Phrenic Nerve

AC-coupled recordings were made of the cord dorsum potentials for incoming afferent volleys and the electroneurograms (ENGs: the phrenic nerve; the external intercostal nerve in one cat; when appropriate, see below, the dissected hindlimb nerves as listed above). Intracellular recordings were DC-coupled, but a high gain output channel high pass filtered at 1 Hz was also included. Intracellular recordings were made from antidromically identified motoneurons, using an Axoclamp 2B amplifier (Axon Instruments) in either standard bridge mode, or in discontinuous current clamp (DCC) mode. Microelectrodes (typical impedance 5 MΩ) were filled with 2 M potassium acetate, and contained the local anesthetic derivative QX-314 (50 mm) to block actions potentials, so as to facilitate the study of the size of EPSPs at different membrane potentials. Note that in several of the records illustrated, a few action potentials survived, showing the QX-314 block to be incomplete at those times. DCC mode was used to allow for more accurate measurements of membrane potential despite changes in electrode resistance with injected current. The DCC cycling rate was typically around 3 kHz with optimal capacitance compensation. Most often slow depolarizing and hyperpolarizing ramps of currents were used (triangular current ramps), but some step changes of constant current levels were also employed. During many of the motoneuron recordings we also recorded efferent discharges from the hindlimb nerves via the same electrodes as used for antidromic identification purposes. This was rarely done in the early experiments, where the focus was on the voltage-dependent amplification of synaptic potentials, but once it was realized that a locomotor drive was sometimes present in the recordings, then these electrodes were switched to their recording mode as soon as antidromic identification had been confirmed. The ENG recordings were done with custom built amplifiers and analog filtering (1–10 kHz) and digitized at a rate of 10 kHz. Full wave rectification and additional filtering was done during analysis so that the onset and the offset of ENG bursts in each nerve were identified by visual inspection of ENG levels crossing a baseline defined by no activity periods. These onset and offset points were used during cycle-based averaging of ENG activity. The data were collected and analyzed with a Canadian software-based QNX-system, developed by the Winnipeg Spinal Cord Research Center to run under a real-time Unix personal computer, usually using separate runs of 200 s duration.

Patients admitted over a period of one year (from May 2013 to May 2014) to the ICU of the University of Bari Academic Hospital were considered for enrollment in the study. The local ethics committee (Azienda Ospedaliero-Universitaria Policlinico di Bari Ethic Committee, protocol number: 257/C.E. March 2013) approved the investigative protocol, and written informed consent was obtained from each patient or next of kin. A physician not involved in the study was always present for patient care. Our clinical trial was registered with clinicalTrials.gov, identifier: NCT02473172.
Patients were eligible for the study if they were older than 18 years, oro-tracheally or naso-tracheally intubated, had been ventilated for acute respiratory failure with CMV (flow-limited, pressure-limited or volume-targeted pressure-limited) for at least 72 hours consecutively and were candidates for assisted ventilation. The criteria for defining the readiness to assisted ventilation were: a) improvement of the condition leading to acute respiratory failure; b) positive end-expiratory pressure (PEEP) lower than 10 cmH₂O and inspiratory oxygen fraction (FiO₂) lower than 0,5; c) Richmond agitation sedation scale (RASS) score between 0 and –1 [23 (link)] obtained with no or moderate levels of sedation and, d) ability to trigger the ventilator, i.e., to decrease pressure airway opening (P_AO) >3–4 cmH₂O during a brief (5–10 s) end-expiratory occlusion test. Other criteria included hemodynamic stability without vasopressor or inotropes (excluding a dobutamine and dopamine infusion <5 gamma/Kg/min and 3 gamma/Kg/min, respectively) and normothermia. Patients were excluded from the study if they were affected by neurological or neuromuscular pathology and/or known phrenic nerve dysfunction, or if they had any contraindication to the insertion of a nasogastric tube (for example: recent upper gastrointestinal surgery, esophageal varices).

Electrophysiology was performed as previously described (Vagnozzi et al., 2020 (link)). Mice were cryoanesthetized and rapid dissection was carried out in 22–26°C oxygenated Ringer’s solution. The solution was composed of 128 mM NaCl, 4 mM KCl, 21 mM NaHCO₃, 0.5 mM NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, and 30 mM D-glucose and was equilibrated by bubbling in 95% O₂/5% CO₂. The hindbrain and spinal cord were exposed by ventral laminectomy, and phrenic nerves exposed and dissected free of connective tissue. A transection at the pontomedullary boundary rostral to the anterior inferior cerebellar artery was used to initiate fictive inspiration. Electrophysiology was performed under continuous perfusion of oxygenated Ringer’s solution from rostral to caudal. Suction electrodes were attached to phrenic nerves just proximal to their arrival at the diaphragm. The signal was band-pass filtered from 10 Hz to 3 kHz using AM-Systems amplifiers (Model 3000), amplified 5,000-fold, and sampled at a rate of 50 kHz with a Digidata 1440A (Molecular Devices). Data were recorded using AxoScope software (Molecular Devices) and analyzed in Spike2 (Cambridge Electronic Design). Burst duration and burst activity were computed from 4 to 5 bursts per mouse, while burst frequency was determined from 10 or more minutes of recording time per mouse. Burst activity was computed by rectifying and integrating the traces with an integration time equal to 2 s, long enough to encompass the entire burst. The maximum amplitude of the rectified and integrated signal was then measured and reported as the total burst activity.

For labeling of phrenic MNs, crystals of carbocyanine dye, DiI (Invitrogen, #D3911) were pressed onto the phrenic nerves of eviscerated embryos at e18.5, and the embryos were incubated in 4% PFA at 37°C in the dark for 4–5 weeks. Spinal cords were then dissected, embedded in 4% low melting point agarose (Invitrogen) and sectioned using a Leica VT1000S vibratome at 100–150 μm.