Thermalert th 5

Ventilatory responses to hypoxia and hypercapnia were measured in unrestrained mice using a whole body barometric plethysmograph (500 ml) modified for continuous flow (Jacky, 1978; Pamenter et al. 2014). Flow was maintained through the chamber while a pressure transducer (mp45 with 2 cm H₂O diaphragm, Validyne, Northridge CA) recorded the changes due to the warming and expansion of inhaled gases. On the experimental day, the mice were weighed and sealed into the plethysmograph chamber along with a temperature and humidity probe (Thermalert TH5, Physitemp, Clifton NJ). A constant gas flow (335 ml/min) was delivered with a rotameter (603, Matheson, Montgomeryville PA) and measured with a flow meter (S-110, McMillan Co., Georgetown TX) upstream of the chamber. Gases exited the chamber through a valve and into a vacuum pump (Model 25, Precision Scientific Co, Chicago IL) to isolate pressure changes from breathing in the chamber during constant flow with high input and output impedances. This also allowed us to maintain chamber pressure near-atmospheric pressure and reference pressure measurements (Validyne, Northridge CA) in the chamber to atmosphere. Inspired and expired O₂ and carbon dioxide (CO₂) fractions were measured in a mass spectrometer (MGA1100, Marquette Electronics, Milwaukee WI) sampling from the chamber.

Ten adult male Sprague-Dawley rats weighing 571 ± 33 g were studied. All experiments were conducted in accordance with the Guideline for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85–23, revised 1996). The study was approved by the Pennsylvania State University College of Medicine Institutional Care and Use Committee. Anesthesia was initiated with 3.5% isoflurane in O₂ followed by a unique injection of urethane (1.2 g/kg, intraperitoneal). Then, the rats were tracheostomized (14G surflo catheter, Terumo, NJ) as previously described (Haouzi and Van de Louw, 2013 (link)). A catheter was inserted into the right femoral artery for systemic blood pressure monitoring and arterial blood sampling (PE-50 tubing) while an additional catheter was inserted into the right femoral vein for infusion of H₂S. Body temperature was monitored with a rectal probe (Thermalert TH-5, Physitemp, Clifton NJ) and maintained at around 36.5°C with a heating pad and lamp. At the end of the experiment, rats were euthanized with CO₂ followed by loading dose of pentobarbital (200 mg/kg) and aortic dissection.

Mouse pup body weight was measured using an analytical balance (Mettler Toledo PB303-S, Columbus, OH). Rectal thermometry (Thermalert TH-5, Physitemp Instruments, Clifton, NJ) was utilized to determine body temperature. Temperature measurements were performed between 9:00 am and 11:00 am in a room maintained at approximately 20°C. All measurements were obtained in awake, hand-restrained mice.