The liver injury following ischemia and reperfusion is worse in experimental knockout heterozygote mouse model for expression of connexin 43

Abstract Purpose: To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury. Methods: We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology. Results: The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group. Conclusions: This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.

Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring / Restrição Proteica Gestacional Aumenta Níveis de RNAm de Conexina 43 no Coração de Filhotes Machos Adultos

Abstract Background: The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. Objectives: The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Methods: Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. Results: LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. Conclusion: GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium.

Resumo Fundamento: A limitação dietética durante a gravidez influencia o crescimento e desenvolvimento do feto e da prole e sua saúde na vida adulta. Os mecanismos subjacentes dos efeitos adversos da restrição proteica gestacional (RPG) no desenvolvimento dos corações da prole não são bem compreendidos. Objetivos: Avaliar os efeitos da RPG sobre a estrutura cardíaca em filhotes machos de ratas aos 60 dias após o nascimento (d60). Métodos: Ratos fêmeas Wistar grávidas foram alimentadas com uma dieta de proteína normal (PN, 17% caseína) ou de baixa proteína (BP, caseína 6%). Os valores de pressão arterial (PA) de descendentes do sexo masculino de 60 dias de idade foram medidos por meio de um método indireto de manguito de cauda usando um eletro esfigmomanômetro. Os corações (d60) foram coletados para avaliação da expressão de RNAm da conexina 43 (Cx43) e análise morfológica e morfométrica. Resultados: A prole BP não mostrou diferença no peso corporal, embora tenha nascido mais leve do que a prole PN. Os níveis de PA foram significativamente mais altos no grupo BP. Observou-se um aumento significativo na área ocupada pelas fibras colágenas, diminuição do número de cardiomiócitos em 104 µm2 e aumento da área de cardiomiócitos associada ao aumento da expressão de Cx43. Conclusão: A RPG altera os níveis miocárdicos de RNAm de Cx43 em ratos adultos jovens, sugerindo que este mecanismo visa compensar o processo fibrótico pelo acúmulo de fibras de colágeno no interstício cardíaco.

Influence of feeder layer on the expression of stem cell markers in cultured limbal corneal epithelial cells.

BACKGROUND & OBJECTIVE: The limbus is enriched with the stem cells of corneal epithelium. Auto- and allograft limbal transplantations are effective in restoring the corneal epithelium and inhibiting inflammation and neovascularization. Preserved human amniotic membrane (AM) is now widely used as a substrate for ocular surface reconstruction. The combination of limbal and AM transplantation has been shown to improve the surgical outcome in patients with total limbal stem cell deficiency (LSCD). The purpose of this study was to compare the expression of putative stem cell markers ATP binding cassette protein (ABCG2) and keratinocyte stem cell marker: p63 and differentiation markers. (connexin 43 and keratin 3 / keratin 12) on the limbal epithelial cells cultured over the denuded AM with and without the 3T3 murine fibroblast cells as feeder layer. METHODS: Human limbal tissues obtained from the cadaveric donor eyes were cultured over the denuded human amniotic membrane in the presence of mitomycin C treated 3T3 fibroblasts and the cultured cells studied for the expression of ABCG2 and p63 by immunohistochemistry and Western blot. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was done on the cultured cells at varying intervals of time for expression of ABCG2, p63, connexin43 (Cnx43), and keratin 3 (K3) and keratin 12 (K12). RESULTS: The growth rates were similar in both denuded AM and denuded AM + 3T3. The cells cultured over AM + 3T3 showed the expression of p63 and ABCG2 till 21 days of incubation by immunohistochemistry and Western blot. The expression of p63 and ABCG2 were retained till 21 days of incubation on the cells cultured over denuded AM + 3T3, whereas it was expressed only till day 8 on the cells cultured over the denuded membrane by semi quantitative RT-PCR. Cnx43 and K3/K12 were observed in both the conditions. INTERPRETATION & CONCLUSION: The limbal epithelial cells cultured in the presence of mitomycin C treated 3T3 feeder layer were able to maintain the expression of putative stem cell markers. Further in vitro studies using feeder layer will enable us to understand the factors, which play a role in maintaining the limbal stem cell niche.