Paced breathing and phrenic nerve responses evoked by epidural stimulation following complete high cervical spinal cord injury in rats

Spinal cord injury (SCI) at the level of cervical segments often results in life-threatening respiratory complications and requires long-term mechanical ventilator assistance. Thus restoring diaphragm activity and regaining voluntary control of breathing are the primary clinical goals for patients with respiratory dysfunction following cervical SCI. Epidural stimulation (EDS) is a promising strategy that has been explored extensively for nonrespiratory functions and to a limited extent within the respiratory system. The goal of the present study is to assess the potential for EDS at the location of the phrenic nucleus (C3–C5) innervating the diaphragm: the main inspiratory muscle following complete C1 cervical transection. To avoid the suppressive effect of anesthesia, all experiments were performed in decerebrate, C1 cervical transection, unanesthetized, nonparalyzed ( n = 13) and paralyzed ( n = 7) animals. Our results show that C4 segment was the most responsive to EDS and required the lowest threshold of current intensity, affecting tracheal pressure and phrenic nerve responses. High-frequency (200–300 Hz) EDS applied over C4 segment (C4-EDS) was able to maintain breathing with normal end-tidal CO2 level and raise blood pressure. In addition, 100–300 Hz of C4-EDS showed time- and frequency-dependent changes (short-term facilitation) of evoked phrenic nerve responses that may serve as a target mechanism for pacing of phrenic motor circuits. The present work provides the first report of successful EDS at the level of phrenic nucleus in a complete SCI animal model and offers insight into the potential therapeutic application in patients with high cervical SCI. NEW & NOTEWORTHY The present work offers the first demonstration of successful life-supporting breathing paced by epidural stimulation (EDS) at the level of the phrenic nucleus, following a complete spinal cord injury in unanesthetized, decerebrate rats. Moreover, our experiments showed time- and frequency-dependent changes of evoked phrenic nerve activity during EDS that may serve as a target mechanism for pacing spinal phrenic motor networks.

Phrenic motoneurons: output elements of a highly organized intraspinal network

pontomedullary respiratory network generates the respiratory pattern and relays it to bulbar and spinal respiratory motor outputs. The phrenic motor system controlling diaphragm contraction receives and processes descending commands to produce orderly, synchronous, and cycle-to-cycle-reproducible spatiotemporal firing. Multiple investigators have studied phrenic motoneurons (PhMNs) in an attempt to shed light on local mechanisms underlying phrenic pattern formation. I and colleagues (Marchenko V, Ghali MG, Rogers RF. Am J Physiol Regul Integr Comp Physiol 308: R916–R926, 2015.) recorded PhMNs in unanesthetized, decerebrate rats and related their activity to simultaneous phrenic nerve (PhN) activity by creating a time-frequency representation of PhMN-PhN power and coherence. On the basis of their temporal firing patterns and relationship to PhN activity, we categorized PhMNs into three classes, each of which emerges as a result of intrinsic biophysical and network properties and organizes the orderly contraction of diaphragm motor fibers. For example, early inspiratory diaphragmatic activation by the early coherent burst generated by high-frequency PhMNs may be necessary to prime it to overcome its initial inertia. We have also demonstrated the existence of a prominent role for local intraspinal inhibitory mechanisms in shaping phrenic pattern formation. The objective of this review is to relate and synthesize recent findings with those of previous studies with the aim of demonstrating that the phrenic nucleus is a region of active local processing, rather than a passive relay of descending inputs.

The role of spinal GABAergic circuits in the control of phrenic nerve motor output

While supraspinal mechanisms underlying respiratory pattern formation are well characterized, the contribution of spinal circuitry to the same remains poorly understood. In this study, we tested the hypothesis that intraspinal GABAergic circuits are involved in shaping phrenic motor output. To this end, we performed bilateral phrenic nerve recordings in anesthetized adult rats and observed neurogram changes in response to knocking down expression of both isoforms (65 and 67 kDa) of glutamate decarboxylase (GAD65/67) using microinjections of anti-GAD65/67 short-interference RNA (siRNA) in the phrenic nucleus. The number of GAD65/67-positive cells was drastically reduced on the side of siRNA microinjections, especially in the lateral aspects of Rexed's laminae VII and IX in the ventral horn of cervical segment C4, but not contralateral to microinjections. We hypothesize that intraspinal GABAergic control of phrenic output is primarily phasic, but also plays an important role in tonic regulation of phrenic discharge. Also, we identified respiration-modulated GABAergic interneurons (both inspiratory and expiratory) located slightly dorsal to the phrenic nucleus. Our data provide the first direct evidence for the existence of intraspinal GABAergic circuits contributing to the formation of phrenic output. The physiological role of local intraspinal inhibition, independent of descending direct bulbospinal control, is discussed.