Spinal circuitry and respiratory recovery following spinal cord injury☆
Introduction
Considerable experimental evidence now exists for respiratory plasticity following spinal cord injury (SCI). This potential for intrinsic recovery has been most extensively described in a rat model of high cervical (C2) spinal hemisection (HS) and is often referred to as the crossed-phrenic phenomenon (CPP) (Goshgarian, 2003). Initial studies of this injury model showed that recovery of activity in the ipsilateral phrenic nerve and hemidiaphragm can be elicited by contralateral phrenicotomy within minutes to hours after injury. It has since been found that recovery of ipsilateral phrenic nerve and diaphragm activity also can occur spontaneously over the course of weeks (Fuller et al., 2003, Golder and Mitchell, 2005, Fuller et al., 2006, Vinit et al., 2007, Fuller et al., 2008) to months (Nantwi et al., 1999, Golder et al., 2001a) following C2HS. For present discussion purposes, this delayed natural recovery is operationally referred to as the “spontaneous crossed-phrenic phenomenon (sCPP)”. While the C2HS lesion model (in which both the CPP and sCPP can be expressed) is not typical of the majority of clinical cases of cervical SCI, it provides a valuable experimental setting for demonstrating proof-of-principle pertaining to respiratory recovery (reviewed in Lane et al., 2008a).
Often lacking in SCI research is detailed identification of neural substrates engaged in functional improvements occurring either naturally or in response to therapeutic interventions. Regarding the CPP and sCPP, the neural substrates may be quite different as suggested by the striking differences in their expression. To date, the CPP has been attributed to activation of a normally silent crossed intraspinal pathway which can restore inspiratory drive to the ipsilateral phrenic nucleus (Goshgarian, 2003). This pathway consists of axonal collaterals from uninjured, contralateral bulbospinal fibers that form monosynaptic projections onto phrenic motoneurons ipsilateral to the C2HS (Goshgarian et al., 1991, Moreno et al., 1992). This basic circuitry, however, does not take into account other neuronal constituents – interneurons in particular – that may affect the nature and extent of spontaneous recovery (i.e., the sCPP) in the chronically injured spinal cord.
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Spinal interneurons and neuroplasticity following SCI
Increasing recognition of the neuroplastic reserve of the injured spinal cord has evolved in recent years (Fouad and Tse, 2008, Harkema, 2008, Stein, 2008, Blesch and Tuszynski, 2009, Darian-Smith, 2009), and spinal interneurons may play prominent roles in neural circuit remodelling and modulation of motoneuron excitability. For example, one emerging concept is that interneurons with axonal projections traversing multiple spinal levels (i.e., long propriospinal interneurons) can provide
Spinal interneurons: projections onto respiratory motoneurons
The diaphragm and the circuitry mediating its function (i.e., phrenic motor system) are key components of inspiration (Feldman, 1986). Direct monosynaptic innervation of phrenic motoneurons by respiratory bulbospinal axons has been extensively documented in the rat (Ellenberger and Feldman, 1988, Ellenberger et al., 1990). In cats and ferrets, however, both monosynaptic and polysynaptic pathways have been observed (Yates et al., 1999, Lois et al., 2009). Pre-phrenic interneurons (neurons with
Neuroanatomical evidence for spinal respiratory interneurons in adult rat
A re-evaluation of anatomical relationships between spinal interneurons and the phrenic nucleus of the rat was recently carried out in an effort to gain a greater understanding of the circuitry that may influence spontaneous recovery of phrenic motoneuron and ipsilateral diaphragm functions after C2HS. Our primary focus was on cervical interneurons projecting onto phrenic motoneurons. The aim of initial studies was to establish a baseline for the C2HS model by obtaining a more complete
Spinal respiratory interneurons: connectivity with supraspinal centers
Electrophysiological studies in the cat have revealed medullary inspiratory drive to interneurons in the cervical (Bellingham and Lipski, 1990) and thoracic spinal cord (Kirkwood et al., 1988). There has been some additional evidence in cat and ferret that respiratory interneurons in the cervical spinal cord receive parallel input from the vestibular nuclei (Anker et al., 2006) and also from medullary centers that are known to regulate diaphragm function (Yates et al., 1999, Lois et al., 2009).
Changes in spinal respiratory interneurons following C2 hemisection: ipsilateral phrenic circuit
The next question addressed was whether any temporal remodeling of the phrenic circuitry occurs following C2HS as reflected by changes in the number or distribution of PRV labeled cells. PRV labeling via ipsilateral hemidiaphragm delivery was performed two weeks after injury (Fig. 3), based upon previous documentation of the initial emergence of the sCPP (Golder and Mitchell, 2005, Fuller et al., 2006, Fuller et al., 2008). As in spinal-intact rats, analysis of the number of PRV-positive
Plasticity, spinal interneurons, and compensation in other respiratory circuits
Although studies of respiratory plasticity following SCI have focused on recovery of ipsilateral phrenic function following C2HS (i.e., “restorative” neuroplasticity), the functional improvement is limited (as noted earlier), and respiratory deficits persist (Fuller et al., 2008). Phrenic nerve recordings (Fuller et al., 2008) and diaphragm EMG data (Lane, Fuller, Reier – unpublished) show that activity ipsilateral to C2HS remains minimal even 12 weeks following injury. In contrast,
Changes in spinal respiratory interneurons following C2 hemisection: contralateral phrenic circuit
Given functional changes occurring contralateral to the injury, the question arises whether remodeling of the contralateral phrenic circuit is associated with compensatory phrenic motoneuron responses to C2HS. To address this issue, we recently initiated studies in which PRV was applied to the contralateral hemidiaphragm to transynaptically label the associated phrenic circuit following C2HS (Lane et al., 2009). This resulted in robust second-order infection of interneurons, similar in laminar
Respiratory interneurons in the rat: neuroanatomical and electrophysiological correlates
A few studies have reported cervical spinal interneurons in the adult rat which show respiratory-related discharge (Lipski et al., 1993, Hayashi et al., 2003), and our recent neuroanatomical data (see above) provide correlative a neural substrate basis for those findings. These and other observations in our laboratory and others, also offer an opportunity to speculate on potential roles of interneurons in post-C2HS and other forms of spinal respiratory plasticity.
Concluding remarks
Increasing attention to neuroplasticity in the injured spinal cord under experimental and clinical conditions has underscored a need for greater understanding of associated neural substrates. Such information can facilitate better definition of therapeutic targets and underlying mechanisms not only in relation to spontaneous recovery processes, but also therapeutically-driven repair. Thus far, the neural circuits mediating most forms of neuroplasticity in the injured spinal cord have proven
Acknowledgements
Support for this work was provided by grants from the National Institutes of Health (NIH): NIH NS054025 (PJR) and NIH HD052682, NIH 1R01HD052682-01A1 (DDF). Additional support was provided by the Craig H. Neilsen Foundation (MAL), the University of Florida (KZL) and the Anne and Oscar Lackner Chair in Medicine (PJR). PRV152 was were provided to us by L.W. Enquist, Princeton University as a service of the National Center for Experimental Neuroanatomy with Neurotropic Viruses (NCRR P40
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2022, Experimental NeurologyCitation Excerpt :Some of these are highlighted in Fig. 1. Given the selective nature of attenuated PRV Bartha strains to move retrogradely (Enquist et al., 2002), they have been very effectively used to map a number of spinal networks, including locomotor (Bareyre et al., 2004; Jovanovic et al., 2010; Kim et al., 2002; Rotto-Percelay et al., 1992), upper extremity (Gonzalez-Rothi et al., 2015), respiratory (Lane et al., 2009a; Lane et al., 2008b; Zholudeva et al., 2017), micturition (Hou et al., 2016; Im et al., 2008; Karnup and De Groat, 2020; Vizzard et al., 1995; Yu et al., 2003), sexual organs (Marson, 1995; Marson and Carson 3rd, 1999; Marson and McKenna, 1996; Marson et al., 1993) and the sympathetic networks (Cano et al., 2004; Gao et al., 2014; Huang et al., 2002; Krout et al., 2003; Lee et al., 2007; Rotto-Percelay et al., 1992; Strack and Loewy, 1990; Strack et al., 1989; Tang et al., 2004) (Fig. 1). Combining these tracers either together (Banfield et al., 2003; Lane et al., 2008b), with other tracing methods (e.g. anterograde tracing of supraspinal projections (Lane et al., 2008b)) or transgenic lines (e.g. Chx10-GFP mouse lines (Zholudeva et al., 2017) has provided extremely detailed mapping of neural networks and how they are integrated with each other (e.g. intercostal and phrenic networks (Lane et al., 2008b)).
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This paper is part of a special issue entitled ‘Spinal cord injury—Neuroplasticity and recovery of respiratory function’, guest-edited by Gary C. Sieck and Carlos B. Mantilla.