Animal surgery

13 male Sprague Dawley rats (170 g–211 g) were obtained from Charles River Laboratories and were allowed to acclimatize in animal care for 1 week before use. Rats were fed Teklad 2018 (Envigo, Indianapolis, IND, United States) standard rodent chow and allowed water ad libitum. All animal protocols were approved by Memorial University Animal Care Committee.

On the day of testing, animals were anesthetized with a 65 mg/kg intraperitoneal injection of sodium pentobarbital (Euthanyl, Bimeda, Cambridge, ON, Canada). Following induction, depth of anesthesia was assessed with palpebral reflex and toe pinch prior to the start of surgery to verify the animal was sufficiently anesthetized. Once in the surgical plane, a rectal temperature probe was inserted to monitor the body temperature of the animal throughout the experiment. Physiological temperatures were maintained between 36–37°C using a heated mat and/or heat lamp as necessary.

The common carotid artery was cannulated to allow continuous monitoring of blood pressure and heart rate (400a Blood Pressure Analyzer, Micro-Med, Louisville, KY, United States). The right jugular vein was cannulated to provide fluid resuscitation (0.5 ml/kg/hr) and maintenance anaesthetic as required. The animal’s heart rate and blood pressure were monitored continuously for variability as well as regular testing the palpebral reflexes and toe pinch to ensure acceptable depth of anaesthesia. Maintenance doses of sodium pentobarbital (22 mg/kg) were administered intravenously when the animal’s mean arterial pressure exceeded 110 mmHg or if the animal responded to adverse stimuli. Animals were tracheotomized and mechanically ventilated (Inspira ASV, Harvard Apparatus, Holliston, MA, United States) with an initial gas mixture of ∼30% O₂ and 70% N₂. Respiratory rates and volumes were automatically determined by the ventilator’s built in software based on the animal’s weight. The right extensor digitorum longus (EDL), a muscle of the lower hind limb, was blunt dissected and isolated as previously described (Tyml and Budreau, 1991; Fraser et al., 2012). The distal tendon was cut, the muscle was lifted and cleared from the remaining tissue without damaging the feed artery and vein. The EDL muscle was reflected over the gas exchange chamber on the stage of an inverted microscope (IX73, Olympus, Tokyo, Japan). The EDL was fixed under slight tension at approximately in situ length, covered with a polyvinylidene chloride film, bathed in warm saline, and gently compressed with a glass coverslip and microscope slide to isolate the EDL from room air, and to aid in establishing a uniform optical interface that is orthogonal to the incident light path. The animal was allowed to acclimatize on the stage for 30 min following positioning. Following the acclimatization, and with the animal’s body temperature between 36–37°C, an arterial blood sample was collected to measure blood gases (VetScan iSTAT, Abbott Point of Care Inc. Princeton, NJ, United States). Arterial PCO₂ and PO₂ were maintained within normal physiological range by adjusting ventilation rate and volume as needed prior to data collection.

The microscopy imaging setup was composed of an Olympus IX73 microscope (Olympus, Tokyo, Japan) fitted for transillumination with a 300 W Xenon light source (Lambda LS-30, Sutter Instruments, Novato, CA, United States). A parfocal beam splitter (Optosplit II Bypass, Cairn Research Ltd. Faversham, United Kingdom) directed light through 420 nm (isosbestic wavelength) and 438 nm (oxygen-sensitive) bandpass filters. Simultaneous and parfocal capture of video sequences were recorded for both wavelengths, with each wavelength on separate halves of the camera chip. Video recordings were made at 16 bit depth 2048 × 2048 resolution using a 10× objective (NA 0.40, Olympus, Tokyo, Japan) using an Orca Flash 4.0 v3 scientific digital camera (Hamamatsu, Hamamatsu City, Japan) and controlled by HCImage Live software (Hamamatsu, Hamamatsu City, Japan) on a desktop computer.