLA2 by posttranslational modification such as phosphorylation, little is known about its epigenetic regulation. In this study, treatment with histone deacetylase (HDAC) inhibitors, trichostatin A (TSA), valproic acid, tubacin and the class I HDAC inhibitor, MS-275, were found to increase cPLA2α messenger RNA (mRNA) expression in SH-SY5Y human neuroblastoma cells. Co-treatment of the histone acetyltransferase (HAT) inhibitor, anacardic acid, modulated upregulation of cPLA2α induced by TSA. Specific involvement of class I HDACs and HAT in cPLA2α regulation was further shown, and a Tip60-specific HAT inhibitor, NU9056, modulated the upregulation of cPLA2α induced by MS-275. In addition, co-treatment of with histone methyltransferase (HMT) inhibitor, 5'-deoxy-5'-methylthioadenosine (MTA) suppressed TSA-induced cPLA2α upregulation. The above changes in cPLA2 mRNA expression were reflected at the protein level by Western blots and immunocytochemistry. Chromatin immunoprecipitation (ChIP) showed TSA increased binding of trimethylated H3K4 to the proximal promoter region of the cPLA2α gene. Cell injury after TSA treatment as indicated by lactate dehydrogenase (LDH) release was modulated by anacardic acid, and a role of cPLA2 in mediating TSA-induced injury shown, after co-incubation with the cPLA2 selective inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3). Together, results indicate epigenetic regulation of cPLA2 and the potential of such regulation for treatment of chronic inflammation.
Assuntos
Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fosfolipases A2 do Grupo IV/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Ácidos Anacárdicos/farmacologia , Anilidas/farmacologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Desoxiadenosinas/farmacologia , Epigênese Genética/efeitos dos fármacos , Imunofluorescência , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , L-Lactato Desidrogenase/metabolismo , Lisina/metabolismo , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tiazóis/farmacologia , Tionucleosídeos/farmacologia , Ácido Valproico/farmacologiaRESUMO
Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi: 10.1007/s12035-015-9314-z , 2015). This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal. In this study, we examined the effects of C. nutans ethanol leaf extracts on epigenetic regulation of cPLA2 mRNA expression in SH-SY5Y human neuroblastoma cells and mouse primary cortical neurons. C. nutans modulated induction of cPLA2 expression in SH-SY5Y cells by histone deacetylase (HDAC) inhibitors, MS-275, MC-1568, and TSA. C. nutans extracts also inhibited histone acetylase (HAT) activity. Levels of cPLA2 mRNA expression were increased in primary cortical neurons subjected to 0.5-h oxygen-glucose deprivation injury (OGD). This increase was significantly inhibited by C. nutans treatment. Treatment of primary neurons with the HDAC inhibitor MS-275 augmented OGD-induced cPLA2 mRNA expression, and this increase was modulated by C. nutans extracts. OGD-stimulated increase in cPLA2 mRNA expression was also reduced by a Tip60 HAT inhibitor, NU9056. In view of a key role of cPLA2 in the production of pro-inflammatory eicosanoids and free radical damage, and the fact that epigenetic effects on genes are often long-lasting, results suggest a role for C. nutans and phytochemicals to inhibit the production of arachidonic acid-derived pro-inflammatory eicosanoids and chronic inflammation, through epigenetic regulation of cPLA2 expression.
Assuntos
Acanthaceae/química , Epigênese Genética/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Fosfolipases A2/genética , Extratos Vegetais/farmacologia , Animais , Benzamidas/farmacologia , Linhagem Celular , Humanos , Neurônios/efeitos dos fármacos , Piridinas/farmacologiaRESUMO
Oxidative stress plays an important role in the pathogenesis of neurodegeneration after the acute central nervous system injury. We reported previously that increased nitric oxide (NO) production following spinal cord hemisection tends to lead to neurodegeneration in neurons of the nucleus dorsalis (ND) that normally lacks expression of neuronal NO synthase (nNOS) in opposition to those in the red nucleus (RN) that constitutively expresses nNOS. We wondered whether oxidative stress could be a mechanism underlying this NO involved neurodegeneration. In the present study, we examined oxidative damage evaluated by the presence of 4-hydroxynonenal (HNE) and iron accumulation and expression of putative antioxidant enzymes heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) in neurons of the ND and RN after spinal cord hemisection. We found that HNE expression was induced in neurons of the ipsilateral ND from 1 to 14 days following spinal cord hemisection. Concomitantly, iron staining was seen from 7 to 14 days after lesion. HO-1, however, was only transiently induced in ipsilateral ND neurons between 3 and 7 days after lesion. In contrast to the ND neurons, HNE was undetectable and iron level was unaltered in the RN neurons after spinal cord hemisection. HO-1, SOD-Cu/Zn and SOD-Mn were constitutively expressed in RN neurons, and lesion to the spinal cord did not change their expression. These results suggest that oxidative stress is involved in the degeneration of the lesioned ND neurons; whereas constitutive antioxidant enzymes may protect the RN neurons from oxidative damage.
Assuntos
Antioxidantes/uso terapêutico , Núcleo Mediodorsal do Tálamo/patologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Núcleo Rubro/patologia , Traumatismos da Medula Espinal , Aldeídos/metabolismo , Animais , Contagem de Células/métodos , Lateralidade Funcional , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Histocitoquímica/métodos , Imuno-Histoquímica/métodos , Ferro/metabolismo , Masculino , Neurônios/patologia , Neurônios/fisiologia , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Coloração e Rotulagem/métodos , Superóxido Dismutase/metabolismo , Fatores de TempoRESUMO
This study investigated the possible involvement of gamma-aminobutyric acid (GABA) in the therapeutic effect of cerebral ischemia by electro-acupuncture (EA) using the rat model with middle cerebral artery occlusion (MCAO). By immunohistochemistry, the changes of GABA expression level in the primary infarct area and its penumbral regions were examined. The changes in infarct area and survival neuron percentages were also assessed using haematoxylin and eosin stained sections after picrotoxin (PTX) injection, a GABA receptor's antagonist. Our results showed that EA markedly decreased the ischemic damaged areas in the cerebral cortex and hippocampus. Concomitant to this was an up-regulation of GABA immunoexpression in MCAO rats with EA treatment (P < 0.05). Furthermore, injection of PTX in rats subjected to MCAO or MCAO followed by EA treatment increased the infarct area and decreased survival cell percentage significantly when compared with those without PTX injection. In the light of these findings, it is suggested that EA on specific and established acupoints that are commonly used in clinical management of cerebral ischemia may have elicited an up-regulated expression of GABA that would have a neuroprotective effect.
Assuntos
Isquemia Encefálica/terapia , Eletroacupuntura/métodos , Infarto da Artéria Cerebral Média/terapia , Ácido gama-Aminobutírico/metabolismo , Animais , Isquemia Encefálica/etiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/efeitos da radiação , Relação Dose-Resposta a Droga , Lateralidade Funcional , Antagonistas GABAérgicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/efeitos da radiação , Imuno-Histoquímica/métodos , Infarto da Artéria Cerebral Média/complicações , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos da radiação , Picrotoxina/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
PURPOSE: The expression of inducible nitric oxide synthase (iNOS) and bcl-2 proteins was evaluated and the prognostic significance determined in nasopharyngeal cancer (NPC) patients treated by radiotherapy. METHODS AND MATERIALS: Tissue sections from 55 patients with NPC were assessed for iNOS and bcl-2 protein expression by immunohistochemistry, immunoelectron microscopy, and in situ hybridization before treatment. The markers were correlated with apoptosis (detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay) and clinicopathologic parameters. RESULTS: All NPC sections exhibited positive iNOS and bcl-2 immunoreactivity, with a mean percentage of 6.24% +/- 0.58% and 17.09% +/- 2.48%, respectively. A significant positive correlation was observed between iNOS expression and the apoptotic index (p < 0.0001, Pearson's r = 0.8518), and bcl-2 expression correlated inversely with apoptosis (p = 0.0001; Pearson's r = -0.6170). A significant inverse correlation was found between iNOS and bcl-2 immunoreactivity (p < 0.0001, Pearson's r = -0.7144). Bcl-2 but not iNOS expression was associated with the stage of the tumor according to the criteria of the American Joint Committee on Cancer (1997) (p < 0.0001). Patients who had recurrence of the tumor and metastasis after radiotherapy had a lower expression of iNOS (p = 0.014 and p = 0.035, respectively), although overall survival was not significantly different statistically. Higher bcl-2 expression was also associated with local tumor recurrence (p = 0.005) but not with metastasis or overall survival. CONCLUSION: It appears that iNOS and bcl-2 expression may be potentially useful biomarkers for predicting the outcome of radiotherapy in NPC patients.
Assuntos
Neoplasias Nasofaríngeas/química , Proteínas de Neoplasias/análise , Óxido Nítrico Sintase/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Masculino , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/radioterapia , Óxido Nítrico Sintase Tipo II , Prognóstico , Estatística como AssuntoRESUMO
Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.
Assuntos
Bulbo/citologia , Proteínas do Tecido Nervoso/análise , Neurônios/química , Receptores Adrenérgicos/fisiologia , Estilbamidinas , Sistema Nervoso Simpático/citologia , Animais , Corantes Fluorescentes , Bulbo/química , Bulbo/enzimologia , Microscopia Eletrônica , Proteínas do Tecido Nervoso/imunologia , Vias Neurais , Neurônios/enzimologia , Neurônios/ultraestrutura , Feniletanolamina N-Metiltransferase/análise , Feniletanolamina N-Metiltransferase/imunologia , Ratos , Medula Espinal/química , Medula Espinal/citologia , Medula Espinal/enzimologia , Sistema Nervoso Simpático/química , Sistema Nervoso Simpático/enzimologia , Tirosina 3-Mono-Oxigenase/análise , Tirosina 3-Mono-Oxigenase/imunologiaRESUMO
This study was aimed to investigate the expression of glutamate receptors and calcium-binding proteins in 1- and 4-month/s (mo) streptozotocin (STZ)-induced diabetic rats. Upregulation of glutamate receptors' [N-methyl-D-aspartate receptor (NMDAR)1 and GluR2/3] immunoreactivities was observed in the ganglion, amacrine and bipolar cells as well as in the inner and outer plexiform layers (IPL and OPL) in 1 mo diabetes and was further enhanced at 4 mo. Immunoreactivity of calcium-binding proteins (calbindin and parvalbumin) was also concomitantly increased. The present results suggest that upregulation of glutamate receptors and calcium-binding proteins may reflect changes of the glutamate and calcium metabolism in the diabetic retina. It is speculated that the above changes in the IPL and OPL may be linked to alteration of synaptic transmission in the diabetic retina.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/metabolismo , Degeneração Neural/metabolismo , Receptores de Glutamato/metabolismo , Retina/metabolismo , Células Amácrinas/metabolismo , Células Amácrinas/patologia , Animais , Calbindinas , Retinopatia Diabética/patologia , Retinopatia Diabética/fisiopatologia , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Imuno-Histoquímica , Masculino , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Parvalbuminas/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/patologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Proteína G de Ligação ao Cálcio S100/metabolismo , Transmissão Sináptica/fisiologia , Regulação para Cima/fisiologiaRESUMO
Ureteric stones are a common cause of obstruction of the urinary tract, usually presenting with characteristic signs and symptoms, such as acute ureteric colic and hematuria. Occasionally, stones may present with non-specific symptoms such as low back pain and remain unidentified, leading to stone growth, chronic ureteric obstruction and complications such as hydronephrosis and renal damage. Here, we report a large ureteric stone in a cadaver with complete obstruction at the left ureterovesical junction, resulting in severe dilatation of the left ureter and renal pelvis.
RESUMO
3-Mercaptopyruvate sulfurtransferase (3MST) is an important enzyme for the synthesis of hydrogen sulfide (H2S) in the brain. We present here data that indicate an exclusively localization of 3MST in astrocytes. Regional distribution of 3MST activities is even and unremarkable. Following permanent middle cerebral artery occlusion (pMCAO), 3MST was down-regulated in both the cortex and striatum, but not in the corpus collosum. It appears that the down-regulation of astrocytic 3MST persisted in the presence of astrocytic proliferation due to gliosis. Our observations indicate that 3MST is probably not responsible for the increased production of H2S following pMCAO. Therefore, cystathionine ß-synthase (CBS), the alternative H2S producing enzyme in the CNS, remains as a more likely potential therapeutic target than 3MST in the treatment of acute stroke through inhibition of H2S production.