Immunohistomorphology of pancreatic islet microvasculature and the immunophenotypic analysis of CEPC in adult diabetic rats / Inmunohistomorfología de microvasculatura de islotes pancreatic y el análisis inmunofenotípico de CEPC en ratas diabéticas adultas

SUMMARY: Hyperglycaemia is one of the main causes for the endothelial cell (EC) damage in diabetic patients. Even though circulating endothelial progenitor cells (CEPC) could be used as a prognosis for microvascular complications, there is very little information on the islet microvasculature. We analysed by immunohistochemistry and by flow cytometric immunophenotyping, the expression of CD34 on EC and the expressions of CD31, CD34, CD45 and CD133 on CEPC in Streptozotocin (STZ)-induced diabetic rats. Peripheral blood and tissue specimens were obtained from rats of different treatment regimens: STZ treatment, control saline (NS) and sodium citrate (CB) treatments. Blood cells were exposed to flow cytometric immunophenotyping for CD133, CD31, CD34, CD45 and CD133. While tissues from the pancreas, liver and kidney were routinely processed and stained immunohistochemically for CD34. There was a tendency of an increased in CD45-/CD133+/CD31+/CD34+ cells (0.04 ± 0.11 %) in diabetic rats compared to the controls (CB: 0.03 ± 0.04 %; Saline: 0.01 ± 0.03 %). But there was no significant statistical difference between them. The expression pattern of CD34 on the EC in the organs' vascular beds including arterioles, venules, capillaries and sinusoids was extremely heterogeneous across and within treatment regimens. The ECs in the sinusoids of the liver presented similar CD34 expression patterns across different treatment regimens, while the expression of CD34 on the ECs of sinusoidal capillaries in the pancreas vary with the treatment regimen. We conclude that the degree of endothelial cell damage is not uniform across organs' vascular beds in the rat, contrary to mice and humans. Furthermore, the sinusoids in the pancreas and the kidney may have the same degree of endothelial damage when exposed to the same deleterious causes.

RESUMEN: La hiperglucemia es una de las principales causas del daño de las células endoteliales (EC) en pacientes diabéticos. A pesar de que las células progenitoras endoteliales circulantes (CEPC) podrían utilizarse como pronóstico de las complicaciones microvasculares, hay muy poca información sobre la microvasculatura de los islotes. Se analizaron por inmunohistoquímica y por inmunofenotipificación citométrica de flujo, la expresión de CD34 en EC y las expresiones de CD31, CD34, CD45 y CD133 en CEPC en ratas diabéticas inducidas por estreptozotocina (STZ). Se obtuvieron muestras de sangre y tejidos periféricos a partir de ratas de diferentes regímenes de tratamiento: tratamiento con STZ, solución salina control (NS) y citrato de sodio (CB). Las células sanguíneas fueron expuestas a inmunofenotipado por citometría de flujo para CD133, CD31, CD34, CD45 y CD133. Mientras que los tejidos del páncreas, el hígado y el riñón fueron rutinariamente procesados y teñidos inmunohistoquímicamente para CD34. Se observó una tendencia a un aumento en las células CD45- / CD133 + / CD31 + / CD34 + (0,04 ± 0,11 %) en ratas diabéticas en comparación con los controles (CB: 0,03 ± 0,04 %; Salino: 0,01 ± 0,03 %). Pero no hubo diferencias estadísticamente significativas entre ellos. El patrón de expresión de CD34 en la EC en los lechos vasculares de los órganos incluyendo arteriolas, vénulas, capilares y sinusoides fue extremadamente heterogéneo a través de y dentro de los regímenes de tratamiento. Las EC en los sinusoides del hígado presentaron patrones de expresión de CD34 similares a través de diferentes regímenes de tratamiento, mientras que la expresión de CD34 en las CE de capilares sinusoidales en el páncreas varía con el régimen de tratamiento. Concluimos que el grado de daño de las células endoteliales no es uniforme en los lechos vasculares de los órganos en la rata, en comparación de los ratones y los seres humanos. Además, los sinusoides en el páncreas y el riñón pueden tener el mismo grado de daño endotelial cuando se exponen a las mismas causas deletéreas.

Análise da expressão de citocinas e dos receptores RAGE no periodonto de ratos diabéticos / Cytokine and RAGE receptor expression analyses in periodontium of diabetic rats

As doenças periodontais (DPs) são alterações inflamatórias crônicas que acometem os tecidos de sustentação do órgão dental. A presença do diabetes é refletida em maior severidade e prevalência das DPs tanto em humanos quanto em modelos experimentais. Contudo, os mecanismos biológicos envolvidos no aumento da prevalência e da severidade permanecem pouco conhecidos. Desta forma, o objetivo deste estudo foi avaliar o número de células marcadas por imunohistoquímica para TNF- , IL-1 , IL-6, RANKL, MMP-2, MMP-9 e para os receptores RAGE na doença periodontal experimental decorrente da indução do diabetes em ratos. Inicialmente, os ratos (n=25) foram submetidos à indução do diabetes através de administração endovenosa de aloxana (42mg/kg) e, juntamente com o grupo controle (n=25), acompanhados por 1, 3, 6, 9 e 12 meses. Em seguida, as hemimandíbulas foram coletadas e submetidas aos procedimentos de imunohistoquímica. Os resultados revelam que a presença do diabetes resulta em alterações significativas no número de células imunomarcadas para diferentes mediadores do processo inflamatório. Nos animais diabéticos, foi observado aumento estatisticamente significativo (p<0,05 ANOVA) no número de células imunomarcadas para TNF- (6 e12 meses), IL-1 (12 meses), IL-6 (9 e 12 meses), RANKL (9 meses) e para os receptores RAGE (6, 9 e 12 meses). Não foram observadas diferenças em relação ao número de células imunomarcadas para MMP-2 e MMP-9 entre os grupos controle e experimental, apesar da tendência a aumento no número de células MMP-9+ nos ratos após 12 meses da indução do diabetes (p>0,05 ANOVA). Assim, a desregulação na expressão de citocinas inflamatórias e fatores osteoclastogênicos parece ser um dos mecanismos biológicos envolvidos no aumento da prevalência e da severidade das doenças periodontais em decorrência do diabetes.

Periodontal diseases (PD) are chronic inflammatory diseases leading the destruction of connective tissue and alveolar bone supporting the teeth. The establishment of diabetes increases PD prevalence and severity in humans and experimental model. However, biological mechanisms regarding to increase of prevalence and severity remains poorly known. The aim of this study was to evaluate the number of immuno-staining cells to TNF- IL-1 , IL-6, MMP-2, MMP-9, RANKL and RAGE receptors in experimental periodontal disease in diabetic rats. Diabetes was induced in Wistar rats (n=25) by endovenous administration of 42 mg/kg of alloxan, and together with control animals (n=25), were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The animals were sacrificed and the jaws were removed and submitted to immunohistochemistry procedures. Our data demonstrated that diabetes induction and progression resulted in significant alterations in number of immuno-staining cells to different mediators of inflammatory process. In diabetic rats, we observed an increased number of immuno-staining cells to TNF- (6 and 12 months), IL-1 (12 months), IL-6 (9 e 12 months), RANKL (9 months) and RAGE receptors (6, 9 and 12 months) (p<0,05 ANOVA). Regarding to MMP-2+ and MMP-9+ cells, we did not found differences between control and experimental groups. However, we found a trend of towards in MMP-9+ cells in diabetic rats after 12 months of diabetes induction (p>0,05 ANOVA). Then, our data demonstrated that diabetes establishment and progression resulted in an increase of immuno-staining cells to TNF- , IL-1 IL-6, RANKL and RAGE receptors. Taken together, desregulation of inflammatory cytokines and osteoclastogenic factor expression seems to be one of biological mechanisms involved in the increase of periodontal disease prevalence and severity associated with diabetes.

Análise da expressão de citocinas e dos receptores RAGE no periodonto de ratos diabéticos / Cytokine and RAGE receptor expression analyses in periodontium of diabetic rats

As doenças periodontais (DPs) são alterações inflamatórias crônicas que acometem os tecidos de sustentação do órgão dental. A presença do diabetes é refletida em maior severidade e prevalência das DPs tanto em humanos quanto em modelos experimentais. Contudo, os mecanismos biológicos envolvidos no aumento da prevalência e da severidade permanecem pouco conhecidos. Desta forma, o objetivo deste estudo foi avaliar o número de células marcadas por imunohistoquímica para TNF- , IL-1 , IL-6, RANKL, MMP-2, MMP-9 e para os receptores RAGE na doença periodontal experimental decorrente da indução do diabetes em ratos. Inicialmente, os ratos (n=25) foram submetidos à indução do diabetes através de administração endovenosa de aloxana (42mg/kg) e, juntamente com o grupo controle (n=25), acompanhados por 1, 3, 6, 9 e 12 meses. Em seguida, as hemimandíbulas foram coletadas e submetidas aos procedimentos de imunohistoquímica. Os resultados revelam que a presença do diabetes resulta em alterações significativas no número de células imunomarcadas para diferentes mediadores do processo inflamatório. Nos animais diabéticos, foi observado aumento estatisticamente significativo (p<0,05 ANOVA) no número de células imunomarcadas para TNF- (6 e12 meses), IL-1 (12 meses), IL-6 (9 e 12 meses), RANKL (9 meses) e para os receptores RAGE (6, 9 e 12 meses). Não foram observadas diferenças em relação ao número de células imunomarcadas para MMP-2 e MMP-9 entre os grupos controle e experimental, apesar da tendência a aumento no número de células MMP-9+ nos ratos após 12 meses da indução do diabetes (p>0,05 ANOVA). Assim, a desregulação na expressão de citocinas inflamatórias e fatores osteoclastogênicos parece ser um dos mecanismos biológicos envolvidos no aumento da prevalência e da severidade das doenças periodontais em decorrência do diabetes.

Periodontal diseases (PD) are chronic inflammatory diseases leading the destruction of connective tissue and alveolar bone supporting the teeth. The establishment of diabetes increases PD prevalence and severity in humans and experimental model. However, biological mechanisms regarding to increase of prevalence and severity remains poorly known. The aim of this study was to evaluate the number of immuno-staining cells to TNF- IL-1 , IL-6, MMP-2, MMP-9, RANKL and RAGE receptors in experimental periodontal disease in diabetic rats. Diabetes was induced in Wistar rats (n=25) by endovenous administration of 42 mg/kg of alloxan, and together with control animals (n=25), were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The animals were sacrificed and the jaws were removed and submitted to immunohistochemistry procedures. Our data demonstrated that diabetes induction and progression resulted in significant alterations in number of immuno-staining cells to different mediators of inflammatory process. In diabetic rats, we observed an increased number of immuno-staining cells to TNF- (6 and 12 months), IL-1 (12 months), IL-6 (9 e 12 months), RANKL (9 months) and RAGE receptors (6, 9 and 12 months) (p<0,05 ANOVA). Regarding to MMP-2+ and MMP-9+ cells, we did not found differences between control and experimental groups. However, we found a trend of towards in MMP-9+ cells in diabetic rats after 12 months of diabetes induction (p>0,05 ANOVA). Then, our data demonstrated that diabetes establishment and progression resulted in an increase of immuno-staining cells to TNF- , IL-1 IL-6, RANKL and RAGE receptors. Taken together, desregulation of inflammatory cytokines and osteoclastogenic factor expression seems to be one of biological mechanisms involved in the increase of periodontal disease prevalence and severity associated with diabetes.

Glutamine or whey-protein supplementation on alloxan-induced diabetic rats. Effects on CD4+ and CD8+ lymphocytes

PURPOSE: To evaluate the effects of glutamine (L-Gln) or whey-protein supplementation on CD4+ and CD8+ lymphocytes in alloxan-induced diabetic rats. METHODS: Thirty-two healthy male Wistar rats were used in the experiment. Eight rats served as baseline controls (G-1). The remaining 24 animals received alloxan 150mg/Kg intraperitonially dissolved in buffer solution and were equally distributed in 3 subgroups, upon induction of diabetes mellitus, and treated as follows: (G2): saline, 2.0ml; (G3): glutamine solution (0.7g/kg), 2.0 ml; and (G4): whey-protein (WPS) solution (0.7g/kg), 2.0 ml. All solutions were administered by daily 7:00 AM gavages during 30 days. Next, arterial blood samples (3.0 ml) were collected from anesthetized rats for CD4+ and CD8+ lymphocyte count through flow cytometry technology. RESULTS: CD4+ and CD8+ counts decreased significantly in all groups compared with baseline values (G1). G2 rats CD4+/CD8+ ratio decreased significantly compared with G1. CD4+/CD8+ ratio increased significantly (>260 percent) in L-Gln treated group (G3) compared with saline-treated rats (G2). There were no statistical differences in lymphocyte counts (CD4+ and CD8+) between L-Gln (G3) and saline-treated (G2) groups. There was a significant reduction in CD8+ cell count compared with CD4+ cell count in L-Gln treated rats (G3). CONCLUSION: The offer of L-Gln to experimental diabetic rats enhances the immunologic response to infection.

OBJETIVO: Avaliar os efeitos da suplementação de glutamina ou proteína do soro de leite ( PSL) sobre os linfócitos CD4+ e CD8+ em ratos diabéticos aloxano induzidos. MÉTODOS: Trinta e dois ratos Wistar machos, saudáveis, foram utilizados no estudo. Oito ratos foram usados como controles basais (G1). Os 24 animais remanescentes foram equitativamente distribuídos em 3 subgrupos, após indução do diabetes mellitus por injeção intraperitonial de aloxano (150mg/Kg) e tratados como se segue: (G2): salina; (G3): 2,0 ml de solução de glutamina (0,75g/Kg);(G4): PSL, (0,7g/Kg), 2,0ml. Todas as soluções foram administradas por gavagem, diariamente, cada 7:00 h, durante 30 dias. Após esse período, foram coletadas amostradas de sangue arterial para contagem de linfócitos CD4+ e CD8+ por citometria de fluxo. RESULTADOS: A população de linfócitos CD4+ e CD8+ diminuiu significantemente em todos os grupos em comparação aos valores encontrados no grupo G1. A razão CD4+/CD8+ foi significantemente maior (>260 por cento) nos ratos tratados com L-Gln que nos ratos tratados com salina (G2). Não se observaram diferenças significantes na população de linfócitos CD4+ e CD8+ entre os grupos G3 e G2. Houve redução significante do número de células CD8+ quando comparado ao número de células CD4+ nos ratos tratados com L-Gln (G3). CONCLUSÃO: A oferta de L-Gln em ratos diabéticos aloxano-induzidos melhora a resposta imunológica à infecção.

Sleep deprivation reduces the lymphocyte count in a non-obese mouse model of type 1 diabetes mellitus

The objective of the present study was to determine whether sleep deprivation (SD) would promote changes in lymphocyte numbers in a type 1 diabetes model (non-obese diabetic, NOD, mouse strain) and to determine whether SD would affect female and male NOD compared to Swiss mice. The number of lymphocytes in peripheral blood after 24 and 96 h of SD (by multiple platform method) or equivalent period of time in home-cage controls was examined prior to the onset of diabetes. SD for 96 h significantly reduced lymphocytes in male Swiss mice compared to control (8.6 ± 2.1 vs 4.1 ± 0.7 10³/æL; P < 0.02). In male NOD animals, 24- and 96-h SD caused a significant decrease of lymphocytes compared to control (4.4 ± 0.3 vs 1.6 ± 0.5; P < 0.001 and 4.4 ± 0.3 vs 0.9 ± 0.1 10³/æL; P < 0.00001, respectively). Both 24- and 96-h SD induced a reduction in the number of lymphocytes in female Swiss (7.5 ± 0.5 vs 4.5 ± 0.5, 4.4 ± 0.6 10³/æL; P < 0.001, respectively) and NOD mice (4 ± 0.6 vs 1.8 ± 0.2, 1.2 ± 0.4 10³/æL; P < 0.01, respectively) compared to the respective controls. Loss of sleep induced lymphopenia in peripheral blood in both genders and strains used. Since many cases of autoimmunity present reduced numbers of lymphocytes and, in this study, it was more evident in the NOD strain, our results suggest that SD should be considered a risk factor in the onset of autoimmune disorders.

Evaluation of the natural killer cytotoxicity and the levels of cytokines in rats with type I diabetes mellitus

Type I diabetes mellitus (insulin-dependent DM = IDDM) is a chronic disease characterized by specific destruction of pancreatic beta cells, resulting in an absolute lack of insulin. Immune mechanisms, genetic susceptibility, and environmental factors are all implicated in the pathogenesis of Type 1 diabetes. This study was aimed at determining the efficiency of cytokines, natural killer (NK) cells in the pathophysiology of IDDM. Therefore, we evaluated the plasma levels of cytokines by specific enzyme-linked immunosorbent assay (ELISA) and the cytotoxicity activity of NK cells by anti-candididal index in rats with type I diabetes. We found that the cytotoxicity activity of NK cells in IDDM groups significantly decreased compared to the control groups. The levels of interferon-g (IFN-g) in IDDM groups were slightly higher than in healthy controls. These results indicate that the changes of T H1 type cytokines such as IFN-g and NK cell activity can play a role in the etiology of IDDM. The data may provide new strategies for the treatment of IDDM.

Mast cell changes in experimental diabetes: focus on attenuation of allergic events

The prevalence of atopic diseases and diabetes is increasing worldwide though the concurrence of these pathologies in individual patients is found less frequent than it would be predicted. Moreover, co-existence of diabetes and allergy is generally marked by attenuation of their respective symptoms, and effective treatment of one disease exacerbates the other. This review gives an update of the state-of-the-art concerning the intercurrence of allergy and diabetes, particularly focusing on the consequences to the allergen-evoked vascular and cellular changes. It is proposed that the reduction in mast cell numbers and reactivity may be a pivotal mechanism behind the mutual exclusion phenomenon.

Effects of cyclosporin A immunosuppression on pancreaticoduodenal transplantation in alloxan-diabetic rats

1. Forty-five outbred Wistar rats were randomly assigned to three experimental groups: GI, 10 non-diabetic control rats; GII, 10 alloxan-diabetic control rats; GIII, 25 alloxan-diabetic rats which received pancreaticoduodenal transplantation (PDT) from normal Wistar donor rats and were immunosuppressed with cyclosporin A (Cy-A), 10 mg kg body weight-1 day-1, administered intraperitoneally for 30 days. 2. In parallel, 15 alloxan-diabetic inbred Wistar rats received isogeneic PDT from normal Wistar donor rats. 3. Cy-A prevented graft rejection in the 15 surviving animals in group III. These observations were confirmed by clinical and biochemical parameters (body weight, urine output, water and food intake, blood and urinary glucose and plasma insulin) and by histology and immunohistochemistry of the pancreas. 4. However, Cy-A was associated with 60% of the infectious complications in transplanted rats leading to 40% mortality. Pulmonary infections were the main cause of death. There were no side effects of immunosuppression on the pancreas. Infections were not significant in inbred rats submitted to PDT