Measurements of plasma levels of leptin were performed at room air (21% O2) in anesthetized rats. After general anesthesia as described above, whole blood samples were taken through a cardiac puncture. Blood samples were drawn into collection tubes containing the anticoagulant EDTA (Sigma-Aldrich, USA) and kept on ice. After centrifugation, the plasma was stored at−80°C for leptin analysis by ELISA kit (#ab100773, Abcam, USA), an in vitro enzyme-linked immunosorbent assay for the quantitative measurement as previously described (Panetta et al., 2017 (link)). The assay was read using a power wave XS2 plate reader (Biotek Instruments, USA).
To confirm whether the chronic activation of leptin signaling pathways played a part in the HVR, subcutaneous injections of leptin (60 μg/kg) or equal volume of vehicle (saline) were carried out once daily for 7 days in CBI LZRs (n = 8 for each group), and breathing parameters were measured after 7 day injections during exposure to room air or hypoxia. To further confirm the CB's role, subcutaneous injections of leptin or saline were performed 7 days after the carotid sinus nerves were sectioned in each group (n = 8 for both).
To confirm whether the chronic activation of leptin signaling pathways played a part in the HVR, subcutaneous injections of leptin (60 μg/kg) or equal volume of vehicle (saline) were carried out once daily for 7 days in CBI LZRs (n = 8 for each group), and breathing parameters were measured after 7 day injections during exposure to room air or hypoxia. To further confirm the CB's role, subcutaneous injections of leptin or saline were performed 7 days after the carotid sinus nerves were sectioned in each group (n = 8 for both).
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