Phrenic motor neuron degeneration compromises phrenic axonal circuitry and diaphragm activity in a unilateral cervical contusion model of spinal cord injury

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Abstract

Respiratory dysfunction is the leading cause of morbidity and mortality following traumatic spinal cord injury (SCI). Injuries targeting mid-cervical spinal cord regions affect the phrenic motor neuron pool that innervates the diaphragm, the primary respiratory muscle of inspiration. Contusion-type injury in the cervical spinal cord is one of the most common forms of human SCI; however, few studies have evaluated mid-cervical contusion in animal models or characterized consequent histopathological and functional effects of degeneration of phrenic motor neuron–diaphragm circuitry. In an attempt to target the phrenic motor neuron pool, two unilateral contusion injury paradigms were tested, a single injury at level C4 and a double injury both at levels C3 and C4, and animals were followed for up to 6 weeks post-injury. Both unilateral cervical injury paradigms are reproducible with no mortality or need for breathing assistance, and are accompanied by phrenic motor neuron loss, phrenic nerve axon degeneration, diaphragm atrophy, denervation and subsequent partial reinnervation at the diaphragm neuromuscular junction, changes in spontaneous diaphragm EMG recordings, and reduction in phrenic nerve compound muscle action potential amplitude. These findings demonstrate significant and chronically persistent respiratory compromise following mid-cervical SCI due to phrenic motor neuron degeneration. These injury paradigms and accompanying analyses provide important tools both for understanding mechanisms of phrenic motor neuron and diaphragm pathology following SCI and for evaluating therapeutic strategies in clinically relevant cervical SCI models.

Highlights

► We characterize 2 new paradigms of unilateral cervical contusion spinal cord injury. ► Both paradigms produce degeneration of respiratory phrenic motor neurons and nerve. ► Both paradigms result in pathology of diaphragm muscle and neuromuscular junctions. ► Diaphragm function is chronically impaired in both injury paradigms. ► We describe important tools for understanding SCI and for evaluating therapies.

Introduction

Approximately half of human traumatic spinal cord injury (SCI) cases affect cervical spinal cord regions (Lane et al., 2008), resulting in debilitating and often chronic respiratory compromise. The majority of these injuries affect mid-cervical spinal cord levels (Shanmuganathan et al., 2008), the location of the important pool of phrenic motor neurons (located approximately at levels C3–C5 in both humans and rodents) that innervates the diaphragm, the primary muscle of inspiration. Following cervical SCI, deficits in respiratory function are often attributed to lesions of afferent connections from bulbospinal neurons of the medullary ventral respiratory group, direct damage to the phrenic motor neuron pool, and/or interruption of efferent phrenic motor nerve projections to the diaphragm. Given that the phrenic motor neuron–diaphragm circuit is directly affected by a large proportion of SCI cases and that ventilation following mid-cervical SCI is significantly determined by phrenic motor neuron loss/sparing (Strakowski et al., 2007), development and characterization of relevant cervical SCI models are crucial both for understanding the mechanisms of phrenic motor neuron loss following SCI and for developing therapies targeting protection and plasticity of this key motor neuron population.

While the use of thoracic SCI animal models has predominated, a number of cervical SCI models have recently been developed (Anderson et al., 2009a, Anderson et al., 2009b, Dunham et al., 2010, Gensel et al., 2006, Khaing et al., 2012, Pearse et al., 2005, Soblosky et al., 2001). Initial models of cervical SCI targeted spinal cord hemisection mostly at level C2 or higher, and have provided key information for understanding and therapeutically targeting regeneration of bulbospinal axons and reinnervation of the phrenic nucleus (Alilain et al., 2011), as well as for characterizing important mechanisms of respiratory plasticity such as the crossed-phrenic pathway (Fuller et al., 2009, Golder et al., 2003, Vinit et al., 2006). Nevertheless, transection-type lesions of the spinal cord are rarely encountered in human SCI; therefore, the use of cervical contusion models is becoming increasingly more relevant to the clinical situation.

Many cervical contusion SCI studies have focused on forelimb motor dysfunction (Anderson et al., 2009a, Anderson et al., 2009b, Dunham et al., 2010, Khaing et al., 2012, Pearse et al., 2005, Soblosky et al., 2001); however, recent work has begun to focus on respiratory outcomes (Baussart et al., 2006, Choi et al., 2005, el-Bohy et al., 1998, Golder et al., 2011, Lane et al., 2012), including analysis of cell populations such as phrenic motor neurons. To date, few studies have reported significant and persistent respiratory deficits under basal respiratory conditions. Weight-drop contusion at a high cervical (C2) level did induce long-lasting unilateral diaphragm deficits under eupnea and asphyxia conditions, as measured by electrophysiology (Baussart et al., 2006). Respiratory circuitry exhibits remarkable plasticity following SCI that allows for the recovery of vital functions such as breathing. The most well-known example of neuroplasticity of the respiratory system is the crossed-phrenic pathway phenomenon (CPP). CPP allows for partial recovery of the paralyzed hemi-diaphragm following a C2 lateral hemisection via activation of contralateral descending bulbospinal input to the ipsilateral phrenic nucleus (Goshgarian, 2009, Goshgarian et al., 1986). Unfortunately, this compensatory phenomenon is often insufficient to mediate recovery following extensive damage to gray matter locations below C3, such as the location of the phrenic motor neuron pool (Goshgarian, 2003, Zimmer et al., 2007).

Using whole-body plethysmography, a recent study demonstrated that a single mid-cervical contusion injury modified the general ventilatory response on a short time frame (i.e. 2 days post-injury) (Golder et al., 2011). Breathing patterns were restored to the levels of the uninjured control group within 2 weeks, demonstrating the neural plasticity of breathing control. In a similar injury paradigm, Lane and colleagues extended these investigations by showing that diaphragm activity was chronically impaired following a bilateral mid-cervical contusion injury; however, general ventilatory parameters were unaffected (Lane et al., 2012). Unfortunately, these two important studies lacked extensive characterization of the changes occurring at all levels of the phrenic motor neuron–diaphragm circuit, including motor neuron cell bodies of the phrenic nucleus, axons of the phrenic nerve, and diaphragm muscle.

Given the central role that phrenic motor neurons play in diaphragm function and respiratory behavior, as well as the significant proportion of human SCI cases that affect mid-cervical spinal cord regions, we extensively characterized histological and functional changes in phrenic motor neuron–diaphragm circuitry in rodent models of mid-cervical contusion SCI. We have specifically examined the time course of phrenic motor neuron and phrenic nerve axonal loss, diaphragm muscle changes, and morphological and functional innervation of the diaphragm by phrenic motor neurons. In an attempt to target a large extent of the phrenic motor neuron pool, we tested two unilateral contusion injury paradigms, a single injury at level C4 and a double injury both at levels C3 and C4. In order to study the long-term evolution of functional innervation of the diaphragm following injury, we characterized functional diaphragmatic changes in both injury paradigms out to 6 weeks post-injury. In addition, we histologically analyzed phrenic nerve–diaphragm circuitry at both 2 and 6 weeks post-injury in order to determine changes at an early time point still within the window of secondary degeneration and at a more chronic time point, respectively. We have demonstrated that our unilateral cervical injury paradigms are reliable, reproducible with no mortality or need for breathing assistance, and accompanied by phrenic motor neuron loss, phrenic nerve axon degeneration, diaphragm atrophy, denervation and subsequent partial reinnervation at the diaphragm neuromuscular junction, and reduction of phrenic nerve compound muscle action potential amplitude. These findings demonstrate significant respiratory compromise following mid-cervical SCI, and these injury paradigms and accompanying analyses provide important tools both for understanding mechanisms of phrenic motor neuron and diaphragm pathology following SCI and for evaluating therapeutic strategies in clinically-relevant cervical SCI models.

Section snippets

Animals and experimental design

Adult female Sprague–Dawley rats (250–300 g) were obtained from Taconic Farm (Rockville, MD) and housed in a controlled (light and temperature) environment with ad libitum access to food and water in the animal facility at Thomas Jefferson University. Care and treatment of animals during all procedures were conducted in compliance with the European Communities Council Directive (2010/63/EU, 86/609/EEC and 87-848/EEC), the NIH Guide for the care and use of laboratory animals, and the Thomas

Decreased ipsilateral forelimb motor performance following C4-only and double C3 + C4 unilateral cervical contusion SCI

In order to target the injury to a significant portion of the ipsilateral phrenic motor neuron pool, rats received either a single unilateral contusion SCI at level C4 or a double unilateral contusion injury at both levels C3 and C4. Retrograde labeling of phrenic motor neurons from the ipsilateral hemi-diaphragm demonstrates that the majority of this motor neuron population is located at level C3 and C4, with the greatest density found at C4 (Fig. 3E). Upon completion of all surgical

Discussion

Due to the urgent need for animal models of cervical SCI that are relevant to the clinical population, we sought to develop a new and reliable paradigm of unilateral, mid-cervical, contusion SCI. Few studies have characterized the pathologic events taking place along the respiratory phrenic motor neuron–diaphragm pathway following contusion SCI, including effects on phrenic motor neuron cell bodies in the cervical spinal cord, phrenic nerve, and diaphragm muscle. Using the Infinite Horizon

Conclusions

In summary, we have described in the present report a new model of mid-cervical spinal cord contusion in the adult rat that induces unilateral diaphragm deficits within 2 weeks post-injury and that persists for at least 6 weeks. With no mortality or no need for respiratory assistance, our model is functionally and histologically reproducible and clinically-relevant to a significant percentage of human SCI cases. Several contusion models at mid-cervical levels (C4/C5) have already been developed

Acknowledgments

This work was supported by the Université Libre de Bruxelles (Bureau des Relations Internationales et de la Coopération, grant BRIC-11/092 to C.N.), the Craig Nielsen Foundation (grant #190140 to A.C.L.) and the Paralyzed Veterans of America (grant #160837 to A.C.L.). We are grateful to Timothy Schneider for technical assistance. We thank Dr. Megan Detloff for valuable advice in setting up the cervical contusion model. We thank Dr. Carlos Mantilla and Dr. Wen-Zhi Zhan for valuable advice with

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