Pancuronium Bromide

The acute electrophysiological experiment was run during the third week after induction of the model. Approaches to the animal preparation and intracellular recording techniques have been reported previously [61 (link),62 (link)]. In brief, each animal was initially anesthetized with ketamine mixture described above. The right jugular vein was catheterized for i.v. infusion of drugs and a cannula was inserted into the trachea. The rat was then fixed in a stereotaxic frame and the vertebral column rigidly clamped at the L2 and L6 vertebrae. The right femur was fixed by a customized clamp onto the stereotaxic frame to minimize movement of the DRG during mechanical searching for receptive fields on the leg. The L4 DRG was selected for study as it contains one of the largest numbers of the hind leg afferent somata. A laminectomy was performed to expose the ipsilateral L4 DRG. The L4 dorsal root was sectioned close to the spinal cord, allowing a 12-15 mm length, and the proximal end was placed on a bipolar electrode (FHC, Bowdoinham, ME, USA) used for stimulation purposes. The exposed spinal cord and DRG were covered with warm paraffin oil at 37°C to prevent drying.
The rat was mechanically ventilated via the tracheal cannula using a Harvard Ventilator (Model 683, Harvard apparatus, Quebec, Canada). The ventilation parameters were adjusted so that end-tidal CO₂ concentration was maintained around 40-50 mmHg, as measured using a CapStar-100 End-Tidal CO₂ analyzer (CWE, Ardmore, PA, USA). Rectal temperature was maintained at ~37°C using a temperature controlled infrared heating lamp. Immediately before the start of recording, the animal was given 20 mg/kg of Na pentobarbital (CEVA SANTE ANIMAL, Libourne, France), and supplemental doses of 10 mg/kg of pentobarbital were given each hour through the jugular catheter to maintain a surgical level of anesthesia. In addition, just before recording, each animal was also given an initial 1 mg/kg dose of pancuronium bromide (Pavulon, Sandoz, Boucherville, QC, Canada) to eliminate muscle tone. Supplemental doses of 1/3 the initial dose of pentobarbital and pancuronium were given about each hour via the jugular catheter.
Intracellular recordings from somata in the exposed DRG were made with borosilicate glass micropipettes (1.2 mm outside diameter, 0.68 mm inside diameter; Harvard Apparatus, Holliston MA, USA). The electrodes were pulled using a Brown-Flaming puller (model p-87; Sutter Instrument CO., Novota, CA, USA) and were filled with 3 M KCl (DC resistance 50-70 MΩ). Signals were recorded with a Multiclamp 700B amplifier (Molecular Devices, Union City CA, USA) and digitized on-line via Digidata 1322A interface (Molecular Devices, USA) with pClamp 9.2 software (Molecular Devices, USA). The microelectrode was advanced using an EXFO IW-800 micromanipulator (EXFO, Montreal, QC, Canada) in 2 μm steps until a hyperpolarization of at least 40 mV suddenly appeared. For any testing to proceed a continuous recording was obtained for at least five minutes after cell penetration; stable recordings were obtained for periods exceeding one hour. For each neuron, once a stable membrane potential had been confirmed a single stimulus was applied to the dorsal roots to provoke an AP; with the aid of the protocol editor function in pClamp 9.2 software, a somatic AP was evoked by stimulation with a single rectangular voltage pulse.

Electrophysiological experiments were conducted as described previously [25 (link)]. Briefly, rats were randomly divided into two groups (n = 10/group) and treated in the same manner as described in the mice experiments. Rats were anesthetized with urethane (1.2–1.4 g/kg, i.p.) on day 14 after the start of treatment. The level of anesthesia and body temperature were maintained with a supplemental dose of urethane and a heating pad, respectively. The left jugular vein was cannulated for the administration of drugs and fluids and the right carotid artery was cannulated for the measurement of blood pressure (BP). The trachea was cannulated to enable artificial ventilation and a 3-lead electrocardiogram was fitted. The vagus nerve was isolated, tied with a silk thread, and cut distally to permit the recording of efferent vagus nerve activity (VNA). Rats were secured in a stereotaxic frame, paralysed (pancuronium bromide; 0.8 mg initially, then 0.4 mg/h) and artificially ventilated with oxygen-enriched room air. Animals were kept hydrated by infusing 5% glucose intravenously. Bipolar silver wire electrodes were used for nerve recordings. The neurograms were amplified (×10,000), bandpass filtered (0.1–2 kHz), and sampled at 3 kHz. Recordings were made using the Spike2 software (v7.1, CED Ltd., Milton, Cambridge, UK).

All of the dogs were euthanized using an intravenous injection of 0.1 mg/kg pancuronium bromide and 0.1 M KCl at the end of the experiment (7 days post-inoculation). At necropsy, gross lesions were examined in the lungs, pharynx, lymph nodes, spleen, and kidneys, and then tissues were collected and fixed in 4% neutral-buffered formalin for 1 week. The tissues embedded in paraffin blocks were sectioned at a thickness of 4 μm and then mounted onto glass slides. The slides were deparaffinized in xylene and rehydrated through a series of graded 100% ethanol to distilled water and then stained with hematoxylin and eosin. For immunohistochemistry, to detect the SARS-CoV-2 antigen in the lung tissue slides, the deparaffinized and rehydrated slides were blocked for endogenous peroxidase with 3% H₂O₂ in phosphate-buffered saline (PBS) for 20 min. The tissue sections were placed in 10 mM citrate buffer (pH 6.0), heated for 1 h, and incubated with SARS Nucleocapsid Protein Antibody (NB100-56576, Novus Biologicals, Centennial, CO, USA) at a 1:200 dilution at 4 °C overnight. For MERS-CoV antigen detection, the tissue sections were digested with proteinase K (P2308, Merck, Darmstadt, Germany) for 30 min at 37 °C and incubated with rabbit polyclonal antiserum against MERS-CoV (Sino Biologicals Inc., Beijing, China) at a 1:1000 dilution at 4 °C overnight. All of the slides were then washed in PBS and incubated with a secondary antibody (RealTM EnvisionTM Detecion system rabbit/mouse, K5007, Dako, Glostrup, Denmark) for 40 min at 37 °C. Color development was performed using 3,3′-diamino-benzidine tetrahydrochloride (DAB; K5007, Dako, Glostrup, Denmark), followed by counterstaining with hematoxylin. Light microscopic examination was performed using a BX53 microscope (Olympus, Tokyo, Japan). In the lung sections, microscopic lesions were evaluated assessing the severity of interstitial, vasculitis and perivasculitis. For the staining of SARS-CoV-2 antigens, the immunohistochemistry assay was performed as described previously [9 (link)].