RESUMEN
The quantification of fibrotic tissue is an important task in the analysis of cardiac remodeling. The use of established fibrosis staining techniques is limited on frozen cardiac tissue sections due to a reduced color contrast compared to paraffin embedded sections. We therefore used FITC-labeled wheat germ agglutinin (WGA), which marks fibrotic tissue in comparable quality as the established picrosirius red (SR) staining, for the staining of post myocardial infarction scar tissue. The fibrosis amount was quantified in a histogram-based approach using the non-commercial image processing program ImageJ. Our results clearly demonstrate that WGA-FITC is a suitable marker for cardiac fibrosis in frozen tissue sections. In combination with the histogram-based analysis, this new quantification approach is i) easy and fast to perform; ii) suitable for raw frozen tissue sections; and iii) allows the use of additional antibodies in co-immunostaining.
Asunto(s)
Fluoresceína-5-Isotiocianato/análogos & derivados , Infarto del Miocardio , Miocardio , Coloración y Etiquetado/métodos , Aglutininas del Germen de Trigo/química , Animales , Fibrosis , Fluoresceína-5-Isotiocianato/química , Inmunohistoquímica/métodos , Masculino , Ratones , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/metabolismo , Miocardio/patologíaRESUMEN
Neuronal nitric oxide-I is constitutively expressed in approximately 2% of cortical interneurons and is co-localized with gamma-amino butric acid, somatostatin or neuropeptide Y. These interneurons additionally express high amounts of glutamate receptors which mediate the glutamate-induced hyperexcitation following cerebral injury, under these conditions nitric oxide production increases contributing to a potentiation of oxidative stress. However, perilesional nitric oxide synthase-I containing neurons are known to be resistant to ischemic and excitotoxic injury. In vitro studies show that nitrosonium and nitroxyl ions inactivate N-methyl-D-aspartate receptors, resulting in neuroprotection. The question remains of how these cells are protected against their own high intracellular nitric oxide production after activation. In this study, we investigated immunocytochemically nitric oxide synthase-I containing cortical neurons in rats after unilateral, cortical photothrombosis. In this model of focal ischemia, perilesional, constitutively nitric oxide synthase-I containing neurons survived and co-expressed antioxidative enzymes, such as manganese- and copper-zinc-dependent superoxide dismutases, heme oxygenase-2 and cytosolic glutathione peroxidase. This enhanced antioxidant expression was accompanied by a strong perinuclear presence of the antiapoptotic Bcl-2 protein. No colocalization was detectable with upregulated heme oxygenase-1 in glia and the superoxide and prostaglandin G(2)-producing cyclooxygenase-2 in neurons. These results suggest that nitric oxide synthase-I containing interneurons are protected against intracellular oxidative damage and apoptosis by Bcl-2 and several potent antioxidative enzymes. Since nitric oxide synthase-I positive neurons do not express superoxide-producing enzymes such as cyclooxygenase-1, xanthine oxidase and cyclooxygenase-2 in response to injury, this may additionally contribute to their resistance by reducing their internal peroxynitrite, H(2)O(2)-formation and caspase activation.
Asunto(s)
Antioxidantes/metabolismo , Apoptosis/fisiología , Isquemia Encefálica/enzimología , Corteza Cerebral/enzimología , Interneuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Isquemia Encefálica/fisiopatología , Corteza Cerebral/fisiopatología , Modelos Animales de Enfermedad , Glutatión Peroxidasa/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Inmunohistoquímica , Masculino , Microscopía Confocal , Degeneración Nerviosa/enzimología , Óxido Nítrico/metabolismo , Estrés Oxidativo/fisiología , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
Cerebral ischemic injury results in the liberation of heme from degenerating heme-containing proteins. The neurotoxic heme is usually detoxified by the constitutive heme oxygenase-2 (HO-2) and its inducible isoform HO-1(heat shock protein 32) resulting in the formation of biliverdin which becomes reduced to bilirubin, carbon monoxide (CO), and iron. Biliverdin and bilirubin have antioxidative properties whereas CO is discussed as a signaling molecule. Iron if it remains free could catalyze Haber--Weiss and Fenton reactions causing the formation of highly toxic radicals. We have studied the alterations of cerebral HO-2 and HO-1 in relation to iron accumulations after defined cortical photothrombosis within the hindlimb area of the rat. HO-2 immunohistochemistry showed that the number of HO-2-positive neurons in most perilesional regions remained constant. However, much stronger systemic immunoreactivity for HO-2 was observed between days 1 and 7 postlesion. For HO-1 a systemic increase of immunoreactivity occurred also between days 1 and 7. In addition HO-1-positive astrocytes and microglia appeared as early as 4 h postlesion and increased up to day 3 followed by a sharp decline toward day 14 within the injured hemisphere. HO-1-positive astrocytes and microglia occurred in ipsilateral cortex, corpus callosum, hippocampus, striatum, and thalamic nuclei. Additionally an increase of HO-1 in myelin-associated globulin-positive oligodendrocytes was found in ipsilateral and contralateral cortex. Next to the lesion iron accumulation occurred after day 3 and increased strongly toward day 14 at times when HO-1 and -2 had decreased, suggesting that HO activity does not directly contribute to postlesional iron deposition.
Asunto(s)
Encéfalo/enzimología , Hemo Oxigenasa (Desciclizante)/biosíntesis , Trombosis Intracraneal/enzimología , Neuroglía/enzimología , Neuronas/enzimología , Animales , Astrocitos/enzimología , Astrocitos/patología , Encéfalo/patología , Isquemia Encefálica/enzimología , Isquemia Encefálica/patología , Inducción Enzimática , Hemo Oxigenasa (Desciclizante)/análisis , Hemo-Oxigenasa 1 , Miembro Posterior/inervación , Inmunohistoquímica , Trombosis Intracraneal/patología , Hierro/metabolismo , Masculino , Microglía/enzimología , Microglía/patología , Neuroglía/patología , Neuronas/patología , Ratas , Ratas Wistar , Rosa Bengala , Factores de TiempoRESUMEN
The coumarin 7-hydroxylase of mice (Coh, CYP2A5) is known to be highly selectively inducible by both a set of heavy metals such as cobalt, indium and cerium and a variety of organic nitrogen-containing heteroaromatic compounds such as 3-amino-1,2,4-triazole, pyrazine and pyrazole. The investigations presented reveal that inorganic divalent tin has to be included in the list of selective inducers. Pretreatment of NMRI-mice with 50 mg SnCl2/kg body weight, daily for 2 days, increases the coumarin hydroxylation 40- and 20-fold in the kidney and liver, respectively. So far, the inducing potency of tin chloride is higher than that of the agents already known. The diagnostic inhibitor metyrapone strongly inhibits the coumarin model reaction. In the kidneys tin generates an almost pure fraction of a cytochrome P450 isozyme catalyzing the metabolism of coumarins, as inhibition experiments reveal.