The effects of magnesium (Mg) on ischaemic complications of pathological cardiac hypertrophy are unclear. In this study, we investigated effects of Mg pretreatment on ischaemia/reperfusion (I/R) injury in isoprenaline (ISO)-induced hypertrophic hearts. Wistar rats were treated for 7 days with different combinations of ISO (1.25 mg/kg) subcutaneously, MgSO (270 mg/kg) intraperitoneally, or vehicle (saline). On the eighth day, hearts were either subjected to regional I/R during Langendorff perfusion or histologically stained with haematoxylin and eosin and Masson's trichrome. Haemodynamic and electrocardiographic parameters were recorded using the PowerLab data-acquisition system. Infarcts were identified by triphenyltetrazolium chloride staining. Plasma Mg was measured using photometric assays. Mg pretreatment significantly decreased I/R-induced infarct size (p = 0.001) and the overall arrhythmia score (p < 0.001) of I/R-induced ventricular ectopics, ventricular tachycardia, and ventricular fibrillation in hypertrophic hearts, but not non-hypertrophied hearts. Mg also improved post-I/R left ventricular developed pressure in hypertrophic hearts. However, Mg did not reverse the ISO-induced myocyte thickening and interstitial fibrosis or increases in heart weight. Plasma Mg was not different among treatment groups. These results suggest that Mg pretreatment may protect against I/R-induced injury and malignant arrhythmias in hypertrophic hearts, possibly via mechanisms unrelated to long-lasting changes in plasma Mg or prevention of structural changes such as fibrosis.
Immunolabeling of tissue sections requires careful optimization of protocols in order to achieve accurate and consistent data. Multiple immunolabeling is desirable when determining the exact location and phenotype of cell populations in the same cellular compartment. 5-bromodeoxyuridine (BrdU)-immunolabeling is commonly used to assess cellular proliferation in vitro. However, the technical limitations of standard methods preclude multiple antigen immunolabeling. The aim was therefore to develop a robust protocol for simultaneous labeling using anti-BrdU and a melanocyte-specific marker in formalin-fixed paraffin-embedded (FFPE) skin samples. Human skin samples were obtained from patients undergoing elective plastic surgery. The tissue was incubated with BrdU, and a standard sample procedure for FFPE tissue was used. Heat-induced antigen retrieval was performed in a conventional pressure cooker, followed by immunolabeling with anti-BrdU and anti-Melan A/MART-1 antibodies. Fluorescent-conjugated secondary antibodies were used for signal detection. We have demonstrated both proliferating cells (BrdU-immunopositive) and melanocytes (Melan A/MART-1-immunopositive) in the basal compartment of the epidermis in our skin samples. Successful double labeling requires heat-induced epitope retrieval to replace the harsh pretreatment protocols of standard BrdU immunolabeling methods. We have optimized a robust protocol for the double labeling of proliferating cells and cells bearing melanocyte-specific antigens (melanocytes and/or melanoblasts) in FFPE human skin samples.
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