Pharmacological disinhibition enhances paced breathing following complete spinal cord injury in rats

Highlights

• Epidural stimulation and intrathecal drug delivery were performed at C4 cervical spinal segment post- complete C1 spinal transection.

• Low doses of GABAzine and strychnine were intrathecally delivered to disinhibit phrenic spinal circuits.

• Phrenic nerve responses, diaphragm electromyography amplitudes, and tidal volume were assessed during experiments.

• Combination of high-frequency EDS and pharmacological disinhibition of phrenic spinal circuits significantly improved paced breathing.

Abstract

Respiratory dysfunction is one of the most devastating and life-threatening deficits that occurs following cervical spinal cord injury (SCI). Assisted breathing with mechanical ventilators is a necessary part of care for many cervical injured individuals, but it is also associated with increased risk of secondary complications such as infection, muscle atrophy and maladaptive plasticity. Pre-clinical studies with epidural stimulation (EDS) have identified it as an alternative/additional method to support adequate lung ventilation without mechanical assistance. The full potential of EDS, however, may be limited by spinal inhibitory mechanisms within the injured spinal cord. The goal of the present work is to assess the potential improvement for EDS in combination with pharmacological disinhibition of spinal circuits following complete high cervical SCI. All experiments were performed in decerebrate, unanesthetized, non-paralyzed (n = 13) and paralyzed (n = 8) adult Sprague-Dawley rats 6 h following a complete C1 transection. The combination of high-frequency EDS (HF-EDS) at the C4 spinal segment with intrathecal delivery of GABA and glycine receptors antagonists (GABAzine and strychnine, respectively) resulted in significantly increased phrenic motor output, tidal volume and amplitude of diaphragm electrical activity compared to HF-EDS alone. Thus, it appears that spinal fast inhibitory mechanisms limit phrenic motor output and present a new neuropharmacological target to improve paced breathing in individuals with cervical SCI.

Introduction

Stimulation of the brain or spinal cord (SC) has been shown to be an effective and clinically viable strategy for treating neural injury and disease. Epidural stimulation (EDS) of the spinal cord is used clinically for the treatment of pain, locomotor, cardio-vascular and autonomic dysfunction (Shealy et al., 1967; Ohta et al., 1992; Pettigrew et al., 2017; Rejc et al., 2017; Dones and Levi, 2018; Harkema et al., 2018; Herrity et al., 2018; Rejc and Angeli, 2019; Calvert et al., 2019; Aslan et al., 2018). Among those is respiratory impairment that arises after cervical spinal cord injury (van Silfhout et al., 2016; Tollefsen and Fondenes, 2012; Baydur and Sassoon, 2018). Breathing is an essential vital function, which is often significantly compromised after high cervical SCI. Even if some muscle function is retained after an injury, it may be insufficient to maintain adequate ventilation and gas exchange.

The standard of care for impaired breathing in most countries is assisted-ventilation with positive pressure mechanical ventilators. While maintaining ventilation, these devices reduce the need for any muscle activity and respiratory muscles can atrophy rapidly (Levine et al., 2008; Powers et al., 2009; Jaber et al., 2011; Gayan-Ramirez, 2013; Gill et al., 2014; Smuder et al., 2016). Intubation required for mechanical ventilation also significantly increases respiratory alkalosis and the risk of secondary infections and complications (van Nieuwenhoven et al., 1999; Watt and Silva, 2001). In contrast, electrical stimulation of the respiratory nerves, muscles, and spinal networks can maintain activity, muscle viability and adequate gas exchange (Sarnoff et al., 1948; Osterholm et al., 1966, 1968; Nochomovitz et al., 1983; DiMarco et al., 1987; Gonzalez-Rothi et al., 2017; Bezdudnaya et al., 2018a; Sunshine et al., 2018; DiMarco and Kowalski, 2019). This has led to the growing use of phrenic nerve and diaphragm pacing clinically, with many reported benefits (Sarnoff et al., 1948; Glenn and Phelps, 1985; DiMarco et al., 1994, 2002, 2005, 2006; Gonzalez-Bermejo et al., 2014; Le Pimpec-Barthes et al., 2013). However, these approaches also carry some limitations: i) surgical implantation of stimulation electrodes risks damage to phrenic axons (Kim et al., 1976; Glenn et al., 1986: DiMarco et al., 2006; Le Pimpec-Barthes et al., 2016), ii) direct diaphragm muscle stimulation requires much higher electrical currents, and in some cases may need to be increased over time as fibrosis occurs around electrodes (Glenn et al., 1986; DiMarco et al., 2006; Le Pimpec-Barthes et al., 2013). Thus, EDS of the spinal cord presents a promising alternative which has been extensively tested pre-clinically using a range of spinal cord injury experimental animal models (DiMarco et al., 1987; Kowalski et al., 2013; Bezdudnaya et al., 2018a; Dimarco and Kowalski, 2019).

Published studies have shown that spinal cord EDS can effectively support respiratory muscle contractions even after complete high cervical (C1-C2) transection (DiMarco et al., 1987, 2007; Kowalski et al., 2013; Bezdudnaya et al., 2018a). Our recent work (Bezdudnaya et al., 2018a has demonstrated that high-frequency epidural stimulation (HF-EDS) at the C4 segment was effective for activation of the phrenic motor system and paced breathing acutely after a C1-transection (C1Tx) in adult rats. However, EDS is also often used in combination with intrathecal (subdural) injection of drugs to treat pain, locomotor disorders and spasticity in patients with SCI (Lind et al., 2004, 2008; Schechtmann et al., 2010; Goto et al., 2013). In fact, combining EDS with neuropharmacological modulation of spinal respiratory circuits may appear to enhance treatment efficacy. An important candidate for neuromodulation of spinal neurons is fast inhibition provided by GABA and glycinergic synapses. It has been demonstrated that GABAA, GABAB and glycine receptors play an important role in the regulation of excitability of phrenic motoneurons (Lalley, 1983; Eldridge et al., 1987; Zhan et al., 1989; Tai and Goshgarian, 1996; Chitravanshi and Sapru, 1999; Goodchild et al., 2017; Marchenko and Rogers, 2009; Cregg et al., 2017). We have also shown an important role of GABA-ergic interneurons in the modulation of phrenic motor output (Marchenko and Rogers, 2009; Marchenko et al., 2015; Ghali and Marchenko, 2016). Thus, we hypothesize that attenuation of spinal inhibitory circuits by GABAa and glycine receptor antagonists during HF-EDS at C4 will improve diaphragm pacing following complete SCI. Therefore, in the current work, we test whether combination of HF-EDS with intrathecal GABAa and glycine receptors antagonists can increase the efficacy of life-supporting paced breathing in the proof-of-principle model (C1Tx) of spinal cord injury.