Blood Pressure Measurement

A subset of each group was subjected to blood pressure measurement during their treatment period using tail plethysmography (Kent Scientific, CT, United States). DOCA-salt, DOCA-salt+OX1R-shRNA, and control rats were all acclimated to the procedure for a week before measurements began through everyday blood pressure measurements, as recommended by the American Heart Association (Kurtz et al., 2005). Briefly, rats were placed in a plastic cylinder with a dark nose-cone, to reduce vision and anxiety. After 10 min of acclimation, the tail cuff apparatus including the occlusion cuff and volume pressure recording cuff were placed on the animal for an additional 5–10 min. Artificial heating was also applied to maintain adequate blood flow to the tail. Following this, blood pressure recording began, and 10 acclimation cycles followed by 20 measurement cycles were conducted. Following blood pressure recording, averages of the 20 measurement cycles were taken and paired with their group to obtain a group mean, which was utilized for statistical analysis. Rats maintained acclimation during the 18-day treatment by daily 20-min sessions in the rat holder/tail cuff apparatus for every rat, to reduce variability and anxiety among the rats.

Tail-cuff plethysmography can be attributed to stress-related fluctuations in blood pressure, due to confinement in a small cylinder for extended periods of time, which may lead to variability of results. However, all necessary precautions were taken to mitigate this effect. Namely, acclimation to the cylinders as well as the occlusion/volume pressure recording apparatus began 2 weeks prior to any actual blood pressure recording was performed. Following 1 week of acclimation, blood pressure measurements were taken to identify when the blood pressure reached a normotensive level in all treatment groups. Once the blood pressure appeared to maintain a consistent, normotensive pressure, the experiment was initiated. In addition, blood pressure recording sessions were done during the same time every day (12:00–4:00 pm), to mitigate circadian influences on orexin system function, and thus to alleviate any fluctuations in blood pressure that may naturally occur with differential orexin system activity throughout the day. Furthermore, during the 3 weeks of blood pressure recording, acclimation was maintained on days when blood pressure measurements were not taken by daily exposure to both confinements in the cylinder as well as attachment of the occlusion/volume pressure recording apparatus for 20 min.