Erythrocyte protoporphyrin fluorescence as a potential marker of diabetes.

Protoporphyrin (PpIX), a porphyrin derivative, is the intermediate metabolic precursor of the heme molecule. Abnormal metabolism of total erythrocyte PpIX has been observed in diseases such as cancer, lead poisoning, psoriasis, iron deficiency anemia and acute porphyries. Diabetes mellitus (DM) is a complex metabolic syndrome in which hyperglycemia is the primary clinical manifestation and contributes to the diabetic complications. The aim of this study was to evaluate the utility of fluorescence spectroscopy of erythrocyte PpIX for monitoring the early stages of diabetes. A total of 14 male C 57BL mice, 6 weeks old, were divided into two groups: diabetic and non-diabetic. Diabetes was induced by intraperitoneal injection of streptozotocin (SZT). Blood cells were cultured with standard and 50 mM supplemented RPMI medium. Blood smears were prepared and stained for qualitative morphology analysis under optical microscopy. Blood porphyrin autofluorescence was analyzed by fluorescence spectroscopy. Characteristic PpIX emission spectra were obtained by exciting the samples at 405 nm. Average blood glucose was lower in the control group than in the diabetic group (156.50 +/- 8.11 mg/dL vs. 371.10 +/- 14.43 mg/dL, P < 0.05). Both diabetic and glucose-cultured erythroblasts showed a significant decrease (around 30.5% and 40%, respectively) in the emission band intensity at 635 nm. Our results indicate that the erythrocyte PpIX profile could be used as a biological monitor for diabetes.

Qualitative and quantitative analysis of rabbit's fat mesenchymal stem cells.

PURPOSE: To present an experimental model of qualitative and quantitative analysis of mesenchymal stem cells from fat of rabbits obtained by lipectomy. The fat could be a great source for obtaining mesenchymal stem cells and to create conditions for repairing injured tissues by bioengineering. METHODS: New Zealand rabbits (n = 10) adipose panicle (2-3 cm) were removed by lipectomy, fragmented and washed with PBS and enzymatically dissociated with trypsin/EDTA. Lately, these cells were incubated in culture medium DMEM and after 20 days, was performed quantitative analysis of the accession of first and second mesenchymal cells in cell culture bottles. RESULTS: The fat total cells (CTF) were 1.62 x10(6) cells/mL and presented 98% of viability. These cells were taken for cultivation and after 20 days were counted 2.88 x10(6) cells/mL MSC. The same was done and after 20 days we quantified 4.28 x10(6) cells/mL MSC. CONCLUSION: The lipectomy of adipose panicule is a very satisfactory method to extract stem cells from fat, quantitatively and qualitatively.

Qualitative and quantitative analysis of rabbit's fat mesenchymal stem cells / Análise quantitativa e qualitativa de células tronco mesênquimais da gordura de coelhos

PURPOSE: To present an experimental model of qualitative and quantitative analysis of mesenchymal stem cells from fat of rabbits obtained by lipectomy. The fat could be a great source for obtaining mesenchymal stem cells and to create conditions for repairing injured tissues by bioengineering. METHODS: New Zealand rabbits (n= 10) adipose panicle (2-3 cm) were removed by lipectomy, fragmented and washed with PBS and enzymatically dissociated with trypsin/EDTA. Lately, these cells were incubated in culture medium DMEM and after 20 days, was performed quantitative analysis of the accession of first and second mesenchymal cells in cell culture bottles. RESULTS: The fat total cells (CTF) were 1.62 x10(6) cells/mL and presented 98 percent of viability. These cells were taken for cultivation and after 20 days were counted 2.88 x10(6) cells/mL MSC. The same was done and after 20 days we quantified 4.28 x10(6) cells/mL MSC. CONCLUSION: The lipectomy of adipose panicule is a very satisfactory method to extract stem cells from fat, quantitatively and qualitatively.(AU)

OBJETIVO: Apresentar um modelo experimental de análise qualitativa e quantitativa de células tronco mesênquimais proveniente da gordura de coelhos obtido por lipectomia. A gordura poderia ser uma grande fonte de obtenção de células tronco mesenquimais, criando condições para a reparação de tecidos lesados. MÉTODOS: Foram removidos os panículos adiposos (2-3 cm) da região cervical de Coelhos Nova Zelândia (n = 10) por lipectomia. Os panículos foram fragmentados e lavados com PBS e, posteriormente, dissociados enzimaticamente com tripsina / EDTA. As células extraídas do panículo adiposo foram incubadas em meio de cultura DMEM e após 20 dias, foi realizada uma análise quantitativa da adesão de primeira e segunda passagem das células mesênquimais em garrafas de cultura. RESULTADOS: Foram extraídas 1,62 x106 cel/ mL células totais de gordura (CTG) with 98 por cento de viabilidade. Essas células foram levadas para o cultivo e após 20 dias, foi realizada a primeira passagem (1pd) sendo quantificadas 2,88 x10(6) cel/mL células tronco mesênquimais (CTM). Na segunda passagem (2pd) foi obtido 4,28 x10(6) cel/mL CTM. CONCLUSÃO: A lipectomia do paniculo adiposo é um método muito satisfatório para extrair células tronco a partir de gordura, quantitativamente e qualitativamente.(AU)

Qualitative and quantitative analysis of rabbit's fat mesenchymal stem cells / Análise quantitativa e qualitativa de células tronco mesênquimais da gordura de coelhos

PURPOSE: To present an experimental model of qualitative and quantitative analysis of mesenchymal stem cells from fat of rabbits obtained by lipectomy. The fat could be a great source for obtaining mesenchymal stem cells and to create conditions for repairing injured tissues by bioengineering. METHODS: New Zealand rabbits (n= 10) adipose panicle (2-3 cm) were removed by lipectomy, fragmented and washed with PBS and enzymatically dissociated with trypsin/EDTA. Lately, these cells were incubated in culture medium DMEM and after 20 days, was performed quantitative analysis of the accession of first and second mesenchymal cells in cell culture bottles. RESULTS: The fat total cells (CTF) were 1.62 x10(6) cells/mL and presented 98 percent of viability. These cells were taken for cultivation and after 20 days were counted 2.88 x10(6) cells/mL MSC. The same was done and after 20 days we quantified 4.28 x10(6) cells/mL MSC. CONCLUSION: The lipectomy of adipose panicule is a very satisfactory method to extract stem cells from fat, quantitatively and qualitatively.

OBJETIVO: Apresentar um modelo experimental de análise qualitativa e quantitativa de células tronco mesênquimais proveniente da gordura de coelhos obtido por lipectomia. A gordura poderia ser uma grande fonte de obtenção de células tronco mesenquimais, criando condições para a reparação de tecidos lesados. MÉTODOS: Foram removidos os panículos adiposos (2-3 cm) da região cervical de Coelhos Nova Zelândia (n = 10) por lipectomia. Os panículos foram fragmentados e lavados com PBS e, posteriormente, dissociados enzimaticamente com tripsina / EDTA. As células extraídas do panículo adiposo foram incubadas em meio de cultura DMEM e após 20 dias, foi realizada uma análise quantitativa da adesão de primeira e segunda passagem das células mesênquimais em garrafas de cultura. RESULTADOS: Foram extraídas 1,62 x106 cel/ mL células totais de gordura (CTG) with 98 por cento de viabilidade. Essas células foram levadas para o cultivo e após 20 dias, foi realizada a primeira passagem (1pd) sendo quantificadas 2,88 x10(6) cel/mL células tronco mesênquimais (CTM). Na segunda passagem (2pd) foi obtido 4,28 x10(6) cel/mL CTM. CONCLUSÃO: A lipectomia do paniculo adiposo é um método muito satisfatório para extrair células tronco a partir de gordura, quantitativamente e qualitativamente.

Characterization of alpha2-adrenoceptors in smooth muscles of the spontaneously hypertensive rat aorta.

Previous works have shown that the alpha(2)-adrenoceptor agonist UK 14,304 induced the relaxation and hyperpolarization of the rat aorta, mediated by alpha(2)-adrenoceptors present in the smooth muscles, through small-conductance, ATP-sensitive K(+) channels. We now report that in spontaneously hypertensive rat (SHR) aortic rings, UK 14,304 induced concentration-dependent hyperpolarizing responses, which were inhibited by yohimbine, an alpha(2)-adrenoceptor inhibitor, and by glibenclamide, a specific inhibitor of small-conductance, ATP-sensitive K(+) channels. The responses were also partially inhibited by iberiotoxin and by apamin. Treatment with N(omega)-nitro-L-arginine (L-NNA) did not affect the response to UK 14,304. These results indicate that alpha(2)-adrenoceptors are present in SHR aortic smooth muscle cell membranes, but differ from those of normotensive animals regarding the K(+) channels involved in their responses. Moreover, the resting membrane potential (RMP) was significantly more negative in SHR than in normotensive rats. This relative hyperpolarized state is probably due to Ca(2+)-dependent K(+) channels being constitutively open in SHR, since the addition of iberiotoxin caused a significant depolarization of the aortic smooth muscle membranes in this strain.

Different mechanism of LPS-induced vasodilation in resistance and conductance arteries from SHR and normotensive rats.

1. The direct and endothelium-dependent effects of lipopolysaccharide (LPS) were investigated on resistance and conductance arteries from normotensive Wistar (NWR) and spontaneously hypertensive (SHR) rats. 2. In both NWR and SHR, LPS induced dose-dependent relaxations of the mesenteric vascular bed, which were inhibited by L-NNA in SHR but not in NWR. Iberiotoxin (IBTX) inhibited the responses to LPS in both groups, indicating the participation of high conductance Ca(2+)-dependent K(+) channels. 3. In mesenteric artery rings, the resting membrane potentials and the hyperpolarizing responses of NWR to LPS did not differ in endothelized and denuded preparations but L-NNA inhibited the responses only in endothelized rings. These responses were reduced by bosentan, suggesting that endothelin release may mask a possible hyperpolarizing response to LPS. The hyperpolarizing responses to LPS were blocked by IBTX in both endothelized and de-endothelized NWR rings. In the SHR only intact rings showed hyperpolarization to LPS, which was inhibited by IBTX and byL-NNA. 4. In SHR aortic endothelized or denuded rings, LPS induced hyperpolarizing responses which, in endothelized rings, were partially blocked by L-NNA, by IBTX or by glibenclamide, but totally abolished by IBTX plus glibenclamide. No response to LPS was observed in NWR aortic rings. 5. Our results indicate that LPS activates large conductance Ca(2+)-sensitive K(+) channels located in the smooth muscle cell membrane both directly and indirectly, through NO release from the endothelium in NWR, whereas NO is the major mediator of the LPS responses in SHR resistance vessels.