CHAPTER

The challenges of
respiratory motor system
recovery following cervical
spinal cord injury
10
Philippa M. Warren, Warren J. Alilain1
Department of Neurosciences, MetroHealth Medical Center, Case Western Reserve University
School of Medicine, Cleveland, OH, USA
1
Corresponding author: Tel.: +01-216-778-8966; Fax: +01-216-778-8720,
e-mail address: warren.alilain@case.edu

Abstract
High cervical spinal cord injury (SCI) typically results in partial paralysis of the diaphragm
due to intrusion of descending inspiratory drive at the level of the phrenic nucleus. The degree
to which such paralysis occurs depends on the type, force, level, and extent of trauma pro-
duced. While endogenous recovery and plasticity may occur, the resulting respiratory compli-
cations can lead to morbidity and death. However, it has been shown that through modification
of intrinsic motor neuron properties, or altering the environment localized at the site of SCI,
functional recovery and plasticity of the respiratory motor system can be facilitated. The pre-
sent review emphasizes these factors and correlates it to the treatment of SCI at the level of the
somatic nervous system. Despite these promising therapies, functional respiratory motor sys-
tem recovery following cervical SCI is often minimal. This review thus focuses on possible
directions for the field, with emphasis on combinatorial treatment.

Keywords
spinal cord injury, respiratory motor system, channelrhodopsin-2, chondroitinase

Abbreviations
5-HT serotonin
A2A adenosine 2A
AIH acute intermittent hypoxia
BBB blood-brain barrier

Progress in Brain Research, Volume 212, ISSN 0079-6123, http://dx.doi.org/10.1016/B978-0-444-63488-7.00010-0

© 2014 Elsevier B.V. All rights reserved.
173
174 CHAPTER 10 The challenges of respiratory motor system recovery

BDNF brain-derived neurotrophic factor

cAMP cyclic adenosine monophosphate
C# cervical level of injury
ChABC chondroitinase ABC
ChR2 channelrhodopsin-2
CIH chronic intermittent hypoxia
CNP cyclic nucleotide phosphodiesterase
CNS central nervous system
CPP crossed phrenic pathway
CSPG chondroitin sulfate proteoglycan
CST corticospinal tract
dAIH daily acute intermittent hypoxia
ECM extracellular matrix
EMG electromyography
EPO erythropoietin
ERK1/2 extracellular regulated kinases 1 and 2
GABA g-aminobutyric acid
GAG glycosaminoglycan
GDNF glial-derived neurotrophic factor
IH intermittent hypoxia
NADPH nicotinamide adenine dinucleotide phosphate
NGF nerve growth factor
NMDA N-methyl-D-aspartic acid
NSCISC National Spinal Cord Injury and Statistics Center
NT neurotrophin
OEC olfactory ensheathing cell
PKA protein kinase A
PKC protein kinase C
pLTF phrenic long-term facilitation
PMF phrenic motor facilitation
PMNs phrenic motor neurons
PNG peripheral nerve graft
PNS peripheral nervous system
PSA polysialic acid
PTEN phosphatase and tensin homolog
rAIH repetitive AIH
Rho Ras homolog gene family
rVRG rostral VRG
Sema semaphorin
SCI spinal cord injury
TrkB tropomyosin-related kinase B
VEGF vascular endothelial growth factor
VRG ventral respiratory group
a alpha
b beta
g gamma
1 Cervical spinal cord injury and the deficit in respiratory motor function 175

1 CERVICAL SPINAL CORD INJURY AND THE DEFICIT

IN RESPIRATORY MOTOR FUNCTION
High cervical spinal cord injury (SCI) has both sensory and motor effects within the
central nervous system (CNS). The consequences of such insult are largely described
and studied in terms of the devastating consequences caused to voluntary movement
and the somatic nervous system. However, it is well established that cervical
SCI has annihilating effects upon other systems (clinically described by Nandoe
Tewarie et al., 2010). Of particular significance are injuries that involve disruption
to respiratory motor function. Indeed, despite meaningful advances, the life
expectancy of ventilator-dependent SCI patients is substantially less than the
uninjured population (NSCISC, 2012), with common causes of death including
pneumonia and septicemia.
More than half of all SCIs occur at the cervical level (NSCISC, 2012) and
prominently feature the loss of inspiration. This is largely caused by disruption
of the motor circuitry to the diaphragm, although injury additionally prevents
innervation to the intercostal and abdominal muscles. Autonomic control of
broncho-pulmonary functions is lost following cervical SCI resulting in abnormal
secretions and airway hypersensitivity (Fein et al., 1998; Grimm et al., 2000;
Singas et al., 1999). Spontaneous recovery has been reported within the respira-
tory motor system (Bluechardt et al., 1992; Linn et al., 2001; Loveridge et al.,
1992; Oo et al., 1999), indicative of plasticity. However, this endogenous effect
is often suboptimal and highly variable. It most likely represents an attempt to
maintain or restore blood-gas homeostasis following SCI by means of an increase
in respiratory drive. Compromised breathing, even in unventilated individuals,
can make a SCI patient increasingly susceptible to pneumonia and atelectasis.
Due to these complications, the search to find treatments and strategies to func-
tionally improve respiratory motor activity following cervical SCIs are of great
importance.
In the present review, we first establish the organization of the respiratory
motor system. We then describe how breathing has been experimentally mod-
eled, the intrinsic and extrinsic mechanisms that drive respiratory motor function
following SCI, and the treatment strategies that have been applied to enhance
this activity. Discussion relates these findings back to spinal cord development
and injury to alternative motor systems, highlighting universal themes for in-
creasing functional recovery and regeneration. We define “functional” recovery
as the restoration, compensation, or use of alternative/novel motor circuits to per-
form a task controlled through the respiratory motor system. Finally, we assess
the use of combination treatment strategies to facilitate respiratory motor func-
tion following high cervical SCI, emphasizing the need for strategy development,
the potential to incorporate currently unexamined treatment stratagems within
the respiratory motor system, and the possibility of assessing multiple outcome
measures.
176 CHAPTER 10 The challenges of respiratory motor system recovery

2 ORGANIZATION OF THE RESPIRATORY MOTOR CIRCUITRY

AND THE CROSSED PHRENIC PHENOMENON
The diaphragm is the major muscle used for inspiration. It is innovated by the phrenic
nerve which originates at C3–C5 (cervical level 3–5) within the spinal cord. Patients
with injuries at or above this level lose the ability to spontaneously breathe. The cir-
cuitry that controls the movement of the diaphragm during breathing was elucidated
through electrophysiological and neuronal tracing studies in rodent and feline
models (Ballanyi et al., 1999; Boulenguez et al., 2007; Dobbins and Feldman,
1994; Ellenberger and Feldman, 1988; Greer et al., 2006; Lipski et al., 1993,
1994; Onai et al., 1987; Tian and Duffin, 1996; Yamada et al., 1988). These models
share significant homology with the primate respiratory motor system. This evolu-
tionary conservation means that animal models of the respiratory motor system can
provide meaningful data regarding the human response to SCI and treatment
(Holstege et al., 1988). The inspiratory premotor neurons that project down through
the phrenic nucleus to the diaphragm originate within the ventral respiratory group
(VRG; Fig. 1A). Located in the ventral lateral medulla, the neurons of the supraspinal
centers, in particular, the pre-Bötzinger complex, control the frequency and rhythm
of respiration (Gray et al., 2001; Onimaru and Homma, 2003; Tan et al., 2008). These
neurons are connected to the propriobulbar and premotor neurons of the VRG in the
ventral lateral medulla (Chitravanshi and Sapru, 1996; Dobbins and Feldman, 1994;
Moreno et al., 1992; Onai et al., 1987). The bulbospinal projections from the left and
right VRG descend through the lateral and ventromedial funiculi of the cervical spinal
cord to innervate the phrenic nucleus (Fig. 1A; Boulenguez et al., 2007; Dobbins and
Feldman, 1994; Ellenberger and Feldman, 1988; Juvin and Morin, 2005; Lipski et al.,
1994; Yamada et al., 1988). Some bulbospinal projections cross the midline in the me-
dulla (Fig. 1A). In both primates and rodents, bulbospinal neurons from the most ros-
tral part of the VRG (rVRG) facilitate the glutamatergic inspiratory drive, while those
from the caudal end facilitate expiration (Bianchi et al., 1995).
The phrenic motor neurons (PMNs) of the phrenic nucleus are located in the ven-
tral horn between C3 and C6 (Fig. 1A; Goshgarian and Rafols, 1984; Routal and Pal,
1999). Inputs to the intact PMNs can be glutamatergic, GABAergic (g-aminobutyric
acid), serotonergic or from norepinephrine neurons (Chitravanshi and Sapru, 1996;
Liu et al., 1990; McCrimmon et al., 1989). However, PMN activity is principally
controlled by excitatory glutamatergic projections from the rVRG in both rodent
and primate models (Chitravanshi and Sapru, 1996; Howard et al., 1992; Nathan
et al., 1996; Vinit et al., 2007). Similarly, PMNs are innervated by cervical spinal
cord interneurons located bilaterally between the brain and spinal cord (Fig. 1A;
Juvin and Morin, 2005; Lane et al., 2008; Lipski et al., 1993, 1994; Lu et al.,
2004a). These interneurons do not mediate respiratory motor system drive in the
uninjured spinal cord, as occurs in the CST (corticospinal tract; Gauthier et al.,
2006; Rossignol et al., 2008; Vinit et al., 2006).
A number of the bulbospinal projections from the rVRG to the PMNs cross the
midline at the level of the phrenic nucleus (C3–C6; Fig. 1A; Vinit et al., 2007). These
FIGURE 1
Schematic of the cervical spinal cord respiratory network before and after C2 hemisection. (A) Basic anatomy of the intact respiratory motor system
detailing the bilateral connections from the rostral ventral respiratory group (rVRG, glutamatergic; blue; dark grey in print version) and caudal raphe nucleus
(serotonergic; yellow; light gray in print version) to those of the phrenic nucleus (PN; red (medium grey in print version)). Dissociations of these pathways
occur in the medulla and spinal cord, the latter comprising the crossed phrenic pathway (CPP), which is retained from development but latent in the adult.
Interneurons (green; light grey in print version) are disbursed through the system creating polysynaptic connections. (B) Following subacute C2
hemisection, connections to the ipsilateral phrenic nucleus and hemidiaphragm are broken causing paralysis. Further to this, axons dieback forming
dystrophic end bulbs, a glial scar develops, and the cells of the immune system invade. (C) However, with an appropriate stimulus generate drive, the latent
CPP may be activated by-passing the site of injury and facilitating activity through the phrenic nucleus to generate activity in the hemidiaphragm ipsilateral
to the hemisection. Stimulus may be varied; the schematic shows the transection phrenic nerve contralateral phrenic to the C2 hemisection and stimulates
ipsilateral hemidiaphragm activity. This is known as the crossed phrenic phenomenon.
178 CHAPTER 10 The challenges of respiratory motor system recovery

spinal decussating pathways do not typically drive PMNs but can be activated under
conditions of stress (Lewis and Brookheart, 1951). The classic example of this effect
involves hemisection of the C2 bulbospinal pathways, causing paralysis of the ipsi-
lateral hemidiaphragm (Fig. 1C). When followed by transection of the contralateral
phrenic nerve below C6, the originally paralyzed hemidiaphragm becomes active,
while the hemidiaphragm ipsilateral to the phrenicotomy is inactivated (Chatfield
and Mead, 1948; Goshgarian, 1981; O’Hara and Goshgarian, 1991; Porter, 1895;
Seligman and Davis, 1941). This is termed the “crossed phrenic phenomenon” as
it involves activation of the crossed phrenic pathway (CPP; defined as the decussat-
ing tract and postsynaptic target; Fig. 1A). This injury archetype has been extensively
used to model recovery of breathing following cervical SCI.
Of course, the diaphragm is not the only musculature involved in breathing. The
respiratory intercostals and abdominal muscles are innervated within the thoraco-
lumbar region of the spinal cord at T1–T11 and T7–L2, respectively. Similar to
the diaphragm, autonomic premotor neurons projecting from the rVRG modulate
the activity of these muscles through innovation of motor neuron pools. However,
while the inputs to the phrenic nucleus are primarily monosynaptic (Ellenberger
and Feldman, 1988; Tian and Duffin, 1996, 1998; Tian et al., 1998), evidence sug-
gests that activation of the intercostals is polysynaptic and relies upon spinal inter-
neurons (Davies et al., 1985; Kirkwood, 1995; Merrill and Lipski, 1987). The
occurrence of these interneurons may act to modulate intercostal activity or drive
independent of the phrenic nerve (Davies et al., 1985; Qin et al., 2002). Due to
the organization of these respiratory motor pathways, patients with incomplete
C3–C5 or lower C6–C8 injuries may have the ability to breathe spontaneously. How-
ever, vital capacity is reduced and respiration impaired due to paralysis of the exter-
nal and parasternal intercostals, scalene, and abdominal musculature.

3 MODELING RESPIRATORY MOTOR FUNCTION FOLLOWING

CERVICAL SPINAL CORD INJURY
3.1 CERVICAL SPINAL CORD INJURY
SCI is a heterogeneous condition where pathological changes can be broadly defined
by location, severity, and the length of time following injury (Fig. 1B; Young and
Koreh, 1986). Briefly, the acute injury is typified by the immediate affect of the
mechanical trauma. The result is direct damage, loss of the blood-brain barrier
(BBB), ischemia, edema, and electrolytic changes (Sandler and Tator, 1976;
Young and Koreh, 1986). These effects cause excitotoxicity and cellular necrosis
within the gray and then white matter during the first 24 h after injury. Glial cells
migrate to the site of SCI and are involved in beneficial phagocytosis of neuronal
debris, conservation of the remaining tissue, and proinflammatory responses
(Bouhy et al., 2006; Davalos et al., 2005; Ha et al., 2005; Rapalino et al., 1998).
However, several days following SCI a process of secondary cell loss occurs
due to Wallerian degeneration (Guth et al., 1999) and apoptotic mechanisms in
3 Modeling respiratory motor function following cervical spinal cord injury 179

oligodendrocytes, oligodendrocyte precursor cells, astrocytes, microglia, and neu-

rons. This is caused by ionic disruption, the expression of free radicals, and inflam-
matory cytokines.
The post-acute/chronic stage of injury typically starts days following the initial
insult and persists over time. The acute affects described initiate inflammatory and
cytotoxic events that cause the formation of fluid filled cysts (Poon et al., 2007),
demyelination (Totoiu and Keirstead, 2005), deposition of myelin debris (Buss
et al., 2004), and axonal loss (Bjartmar et al., 2001). In addition, glial cells are
activated and form a protective barrier around the injury site by reactive gliosis. This
is primarily composed of chondroitin sulfate proteoglycans (CSPGs) and is known as
the “glial scar” (McKeon et al., 1999; Tang et al., 2003). Acutely, this scar limits
damage and preserves function within the damaged cord ( Jones et al., 2003;
reviewed in Rolls et al., 2009). However, in the chronic stages of SCI, the glial scar
acts as both a physical (Windle et al., 1952) and chemical (Snow and Letourneau,
1992) barrier to endogenous growth and regeneration (Windle et al., 1952).
Similarly, the immune cells of the CNS, microglia, and macrophages, are activated
following SCI, and have been shown to promote regeneration through the secretion
of neurotrophic factors (Bessis et al., 2007; Coull et al., 2005). However, microglia
are also associated with an increase in neuropathic pain (Coull et al., 2005;
Hulsebosch, 2008) and thus have detrimental effects, particularly in chronic SCI.

3.2 ACUTE MODELS OF CERVICAL SCI AND THE EFFECT UPON

THE RESPIRATORY MOTOR SYSTEM
Rat models are widely employed to study the effect of SCI on breathing with the
activation of the CPP being the classic paradigm (Goshgarian, 1981; Goshgarian
et al., 1991; O’Hara and Goshgarian, 1991; Porter, 1895). The mechanism behind
the crossed phrenic phenomenon is an increase in respiratory drive due to the
asphyxia caused by the contralateral phrenicotomy following complete ipsilateral
hemisection (Fig. 1C; Goshgarian and Guth, 1977). Of course, the aim of most stud-
ies is to induce an increase in respiratory drive that activates the CPP without per-
forming the contralateral phrenicotomy. For this reason, complete high cervical
hemisection is used as an acute model to assess functional recovery within the respi-
ratory motor system following injury (Fig. 1B). This lesion causes ipsilateral hemi-
diaphragm paralysis, but the animal survives as breathing is maintained through
intact pathways to the contralateral phrenic nucleus (Decherchi and Gauthier,
2002). Inspiratory volume is diminished and frequency of breathing enhanced,
following this lesion (Fuller et al., 2006). However, minute ventilation in rodents
is unaffected (Fuller et al., 2006) and is only apparent upon variation in levels of car-
bon dioxide (Fuller et al., 2008). Of course, the cervical hemisection additionally
affects locomotor and sensory systems. It is suggested that an incomplete lesion
may transiently produce the same effect as a complete hemisection while saving
other motor systems (Goshgarian, 1981; Vinit et al., 2007). However, such injuries
spare bulbospinal axons innervating the phrenic nucleus resulting in more favorable
180 CHAPTER 10 The challenges of respiratory motor system recovery

respiratory outcomes (Fuller et al., 2009; Lipski et al., 1994; Schucht et al., 2002;
Vinit et al., 2006). Nonetheless, such studies clearly demonstrate how spared path-
ways may contribute to post-lesion plasticity.
The CPP can activate spontaneously following acute SCI in the absence of respi-
ratory drive resulting in partial restoration of PMN activity (Fuller et al., 2008;
Golder and Mitchell, 2005; Vinit et al., 2007). However, similar to locomotor and
sensory motor systems, assessment of hemidiaphragm electromyography (EMG) ac-
tivity shows spontaneous recovery within the phrenic nucleus to be modest (Alilain
and Goshgarian, 2008; Fuller et al., 2008; Mantilla et al., 2007; Miyata et al., 1995;
Nantwi et al., 1999) despite the improvement in neuromuscular transmission
(Mantilla et al., 2007; Prakash et al., 1999). Nonetheless, the potential for endoge-
nous recovery is certainly present and the reasons why it fails to be functionally sig-
nificant has yet to be fully understood.

3.3 CONTUSION AND CHRONIC MODELS OF CERVICAL SCI

AND THE AFFECT UPON THE RESPIRATORY MOTOR SYSTEM
Despite the wealth of information generated through the acute and chronic C2 hemi-
section injury model regarding the respiratory motor system, the translational rele-
vance of these data can be questioned when one considers that analogous lacerations
are rare in humans (NSCISC, 2012). For this reason, the effects of the more sever
C2–C5 lateral or midline cervical contusion has been assessed (Fig. 2A). They yield
a complementary system to the lateral C2 hemisection model; however, hold in-
creased clinical relevance. These reproducible injuries do not require long-term
post-injury ventilation for survival but cause PMN loss and degeneration, depressed
diaphragm EMG recordings, reduced phrenic nerve activity, impaired response to
respiratory challenge, and limited endogenous recovery (Awad et al., 2013;
Baussart et al., 2006; Choi et al., 2005; El-Bohy et al., 1998; Lane et al., 2012;
Nicaise et al., 2012). Golder et al. (2011) have recently shown that recovery of
breathing patterns following contusion is, in part, dependent upon mechanisms of
endogenous plasticity (Golder et al., 2011). Further to this, Lane et al. (2012) used
a bilateral C3/C4 contusion model to show that damage to gray matter after injury
could chronically impair hemidiaphragm activity under conditions of respiratory
stress while ventilation remains constant. These data are suggestive of plasticity
and remodeling within the contused animal (Alilain et al., 2008; Hayashi et al.,
2003; White et al., 2010). Most recently, Awad et al. (2013) have developed a dual
injury contusion model to certify the robustness of the respiratory motor deficit.
Their moderate 150 kD C3 contusion causes extensive damage to white and gray
matter. Nonetheless, the injured or spared bulbospinal fibers following trauma en-
able partial ipsilateral hemidiaphragmatic activity to be retained as is shown in other
contusion models. However, Awad et al. (2013) combine the contusion injury with
the elimination of modulatory decussating inputs from the non-injured side through a
contralateral C2 hemisection, revealing the highly weakened state of the contused
pathways at acute and chronic time points (Fig. 2B). The authors suggest that this
FIGURE 2
Schematic of the cervical spinal cord respiratory network after lateral C3 contusion. (A) Following lateral C3 contusion, connections to the
ipsilateral phrenic nucleus and hemidiaphragm are weakened or injured causing reduced activity in the diaphragm. The functional deficit of
this injury is not readily shown in many contusion models. A glial scar develops and the cells of the immune system invade. (B) The C3 contusion
model produced by Awad et al. (2013). This model combines the initial injury with a delayed contralateral C2 hemisection to reveal the full
extent of the functional deficit caused by the contusion injury. The application of treatment strategies for SCI may be applied between the initial
contusion and the deferred contralateral C2 hemisection.
182 CHAPTER 10 The challenges of respiratory motor system recovery

new model of injury may enable further examination of the degree to which any SCI
treatment strategy directs affects the pathways damaged in the initial injury. As such,
this model holds substantial clinical relevance as it can highlight functional recovery
within these pathways following treatment.
The effect of SCI upon the respiratory motor system must also be assessed chron-
ically to facilitate clinical applicability. Similar to human studies (Oo et al., 1999),
rodent models of high cervical injury have shown an increase in ipsilateral PMN ac-
tivity at chronic time points following injury (Fuller et al., 2006; Golder and
Mitchell, 2005; Nantwi et al., 1999; Vinit et al., 2006, 2008). Whether this results
in a functional improvement to diaphragmatic function is variable (Alilain and
Goshgarian, 2008; Goshgarian, 1981; Nantwi et al., 1999; Polentes et al., 2004).
The CPP can still activate in a chronic injury model (Vinit et al., 2006, 2008) al-
though it is possible that endogenous functional recovery at these time points can
be a result of CPP remodeling (Vinit et al., 2008) and is likely reinforced by the spar-
ing of ventromedial tissue (Darlot et al., 2012; Minor et al., 2006). This has been
shown to occur within the CST (Fouad et al., 2001; Ghosh et al., 2010) and may sug-
gest that the mechanisms to respiratory motor system recovery in a chronic model are
divergent from that of an acute injury.

3.4 ENDOGENOUS RESPIRATORY MOTOR SYSTEM RECOVERY

FOLLOWING SUBACUTE/CHRONIC CERVICAL SPINAL CORD INJURY
Endogenous remodeling and activation of the CPP following subacute/chronic C2
hemisection produces a modest return of function (Fuller et al., 2008; Nantwi
et al., 1999; Pitts, 1940) but may hold significant implications for treatment strategies.
This return of function correlates with synaptic and receptor changes around the
PMNs and involves receptor and synaptic plasticity. For example, there is an increase
in presynaptic serotonin (5-HT) terminals and their corresponding receptors, includ-
ing 5-HT2A around PMNs ipsilateral to the C2 hemisection (Fuller et al., 2005; Golder
and Mitchell, 2005; Tai et al., 1997). This endogenous response facilitates induction
of the crossed phrenic phenomenon, which is dependent on 5-HT (Hadley et al.,
1999a,b). In fact, infusion of 5-HT receptor agonists is sufficient to promote func-
tional recovery of respiratory motor function (Choi et al., 2005; Ling et al., 1994;
Zhou and Goshgarian, 1999, 2000; Zhou et al., 2001; Zimmer and Goshgarian, 2006).
Alilain and Goshgarian (2008) demonstrated that spontaneous activity within
the PMNs is correlated with an increase in the 2A subunit of the NMDA
(N-methyl-D-aspartic acid) receptor and a decrease in the AMPA (2-amino-3-(3-
hydroxy-5-methyl-isoxazol-4-yl)propanoic acid) glutamate receptor 1 subunit on
motor neurons. These studies are important because, while 5-HT may modulate re-
spiratory motor output, glutamate is an excitatory neurotransmitter that causes the
neuron to fire (Chitravanshi and Sapru, 1996). Additionally, GABAergic signaling
has been shown to decrease at chronic stages of cervical SCI by reducing inhibition
( James and Nantwi, 2006; Zimmer and Goshgarian, 2007). These spontaneous changes
in the 5-HT, glutamatergic and GABAergic systems following SCI indicate an endog-
enous mechanism to facilitate respiratory motor system recovery.
 4 Intrinsic factors 183

The interneuron population present at the site of the phrenic nucleus may confer
some additional support to endogenous recovery following SCI, although evidence
to this effect is limited. Following acute C2 hemisection, Lane et al. (2008) have
shown the number of interneurons at the level of the PMNs is unaltered from unin-
jured controls, although motor neuron numbers significantly decrease. It is suggested
that these interneurons integrate with phrenic circuits on opposite sides of the cord
and facilitate synchronized activity after injury (Alilain et al., 2008; Lane et al., 2008;
Sandhu et al., 2009). Indeed, an increase in the number of interneurons at C2 post
hemisection suggest a pool of recruited cells positioned to mediate an increase in
drive contralateral to the site of injury and may facilitate intercostal or abdominal
motor circuits (Fig. 1B; Lipski et al., 1994). However, in the chronic setting, both
interneuron and motor neuron numbers are diminished. This suggests that these cells
alone are insufficient to facilitate robust endogenous recovery. Indeed more informa-
tion is required to establish the role of the interneuron population in respiratory
recovery following cervical SCI.

3.5 THE LIMITATIONS OF SPINAL CORD INJURY MODELS

Functional recovery following SCI is typically deemed the most clinically relevant
outcome measure when assessing the efficacy of a treatment strategy. However, such
measures are difficult to assess due to the high variability inherent within CNS in-
juries. This variability cannot be extinguished even when they are mechanically pro-
duced through the same processes. One reason for this is that the hormone, blood
sugar, and hydration levels of each animal vary when surgery is performed as well
as vascularity at the site of injury and level of anesthesia (Gruner, 1992; Kwo et al.,
1989). Furthermore, animal behavior following injury may be altered depending on
their circadian rhythm and the time of year (Dauchy et al., 2010; O’Bryant et al.,
2011). Additionally, different strains of rodents are known to demonstrate wide dif-
ferences in motor control (Webb et al., 2003), indeed the sex of rodent has been
shown to have variation on respiratory motor activity following cervical SCI
(Doperalski et al., 2008). Further, long-lasting increase in respiratory motor drive
(see Section 4.2) is effected by the rodents age, gender, and substrain (Fuller
et al., 2001a; Zabka et al., 1985, 2001). These variations inherent within models
of SCI mean that the anatomical and functional assessment of the effect and treat-
ment of any trauma is potentially problematic and must be considered when asses-
sing the outcome of any one study.

4 INTRINSIC FACTORS CONTROLLING RESPIRATORY

MOTOR RECOVERY FOLLOWING CERVICAL SCI
4.1 ADENOSINE A1 AND CAMP
Levels of adenosine within the neonatal CNS are characteristically high due to fetal
ischemic or hypoxic conditions (Rudolphi et al., 1992; Winn et al., 1981a,b).
Adenosine acts as a neuroprotectant causing dilation of blood vessels in the
184 CHAPTER 10 The challenges of respiratory motor system recovery

CNS when flow is compromised (Phillis et al., 1985), a decrease in neurotrans-

mitter release (Fredholm and Hedquist, 1980), and reduced oxygen consumption
(Gross et al., 1976; Raberger et al., 1970). However, high levels of adenosine
decrease neonatal respiration, an effect that may be counteracted though A1
receptor-mediated antagonism (reviewed in Herlenius and Lagercrantz, 2004).
Similar effects occur to respiratory motor function following treatment of C2
hemisection with adenosine antagonism (Fig. 3A). Through administration of the
methylxanthine theophylline, nonspecific antagonism of central and peripheral aden-
osine A1 receptors partially restored function to the paralyzed hemidiaphragm. This
was caused by increased respiratory drive (Nantwi, 2009; Nantwi and Goshgarian,
1998; Nantwi et al., 1996). Further to this, Nantwi et al. (2003) demonstrated that
chronic theophylline treatment could amplify respiratory motor function beyond
the period of drug administration. This illustrates the pharmaceutical-induced plas-
ticity of the respiratory motor circuitry. High levels of adenosine may be required in
the acute phases of cervical SCI to act as a neuroprotectant when blood flow may be
compromised. However, the antagonism of receptor-mediated transmission within
subacute and chronic phases may generate the drive to increase functional respiratory
motor activity.
Alternatively, theophylline use could increase respiratory drive through the inhi-
bition of cyclic nucleotide phosphodiesterase (CNP), which causes an increase in cy-
clic adenosine monophosphate (cAMP) independent of A1 receptor antagonism
(Fig. 3A). cAMP is known to be a key intracellular signaling molecule and elevated
amounts have facilitated axonal regeneration in numerous injury models (Cai et al.,
1999; Chierzi et al., 2005; Lu et al., 2004b, 2012; Qiu et al., 2002a,b) including the
increase in plasticity and functional recovery of the respiratory motor system (Kajana
and Goshgarian, 2008a,b). This effect is likely caused by enhancing growth factor
receptor translocation (Meyer-Franke et al., 1998). Alternatively, it may be mediated
through activation of protein kinase A (PKA) and the cAMP response element-
binding protein (CREB) transcription factor (Gao et al., 2004; Sands and Palmer,
2008) which arbitrates the neurogenic effects of neurotrophic factors (Gao
et al., 2003).

4.2 GQ PROTEIN SIGNALING CASCADES: INTERMITTENT HYPOXIA,

5-HT2, AND PHRENIC LTF
Originally described by Millhorn et al. (1980a,b), the induction of phrenic long-term
facilitation (pLTF) is the long-lasting increase in respiratory motor drive. This phe-
nomenon is typically observed as an increase in nerve burst amplitude that similarly
effects inspiratory motor output (Bocchiaro and Feldman, 2004; Fuller et al.,
2001a,b, 2002) and is elicited by repeated carotid sinus nerve stimulation or inter-
mittent hypoxia (IH; Hayashi et al., 1993). pLTF is hypothesized to facilitate plas-
ticity following cervical SCI. The most studied paradigm of IH is acute intermittent
hypoxia (AIH), which describes 5-min exposure to the hypoxic environment,
repeated three to five times (Hayashi et al., 1993). Interestingly, Golder and
FIGURE 3
Current models of intrinsic pathways to facilitate phrenic motor activity and drive. (A) Adenosine A1 and cAMP (red; dark gray in print version).
Theophylline acts to inhibit adenosine A1 activity and/or increase intracellular concentrations of cAMP via the inhibition of CNP. These processes
may act to increase PMN drive. The latter pathway acts through an increase in growth factor translocation or through the activation of protein
kinase A (PKA) and the cAMP response element-binding protein (CREB) transcription factor. (B) Intermittent hypoxia, 5-HT2 and phrenic long-
term facilitation (pLTF; green (gray in print version)). Induced through intermittent hypoxia, Gq-coupled metabotropic receptors 5-HT2, or a1 are
activated, stimulating the activity of protein kinase C (PKC) which causes BDNF synthesis and NADPH oxidase (NOX) activity. The release of
BDNF activates TrkB and, subsequently, the ERK kinase (pERK). The pathway is regulated through the action of NOX-dependent reactive oxygen
species and protein phosphatases (PP2/5). (C) Adenosine and 5-HT7 (blue; light gray in print version). Following stimulation of Gs-coupled
metabotropic receptors 5-HT7 and A2A, PKA is activated and induces the synthesis of immature TrkB. The TrkB dimerizes, autophosphorylates
and signals via Akt activation. The figure also demonstrates how downstream of intermittent hypoxia PTEN may be inhibited leading to the
increase in mTOR which may act intrinsic to the neuron to facilitate plasticity and regeneration.
Parts of schematic modified from Dale et al. (2014).
186 CHAPTER 10 The challenges of respiratory motor system recovery

Mitchell (2005) demonstrated that a program of AIH in the chronically injured (4–8
weeks) C2 hemisected animal could produce a functionally relevant increase in re-
spiratory drive to aid recovery. This indicated that, if harnessed, AIH treatment could
be effective regardless of time post-injury.
Of course, there are many other protocols used to induce pLTF through IH includ-
ing chronic IH (CIH; IH for 5–10 min intervals lasting 4 days to 4 weeks; Fletcher
et al., 1992; Gozal et al., 2001), daily AIH (dAIH; 10 AIH episodes lasting 7 days;
Lovett-Barr et al., 2012; Wilkerson and Mitchell, 2009), repetitive AIH (rAIH; AIH
repeated over days to weeks; Golan et al., 2009), repeated AIH (10 AIH episodes 3
days a week for 4–10 weeks; Satriotomo et al., 2012), and sustained hypoxic expo-
sure (Powell et al., 1998). These protocols produce contrasting effects, although they
operate in a mechanistically similar fashion.
5-HT receptor activation is required for the induction of pLTF (Fig. 3B; Millhorn
et al., 1980a,b). Exposure to IH activates the serotonergic medullary raphe neurons
causing the release of 5-HT at the level of the phrenic nucleus and subsequent acti-
vation of Gq protein-coupled 5-HT receptor subtypes (Erickson and Millhorn, 1994).
Specifically, Kinkead and Mitchell (1999) identified the 5-HT2A/B/C receptor activa-
tion is necessary for pLTF initiation (MacFarlane et al., 2011). MacFarlane and
Mitchell (2008, 2009) showed that 5-HT receptor activation was sufficient to cause
long-lasting facilitation in the motor output of the phrenic nerve (phrenic motor
facilitation; PMF) using periodic intraspinal injections of the neurotransmitter and
5-HT receptor agonists without AIH. However, 5-HT receptor activation is not
required to maintain the response in either acute or chronic models (Fuller et al.,
2001a,b, 2002). As the CPP shows an increase in 5-HT2A receptor activation follow-
ing C2 hemisection in subchronic animals (Fuller et al., 2003), an effect exacerbated
following CIH (Fuller et al., 2005), the pathway is believed to be activated following
the increase in drive caused by pLTF.
Baker-Herman and Mitchell (2002) demonstrated that the intermittent activation
of 5-HT receptors causes protein synthesis required for pLTF through the activation
of PKC (protein kinase C). Of particular note is the synthesis of brain-derived neu-
rotrophic factor (BDNF; Fig. 3B; Baker-Herman et al., 2004). The subsequent acti-
vation of tropomyosin-related kinase B (TrkB; the high-affinity receptor for BDNF)
has been shown both necessary and sufficient for induction of pLTF (Baker-Herman
et al., 2004). The significance of BDNF and TrkB has recently been confirmed in the
chronic C2 hemisected model using immunohistochemistry following dAIH (Lovett-
Barr et al., 2012). However, while the exogenous application of BDNF can produce
PMF, it has a short half-life and poor BBB penetration (Poduslo and Curran, 1996),
limiting its use as a treatment for SCI.
Kishino and Nakayama (2003) and Wilkerson and Mitchell (2009) have respec-
tively shown that BDNF increases extracellular regulated kinases 1 and 2 (ERK1/2)
phosphorylation in PMNs. This suggests that these kinases are involved downstream
of TrkB (Fig. 3B). The events which occur downstream of ERK are less clear.
NMDA receptor antagonism is known to prevent pLTF in the anesthetized and
 4 Intrinsic factors 187

unanesthetized animal (McGuire et al., 2005, 2008). Further to this, Slack et al.
(2004) demonstrated that BDNF modulates the phosphorylation of the NR1 subunit
on NMDA receptors though the ERK pathway. This suggests that glutamate receptor
phosphorylation, or membrane insertion, may account for NMDA-mediated induc-
tion and maintenance of pLTF in the phrenic nucleus (Fig. 3B; Bocchiaro and
Feldman, 2004). pLTF is regulated by serine/threonine protein phosphatases
PP2A and PP5 (Wilkerson et al., 2008); which are, in turn, regulated by the formation
of reactive oxygen species by NADPH (nicotinamide adenine dinucleotide phos-
phate) oxidase activity (Abramov et al., 2007; MacFarlane et al., 2008, 2009;
Wilkerson et al., 2008). Wilkerson et al. (2007) and MacFarlane et al. (2008) suggest
that this regulatory mechanism facilitates sensitivity of pLTF expression (Fig. 3B).
However, MacFarlane et al. (2011) have recently shown that NADPH oxidase inhib-
itors block PMF only when induced through 5-HT2B not 5-HT2A receptors. These
findings suggest that the multiple receptors capable of eliciting this activity may have
distinct pathways through which they are regulated.
The insight these studies give into the mechanism through which IH induces
respiratory drive is, perhaps, not surprising when the development of the respiratory
motor system is considered. During critical periods of CNS development 5-HT levels
increase to affect neuronal proliferation, differentiation, migration, and synaptogen-
esis (reviewed in Lauder, 1993; Levitt et al., 1997; Lipton and Kater, 1989) although
knocking out receptors and genes does not cause marked alteration in brain histology
(Gaspar et al., 2003). Further to this, the effect of neurotrophins (including nerve
growth factor (NGF), BDNF, neurotrophin-3 (NT-3) and NT-4), and their corre-
sponding Trk receptors, has been extensively studied during development
(Erickson et al., 1996; Funakoshi et al., 1993; Griesbeck et al., 1995; Ip et al.,
2001). BDNF and NT-3 are known to be present in large neurons, like PMNs, in
the ventral cervical spinal cord during development ( Johnson et al., 2000). In
the adult, CNS neurons may continue to express neurotrophic receptors, a fact
which is exploited following SCI. Altered expression of neurotrophic factors
has been shown within multiple models of SCI. NGF, BDNF, NT-3, and glial-
derived neurotrophic factor (GDNF) are known to promote axon regeneration or
sprouting postinjury (Blesch and Tuszynski, 2003; Grill et al., 1997; Jin et al.,
2002; Liu et al., 1999), while an endogenous or exogenous increase in NT concen-
tration at the site of injury significantly enhances regeneration within both the pe-
ripheral and CNSs (Blesch and Tuszynski, 2003; Kobayashi et al., 1997; Park and
Hong, 2006; Schnell et al., 1994). BDNF specifically has been shown to facilitate
the regeneration of raphe, reticilospinal, rubrospinal, and vestibulospinal neurons
following lateral hemisection ( Jin et al., 2002; Liu et al., 1999). The use of trophic
factors to support axon growth following SCI increases the likelihood that the
micro-environment at the site of injury will be permissible for functional recovery.
The fact that IH endogenously produces these effects to enhance spinal plasticity,
and specifically respiratory motor function, highlights the importance of this
treatment strategy.
188 CHAPTER 10 The challenges of respiratory motor system recovery

4.3 GS PROTEIN SIGNALING CASCADE: ADENOSINE AND 5-HT7

The model described for pLTF induced by IH requires activation of spinal Gq
protein-coupled 5-HT2 receptors and the synthesis of BDNF to activate TrkB
(Baker-Herman et al., 2004; Fuller et al., 2001a,b, 2002). However, Golder et al.
(2008) and Hoffman and Mitchell (2011) report a BDNF-independent mechanism
to achieve PMF without the need for IH (Fig. 3C). Activation of either Gs
protein-coupled adenosine 2A (A2A) or 5-HT7 receptors induces the synthesis of im-
mature TrkB receptors that autodimerize and autophosphorylate. Once activated, in-
tracellular TrkB (Golder et al., 2008) signals via PI3 kinase, which increases the
phosphorylation of protein kinase B or Akt, and is believed to mediate the effects
of PMF (Fig. 3C; Chao, 2003; Golder et al., 2008). This pathway is thus independent
of BDNF and ERK signaling. The ability to initiate PMF without the use of IH could
be useful in the clinical setting where patients’ respiratory motor output is often im-
paired. However, A2A receptor activation has been shown to cause neural and car-
diovascular morbidity (Minghetti et al., 2007; Mojsilovic-Petrovic et al., 2006;
Pedata et al., 2001). 5-HT7 receptor antagonism may be the only means of clinically
mediating this response.
The relationship between the two G-coupled protein-mediated methods to induce
PMF is interesting. Hoffman and Mitchell (2011) demonstrated that inhibition of
ERK delayed, but did not block, 5-HT7-induced PMF. Similarly, Hoffman et al.
(2007) describe that the application of A2A receptor antagonists following AIH in-
creased pLTF. Satriotomo et al. (2012) have recently described that rAIH showed
upregulation of molecules known to be involved in PMN plasticity including both
phosphorylated ERK and phosphorylated Akt. These data suggest that both the
G protein-signaling cascades described are initiated following AIH, but each mutu-
ally inhibit the other. The growth/trophic factors vascular endothelial growth factor
(VEGF) and spinal erythropoietin (EPO) have both been shown to induce PMF
through an ERK- and Akt-dependent mechanism (Dale et al., 2012; Dale-Nagle
et al., 2011) and are known to be hypoxia sensitive (Liu et al., 1995; Stohlman,
1959). However, Dale and Mitchell (2013) have recently shown that PMF induced
by injection of these growth factors into the spinal cord is not amplified following
rAIH pre-conditioning. The implications of this study are intriguing as they imply
that the functional changes evoked by VEGF and EPO are independent of hypoxia
despite the latter causing similar plastic changes at the level of the phrenic nucleus
(Dale et al., 2012; Dale-Nagle et al., 2011; Satriotomo et al., 2012). CNS axon re-
generation is known to involve multifaceted signaling mechanisms that are typically
upregulated following injury including the kinases ERK (Atwal et al., 2000; Liu and
Snider, 2001) and Akt (Atwal et al., 2000; Chierzi et al., 2005; Gallo and Letourneau,
1998; Markus et al., 2002). Through further understanding of these pathways, treat-
ment strategies to enhance PMF may be elucidated to aid functional respiratory mo-
tor activity following cervical SCI.
However, modulation in the expression of 5-HT, BDNF, and adenosine might not
be the only mechanism through which IH treatment can facilitate functional recovery
of the respiratory motor system following cervical SCI. Gutierrez et al. (2013) have
 5 Extrinsic factors 189

recently shown that following acute C2 hemisection, PMNs show decreased expres-
sion of PTEN (phosphatase and tensin homolog) and increased levels of mTOR
(mammalian target of rapamycin) and S6 (Fig. 3B). Recent studies inhibiting PTEN
in the CNS have demonstrated significant regeneration in the spinal cord and visual
system (Kwon et al., 2006; Liu et al., 2010; Park et al., 2008). While in need of fur-
ther examination and the mechanism of action fully elucidated, these data suggest
that IH may mediate functional regeneration and plasticity following SCI through
a multitude of mechanisms.

4.4 OPTOGENETICS
Perhaps the most impressive mechanism though which functional recovery of the respi-
ratory motor system occurs is the use of optogenetics. Neuronal depolarization and ac-
tion potentials can be specifically stimulated and controlled using photostimulation of
the light-sensitive cation channel channelrhodopsin-2 (ChR2; Zheng et al., 2007), fol-
lowing its expression in motor neurons. Such a tool is advantageous following SCI,
where neuronal loss and denervation is extensive. For example, following C2 hemisec-
tion Alilain et al. (2008) were able to achieve near complete restoration of respiratory
motor activity using photostimulation of ChR2 expressed in motor neurons, interneu-
rons and spinal glial cells at the level of the PMNs under a CMV promoter (Fig. 4A).
Significantly, this activity was retained in absence of stimulation (lasting for at least
24 h) and was synchronized to the endogenous activity of the hemidiaphragm
contralateral to the hemisection and not light stimulation. Use of the NMDA receptor
antagonist MK-801 abolished this activity. These data indicate both the importance
of NMDA receptors to the activity of the respiratory motor system after injury and
increasing sensitivity of motor neurons and interneurons to weak, but spared tracts.
Nonetheless, while the use of optogenetics can provide substantial functional re-
sults and illustrate the mechanism of motor system recovery, direct translation of this
technique to the clinic would be challenging. While modern surgical techniques
could facilitate a minimally invasive method to introduce a potential light source
within the human spinal cord, the viral methods currently employed to express
ChR2 within motor neurons would need substantial modification before clinical trial.
For example, it would be necessary to determine the length of time ChR2 is expressed
and the specific cell type within which this occurs. However, this technique remains a
valuable tool for the study of respiratory motor function following cervical SCI.

5 EXTRINSIC FACTORS CONTROLLING RESPIRATORY

MOTOR RECOVERY FOLLOWING CERVICAL SCI
5.1 INFLAMMATION
In response to SCI, both macrophages and microglia are activated each producing
both neuroprotective and proinflammatory effects likely to be caused by cells of dis-
tinct lineages (Kigerl et al., 2009). Augmenting macrophage and microglia activation
FIGURE 4
Intrinsic and extrinsic modification of the cervical spinal cord following C2 hemisection (see Fig. 1) to facilitate the functional regeneration of
the respiratory motor system. (A) Infection of cells at the site of the phrenic motor nucleus with ChR2 can, following light stimulation, case
neuronal depolarization/action potentials that will transiently enable activity of the paralyzed hemidiaphragm. The cells mediating such activity
are possibly spared motor neurons, such as the CPP, or more likely interneurons. (B) Implantation of a peripheral nerve graft (PNG) facilitates
the growth of regenerating neurons which can by-pass the site of injury and form functional synapses with spared tissue at the phrenic
nucleus enabling activity of the paralyzed hemidiaphragm. (C) Application of exogenous chondroitinase ABC (ChABC) breaks down major
components of the glial scar to facilitate the endogenous growth of neurons through the site of injury. The enzyme additionally promotes
neuroprotection, plasticity, and reduces the size of the lesion cavity. This restores minimal activity to the paralyzed hemidiaphragm although
whether this is through regenerated fibers or spared tissue is unknown.
 5 Extrinsic factors 191

has been shown to enhance axon regeneration (Schwartz et al., 1999). This occurs
through enhanced phagocytosis, the production of cytokines and growth factors,
remyelination, angiogenesis, oligodendrogenesis, prevention of excitotoxicity, and
secretion of NTs (Kerschensteiner et al., 1999; Li et al., 2005). Such responses
are likely produced by M2 macrophages, although this has not been proven in
SCI models (Kigerl et al., 2009). Conversely, preclinical models of SCI have shown
that blocking the actions of macrophage and microglia facilitates functional recovery
by preventing axonal dieback and the death of neurons and oligodendrocytes (Blight,
1994; Kaushal et al., 2007; Lopez-Vales et al., 2005; Popovich et al., 1999; Stirling
et al., 2004; Wong et al., 2010). Additionally, inhibition of integrins that mediate the
immune response following injury have been shown to facilitate neuroprotection and
functional recovery (Fleming et al., 2008, 2009; Gris et al., 2004), although aspects
of these studies have been hard to replicate (Hurtado et al., 2012). Macrophage of the
M1 lineage are believed to produce this neurotoxic effect causing the retraction of
axons, secretion of inhibitory CSPGs, and the breakdown of growth promoting ECM
(extracellular matrix) substrates, such as laminin (Busch et al., 2009; Horn et al.,
2008; Martinez et al., 2006).
The effect of the activated immune system upon the functional recovery of the
respiratory motor system following cervical SCI has only recently been assessed.
In a preliminary study, Windelborn and Mitchell (2012) have shown, following in-
complete C2 hemisection, microglia and astrocytes are activated in the phrenic nu-
cleus caudal to the site of injury, peaking at 3 days postlesion but remaining 28 days
following injury. Activation of these cells is retained up to 180 days following SCI in
alternative areas of the cord (Gwak et al., 2012). This suggests that these glial cells
facilitate in the recovery or deficit of the respiratory PMNs following SCI. Indeed the
importance of glia to the correct functioning of the respiratory system has been
widely reported (Gourine et al., 2010; Huckstepp et al., 2010; Parsons and
Hirasawa, 2010; Shimizu et al., 2007; Young et al., 2000). Adding to this hypothesis
is the recent work conducted by Vinit et al. (2011) demonstrating that the initiation of
inflammation through a single injection of lipopolysaccharide can prevent the induc-
tion of pLTF. Whether this response is mediated by activated microglia is not yet
determined (Huxtable et al., 2011). However, such effects have been exhibited else-
where in the CNS (Bessis et al., 2007; Hennigan et al., 2007).

5.2 GRAFTING TISSUE

Unlike the CNS, the peripheral nervous system (PNS) exhibits an exceptional facility
for regeneration following injury. PNS cells mediating growth have historically been
prime targets for transplantation into the injured CNS to facilitate functional regen-
eration (David and Aguayo, 1981; Richardson et al., 1980). One of the most success-
ful uses of peripheral nerve grafts (PNGs) to achieve functional recovery of axonal
pathways has been demonstrated in the respiratory motor system (Fig. 4B; Gauthier
and Rasminsky, 1988). Using a model where the proximal end of a peritoneal nerve
was placed in close proximity to the rVRG or into the funiculi of the descending
192 CHAPTER 10 The challenges of respiratory motor system recovery

respiratory tracts, Gauthier and colleagues were able to demonstrate growth of respi-
ratory bulbospinal axons into the graft (Decherchi et al., 1996; Gauthier and
Lammari-Barreault, 1992; Gauthier and Rasminsky, 1988; Lammari-Barreault
et al., 1991). Specifically, respiratory-related axons within the graft showed sponta-
neous, respiratory-related activity (Gauthier and Rasminsky, 1988; Lammari-
Barreault et al., 1994). This was only maintained in chronic animals if a functional
reconnection with an appropriate target could be established. Further studies in-
cluded implantation of the PNGs distal end at the level of the phrenic nucleus fol-
lowing C3 hemisection (Decherchi and Gauthier, 2002; Gauthier et al., 2002).
These data demonstrated the success of PNGs at acute/subacute time points indicat-
ing that regenerated axons from central respiratory neurons can remain in graft tissue
for 3 weeks and make functional connections with a denervated target.
However, PNGs have minimal impact on the restoration of phrenic nerve activity
following SCI (Gauthier et al., 2002). This result is typical of bridge-graft studies
where anatomical repair does not reflect functional regeneration. This is likely
caused by poor entry of the growing axon from graft tissue into the host spinal cord
due to the inhibitory nature of the glial scar. A multitude of literature demonstrates
that high concentrations of either endogenous or synthetic CSPGs at cellular inter-
faces create a barrier to axon regeneration (Petersen et al., 1996; Pindzola et al.,
1993; Plant et al., 2001; Snow et al., 1990, 2002). While acutely this scar tissue pre-
serves function, chronically it negatively regulates the outgrowth of regenerating
axons whether from graft tissue or endogenous plasticity (McKeon et al., 1995;
Snow et al., 1990, 2002). Due to the presence of this scar tissue, the use of PNS grafts
following cervical SCI, may be functionally limited.

5.3 REDUCTION OF THE GLIAL SCAR

The neuroprotective glial scar, in chronic injuries, has been shown to inhibit the
regeneration and plasticity of the CNS. Part of this inhibition can be attributed to
the ephrins and semaphorin 2A (Sema2A) released from the cells of the spinal cord
following injury (De Winter et al., 2002). Sema3A is constitutively expressed on mo-
tor neurons and, through repellant signaling, acts to guide descending supraspinal
and reflex pathways from sensory afferents expressing the Sema receptor
(Gavazzi et al., 2000; Giger et al., 1998). A series of recent experiments have dem-
onstrated that inhibition or suppression of Sema3A facilitates neuronal growth
(Castellani et al., 2004; Minor et al., 2011). However, this may not be sufficient
to cause functional regeneration (Mire et al., 2008) perhaps indicating that other fac-
tors in the scar, for example, CSPGs, are more significant mediators of inhibition.
It has been demonstrated both in vitro (Dou and Levine, 1994; Fidler et al., 1999;
Smith-Thomas et al., 1994, 1995; Snow et al, 1990; Tom et al., 2004) and in vivo
(Borisoff et al., 2003; Davies et al., 1997, 1999; Dou and Levine, 1994;
Friedlander et al., 1994; Snow et al., 1990; Tang et al., 2003) that CSPGs limit axon
growth and regeneration, and that these molecules are intensely upregulated follow-
ing SCI (Iaci et al., 2007; Jones et al., 2003; Tang et al., 2003). During development,
 5 Extrinsic factors 193

the occurrence of CSPGs within the ECM coincides with a cessation of developmen-
tal plasticity (Pizzorusso et al., 2002, 2006). The major inhibitory component of
CSPGs are the individual disaccharide moieties present on the glycosaminoglycan
(GAG) chains (Rolls et al., 2004). Several methods have been employed to inhibit
or reduce the expression of CSPGs following injury (Laabs et al., 2007; Larsen
et al., 2003; Lemke et al., 2010; Nigro et al., 2009; Schwartz et al., 1974). An im-
portant example of which is the use of chondroitinase ABC (ChABC; Bradbury
et al., 2002; Campbell et al., 1990).
ChABC is a bacterial enzyme isolated from Proteus vulgaris (Yamagata et al.,
1968) that acts to cleave GAG polymers into their component tetrasaccharides
and disaccharides preventing matrix–glycoprotein interactions (Huang et al.,
2000, 2003; Prabhakar et al., 2005; Yamagata et al., 1968). Over the last decade,
ChABC application has been used to stimulate the growth of axons due to the break-
down of CSPGs both in vitro (Asher et al., 2002; McKeon et al., 1991, 1995;
Nakamae et al., 2009; Rudge and Silver, 1990; Vahidi et al., 2008; Zou et al.,
1998) and in vivo (Barritt et al., 2006; Bradbury et al., 2002; Cafferty et al., 2008;
Garcia-Alias et al., 2009; Houle et al., 2006; Jefferson et al., 2011; Lemons et al.,
1999; Moon et al., 2001; Tom et al., 2009a). A wealth of evidence has shown that
the mode and mechanism though which ChABC operates causes axonal regenera-
tion, plasticity, and neuroprotection (Barritt et al., 2006; Bradbury et al., 2002;
Cafferty et al., 2007; Carter et al., 2008; Massey et al., 2006).
Recently, Alilain et al. (2011) have demonstrated the presence of the CSPG com-
posed glia scar at the level of the phrenic nucleus following C2 hemisection
(Fig. 4C). ChABC treatment increased 5-HT fibers about the PMNs indicative of
functional respiratory plasticity. However, while ChABC alone-treated animals
may have demonstrated an increase in functional recovery quicker than controls,
the ultimate result was insubstantial as inspiratory bursts did not exceed 10–20%
of the peak amplitude displayed by controls at 12 weeks postinjury (Alilain et al.,
2011). The reason ChABC failed to induce a significant functional effect in this
instance could be due to the quantity and time frame of enzyme administration.
Our laboratory is currently pursuing the assessment of optimized ChABC treatment
within the respiratory motor system following acute and chronic cervical SCI. Fur-
ther, the advent of virally delivered or thermostabilized chondroitinase could solve
these issues (Curinga et al., 2007; Jin et al., 2011; Lee et al., 2010; Muir et al., 2010;
Zhao et al., 2011). Nevertheless, the efficacy and safety of these modified enzymes
has not yet been established. Further, it is known that the effects of ChABC on long-
distance axon regeneration are relatively modest (Bradbury et al., 2002; Cafferty
et al., 2007; Iseda et al., 2008; Shields et al., 2008). The main functional consequence
of using the enzyme is the increase in sprouting and plasticity. Indeed, two recent
studies in divergent models of the CNS have shown that the neuronal plasticity in-
duced by ChABC application does not uniformly result in functional recovery
(Harris et al., 2010; Vorobyov et al., 2013).
Alilain and colleagues (2011) sought to overcome the lack of functional recovery
in the ipsilateral hemidiaphragm following ChABC treatment by combining it with a
194 CHAPTER 10 The challenges of respiratory motor system recovery

PNG. The removal of CSPGs about the bridge graft enhanced the entry and exit of
axons from host tissue while facilitating axonal sprouting. The use of ChABC upre-
gulated the expression of 5-HT and retrograde labeling showed a small proportion of
the neurons that projected through the graft were from the raphe nuclei and rVRG.
Interestingly, the use of ChABC with the PNG caused the alignment of glial fibrillary
acidic protein positive astrocytes to the regenerating axons. At 12 weeks post-injury,
it was the combined treatment group that showed the maximal return of functional
diaphragmatic muscle activity with both peak inspiratory amplitude and phrenic
nerve activity of the lesioned side often surpassing controls. Significantly, transec-
tion of the graft caused the transient increase in EMG of the ipsilateral hemidiaph-
ragm and suggested that this treatment combination was able to significantly rewire
the cervical spinal cord leading to functional restoration of respiratory motor activity.
The combined use of PNGs and ChABC has been used previously to establish func-
tional regeneration of the locomotor system following acute and chronic SCI. The
degradation of CSPGs in the lesion and graft (Lemons et al., 1999) allows axons
to grow through graft tissue and form functional synaptic connections (Houle
et al., 2006; Tom and Houle, 2008; Tom et al., 2009b, 2013). Similar data have been
achieved by combining ChABC with grafts of Schwann or neural stem/progenitor
cells (Chau et al., 2004; Fouad et al., 2005, 2009; Karimi-Abdolrezaee et al.,
2010; Vavrek et al., 2007).

6 FUTURE DIRECTIONS: INTEGRATION OF TREATMENT

STRATEGIES AND OUTCOME MEASURES
6.1 INTEGRATION OF TREATMENT STRATEGIES
The success of the Alilain et al. (2011) study has shown that a combinatorial
approach to treating respiratory function after SCI is a promising method to achieve
maximal functional recovery. Recent experiments in the treatment of locomotor
function using some of the therapeutic strategies known to have functional effects
for the recovery of the respiratory motor system lend support to this idea. For exam-
ple, Bai et al. (2010) combined the use of ChABC and the b2-adrenoceptor agonist,
clenbuterol, to increase cAMP levels following a chronic thoracic transection of the
spinal cord. Only with the combined treatment was significant anatomical and partial
functional recovery observed. Similarly, Tropea et al. (2003) described how com-
bined BDNF and ChABC treatment worked synergistically to promote plasticity,
regeneration, and synaptogenesis of retinal afferents in a model of acute partial ret-
inal lesion. The combination of BDNF with an olfactory ensheathing cell (OEC)
graft following subacute C5/6 over-hemisection showed significant anatomical
regeneration and substantial functional recovery of skilled motor function (Iarikov
et al., 2007; Lynskey et al., 2006). However, it is important to cast the correct treat-
ment combination spatially and temporally post-injury. Bretzner et al. (2008, 2010)
have shown that the combination of an OEC bridge graft and increase in cAMP
(through the application of the phosphodiesterase inhibitor rolipram) but not BDNF,
 6 Future directions 195

was able to promote anatomical and functional plasticity of rubrospinal axons,

reduce lesion size, and attenuate thermal sensitivity following cervical crush.
Lu et al. (2012) have recently described the application of cAMP and BDNF fol-
lowing either partial midcervical or complete upper thoracic spinal cord transections.
The combined treatment generated significant axon regeneration and synaptogenesis
beyond both the C5 hemisection and the T3 transection. However, both locomotor
function and spasticity worsened. These data highlight the need not only for an op-
timized treatment combination but also additional control in shaping the process of
axonal regeneration. This idea was emphasized through the work of Garcia-Alias
et al. (2009). Following acute dorsal column injury, the combination of ChABC with
task-specific physical rehabilitation enhanced functional recovery of forepaw motor
function further than either treatment strategy alone or ChABC combined with a non-
specific rehabilitation program (Garcia-Alias et al., 2009). These data suggest, fol-
lowing SCI, driving plasticity through specific rehabilitation to form functional
neuronal connections can enhance the benefit attained. These data were furthered
by Wang et al. (2011) whom showed similar effects following a chronic crush injury
model, highlighting the clinical applicability of this strategy. Furthermore,
Weishaupt et al. (2013) used a C3/4 dorsal quadrant spinal lesion to demonstrate that
viral administration of BDNF combined with task-specific rehabilitation provides a
beneficial synergistic effect on functional recovery of forepaw motor function than
either strategy alone. Significantly, the anatomical regeneration of the corticospinal
and rubrospinal tracts was not different between treatment groups. This indicates
that, following cervical SCI, rehabilitation directs the plasticity induced by specific
treatment strategies into a functional rather than maladaptive response, shaping the
outcome of recovery (Ferguson et al., 2012).
The functional use of rehabilitation following SCI is not unusual. It has long been
known that locomotor training enhances messenger ribonucleic acid and protein ex-
pression of neuronal growth and plasticity factors including BDNF, NT-3, fibroblast
growth factor-2, and insulin-like growth factor (Hutchinson et al., 2004; Ying et al.,
2005). In particular, the level of BDNF and its receptor TrkB are specifically regu-
lated in subpopulations of motor neurons following SCI rehabilitation and may mod-
ulate the efficacy of synaptic functions (Gomez-Pinilla et al., 2002; Macias et al.,
2007, 2009; Skup et al., 2002; Ying et al., 2005, 2008). Exercise has also been shown
to restore levels of glycine and GAD-67, a synthetic enzyme for GABA, to postinjury
levels facilitating neuronal functioning (Edgerton et al., 2001; Khristy et al., 2009;
Tillakaratne et al., 2000). However, locomotor training post-SCI acts to increase
plasticity, but is not sufficient to mediate gross structural reorganization (De Leon
and Acosta, 2006; De Leon et al., 2002; Dietz et al., 1994; Leblond et al., 2003;
Petruska et al., 2007). It is the combination of task-specific rehabilitation and the
induction of neural plasticity that mediates these effects. In the case of respiratory
motor function following cervical SCI, such rehabilitation could take the form of
IH or the use of ChR2 transfection in the C4 ventral horn with light stimulation. Both
treatment strategies induce functional effects upon respiratory motor function in their
own right and are known to specifically drive respiratory activity. However, this
196 CHAPTER 10 The challenges of respiratory motor system recovery

effect could be amplified if neural plasticity was further induced through the appli-
cation of ChABC, cAMP, or neurotrophic factors. Such strategies may result in a
rapid or higher level of functional recovery than that previously demonstrated and
are currently being pursued in our laboratory.

6.2 ASSESSMENT OF TREATMENT STRATEGIES CURRENTLY

OVERLOOKED IN THE RESPIRATORY MOTOR SYSTEM MODEL
There exists a significant body of research into the intrinsic and extrinsic mecha-
nisms that can facilitate functional respiratory motor system recovery following cer-
vical SCI. However, a number of other highly successful treatment strategies have
been identified in alternative injury models that have yet to be assessed within the
respiratory motor system. For example, the transmembrane Eph receptors
(EphA3, EphA4, EphA6, EphA8, and EphB3) and ephrins (ephrin-B2) are upregu-
lated in astrocytes, oligodendrocytes, motor neurons, and meningeal fibroblasts fol-
lowing SCI (Bundesen et al., 2003; Goldshmit et al., 2004; Miranda et al., 1999;
Willson et al., 2002, 2003) some of which have been shown to facilitate regeneration
failure (Fabes et al., 2007). The down-regulation of these receptors has been shown
to promote functional recovery by mediating inflammation and facilitating neuronal
extension (Benson et al., 2005; Goldshmit et al., 2004). A similar intrinsic factor,
specific integrin receptors have been implicated in facilitating axonal regeneration.
For example, a7-deficient animals exhibit impaired axon regeneration (Ekström
et al., 2003), while lentivirus-mediated overexpression of a9 facilitated axonal
regeneration in vivo (Andrews et al., 2009). Similarly, inhibition of RhoA (Ras
homolog gene family A), or its downstream effector Rho-associated kinase, have
generated significant neuronal regeneration following CNS injury (Dergham
et al., 2002; Lehmann et al., 1999). This treatment has exhibited promising results
during initial clinical trial (Fehlings et al., 2011). Additionally, it has recently been
shown that taxol (the clinically approved anticancer drug Paclitaxel) can facilitate
axon regeneration and neurite outgrowth in the adult spinal cord and visual system
through the stabilization of microtubules (Erturk et al., 2007; Hellal et al., 2011;
Sengottuvel et al., 2011). Our laboratory is currently pursuing the assessment of taxol
within the respiratory motor system following cervical SCI.
In terms of extrinsic factors not currently assessed within the respiratory motor
system, myelin-derived molecules express a multitude of inhibitory factors including
NogoA (Caroni and Schwab, 1988, 1989), myelin-associated glycoprotein
(McKerracher et al., 1994; Mukhopadhyay et al., 1994), and oligodendrocyte–
myelin glycoprotein (Kottis et al., 2002). Through the use of antibodies, knockout
models, and enzyme-related inhibition to NogoA (Brosamle et al., 2000; Caroni
and Schwab, 1988; GrandPre et al., 2002; Schnell and Schwab, 1990) long-distance
functional regeneration and plasticity of motor neurons following SCI has been
achieved (Fouad et al., 2004; Freund et al., 2007; Maier et al., 2009; Schnell and
Schwab, 1990). The proven efficacy of anti-NogoA antibody treatment in nonhuman
primate models of SCI (Fouad et al., 2004; Freund et al., 2006, 2007) have lead to
 7 Concluding remarks 197

Phase 1 clinical trials as an acute treatment for human SCI patients (Abel et al.,
2011). Alternatively, application of molecules such as polysialic acid (PSA) could
aid functional regeneration of the injured respiratory motor system. PSA is a
cell-surface glycan added to glycoproteins during post-translational modification
(Rutishauser, 2008) which, when attached to the cellular surface, prevents adhesion
due to the large hydrated volume (Rutishauser, 2008). Following injury, axonal
growth occurs in close proximity to astrocytes expressing PSA (Dusart et al.,
1999), possibly facilitating regeneration by shielding axons from inhibitory cues.
Expression of the molecule, or mimetic peptides, in vivo facilitates functional regen-
eration (El Maarouf et al., 2006; Marino et al., 2009; Zhang et al., 2007). None of
these factors alone have caused complete functional regeneration of the somatic
motor system following injury. However, neglecting to assess them in the context
of the respiratory motor system is imprudent as it represents a significant gap in
our understanding and knowledge base.

6.3 ASSESSMENT OF MULTIPLE OUTCOME MEASURES

Throughout the course of this review, it has been demonstrated how the advent of
cervical SCI has multiple affects including impairing both the respiratory and loco-
motor systems. However, the strategies employed to treat this injury have universal
effects on all systems. These often involve the induction of plasticity, neuroprotec-
tion, axonal growth, stimulation of drive, and the construction of a permissive envi-
ronment in which functional regeneration can occur. Trumbower et al. (2012)
described the use of 15 episodes of dAIH in a cohort of 13 patients with chronic,
incomplete SCI that caused a functional increase in voluntary ankle movement. Fur-
ther, Hayes et al. (2014) used a similar protocol of dAIH on a cohort of 19 patients
with chronic, incomplete SCI to show that combining this treatment with rehabili-
tation could increase the speed and endurance of overground walking. Lovett-
Barr et al. (2012) described the use of dAIH following chronic C2 hemisection in
a rodent model of SCI to facilitate plasticity and functional recovery of the respira-
tory motor system. However, without somatic motor rehabilitation, the authors ad-
ditionally report a functional improvement in forelimb motor function and seemed to
be related to the anatomical increase in BDNF and TrkB generated within the rele-
vant motor neurons. While further research is required, collectively, these studies
suggest that the treatment strategy applied following cervical SCI may be effective
at improving function in multiple motor systems simultaneously. Subsequently,
when combined, our current methods of repairing the spinal cord following injury
may be more effective then we imagine.

7 CONCLUDING REMARKS
Complete functional recovery of the respiratory motor system following cervical SCI
is, at present, unattainable. Attempts to enhance endogenous functional activity
involve modifying the pharmaceutical profile of the spinal cord, stimulating or
198 CHAPTER 10 The challenges of respiratory motor system recovery

enhancing endogenous activity, and transforming the microenvironment of the injury

site. It is clear that both intrinsic and extrinsic factors play significant roles in the
function of the respiratory motor system. Through manipulation of these systems,
recovery and plasticity can be achieved. However, these accomplishments are
incomplete as functional restoration of activity seldom approaches that demon-
strated before injury. Further to this, the models we use to assess treatment outcomes
are often disparate from that observed clinically being acute lesions and not the
more relevant and severe chronic contusion. Nonetheless, the advent of new models
and investigations of combinatorial treatment strategies provides expectation for
the achievement of functional activity which mirrors that of the uninjured animal.
Additionally, we must learn and remember that cervical SCI is not a series of
individually damaged motor and sensory systems but as a holistic, coordinated
scheme.

ACKNOWLEDGMENTS
This work was supported by funding to W. J. A. from the International Spinal Research Trust
in the UK, the Craig H. Neilsen Foundation, and the MetroHealth Medical Center in Cleve-
land, Ohio, USA. We thank Drs. B. Awad, D. Gutierrez, and K. Hoy for their constructive
comments. The authors also acknowledge Drs. Gert Holstege, Mathias Dutschmann, and Hari
Subramanian, the organizers of the XIIth Oxford Conference on “Breathing, Emotion, and
Evolution,” and Dr. Gordon Mitchell, the Chair of the Spinal Cord Mechanisms session,
for their kind invitation and oversight.

REFERENCES
Abel, R., Baron, H.-C., Casha, S., Harms, J., Hurlbert, J., Kucher, K., Maier, D., Thietje, R.,
Weidner, N., Curt, A., 2011. Therapeutic anti-Nogo-A antibodies in acute spinal cord
injury: safety and pharmacokinetic data from an ongoing first-in-human trial. In: The
International Spinal Cord Society (ISCoS) (Ed.), International Conference on Spinal Cord
Medicine and Rehabilitation, Washington, D.C., USA, p. 16.
Abramov, A.Y., Scorziello, A., Duchen, M.R., 2007. Three distinct mechanisms generate ox-
ygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation.
J. Neurosci. 27, 1129–1138.
Alilain, W.J., Goshgarian, H.G., 2008. Glutamate receptor plasticity and activity regulated cy-
toskeletal associated protein regulation in the phrenic motor nucleus may mediate spon-
taneous recovery of the hemidiaphragm following chronic cervical spinal cord injury. Exp.
Neurol. 212, 348–357.
Alilain, W.J., Li, X., Horn, K.P., Dhingra, R., Dick, T.E., Herlitze, S., Silver, J., 2008. Light
induced rescue of breathing after spinal cord injury. J. Neurosci. 28, 11862–11870.
Alilain, W.J., Horn, K.P., Hu, H., Dick, T.E., Silver, J., 2011. Functional regeneration of re-
spiratory pathways after spinal cord injury. Nature 475, 196–200.
Andrews, M.R., Czvitkovich, S., Dassie, E., Vogelaar, C.F., Faissner, A., Blits, B., Gage, F.H.,
ffrench-Constant, C., Fawcett, J.W., 2009. Alpha9 integrin promotes neurite outgrowth on
tenascin-C and enhances sensory axon regeneration. J. Neurosci. 29, 5546–5557.
 References 199

Asher, R.A., Morgenstern, D.A., Shearer, K.H., Adcock, K.H., Pesheva, P., Fawcett, J.W.,
2002. Versican is upregulated in CNS injury and is a product of oligodendrocyte lineage
cells. J. Neurosci. 22, 2225–2236.
Atwal, J.K., Massie, B., Miller, F.D., Kaplan, D.R., 2000. The TrkB-Shc site signals neuronal
survival and local axon growth via MEK and P13-kinase. Neuron 27, 265–277.
Awad, B.I., Warren, P.M., Steinmetz, M.P., Alilain, W.J., 2013. The role of the crossed
phrenic pathway after cervical contusion injury and a new model to evaluate therapeutic
interventions. Exp. Neurol. 248, 398–405.
Bai, F., Peng, H., Etlinger, J.D., Zeman, R.J., 2010. Partial recovery after complete spinal cord
transection by combined chondroitinase and clenbuterol treatment. Pflugers Arch.
460, 657–666.
Baker-Herman, T.L., Mitchell, G.S., 2002. Phrenic long-term facilitation requires spinal se-
rotonin receptor activation and protein synthesis. J. Neurosci. 22, 6239–6246.
Baker-Herman, T.L., Fuller, D.D., Bavis, R.W., Zabka, A.G., Golder, F.J., Doperalski, N.J.,
Johnson, R.A., Watters, J.J., Mitchell, G.S., 2004. BDNF is necessary and sufficient
for spinal respiratory plasticity following intermittent hypoxia. Nat. Neurosci.
7, 48–55.
Ballanyi, K., Onimaru, H., Homma, I., 1999. Respiratory network function in the isolated
brainstem-spinal cord of newborn rats. Prog. Neurobiol. 59, 583–634.
Barritt, A.W., Davies, M., Marchand, F., Hartley, R., Grist, J., Yip, P., McMahon, S.B.,
Bradbury, E.J., 2006. Chondroitinase ABC promotes sprouting of intact and injured spinal
systems after spinal cord injury. J. Neurosci. 26, 10856–10867.
Baussart, B., Stamegna, J.C., Polentes, J., Tadie, M., Gauthier, P., 2006. A new model of upper
cervical spinal contusion inducing a persistent unilateral diaphragmatic deficit in the adult
rat. Neurobiol. Dis. 22, 562–574.
Benson, M.D., Romero, M.I., Lush, M.E., Lu, Q.R., Henkemeyer, M., Parada, L.F., 2005.
Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proc. Natl. Acad. Sci. U.S.A
102, 10694–10699.
Bessis, A., Béchade, C., Bernard, D., Roumier, A., 2007. Microglial control of neuronal death
and synaptic properties. Glia 55, 233–238.
Bianchi, A.L., Denavit-Saubie, M., Champagnat, J., 1995. Central control of breathing in
mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiol.
Rev. 75, 1–45.
Bjartmar, C., Kinkel, R.P., Kidd, G., Rudick, R.A., Trapp, B.D., 2001. Axonal loss in normal
appearing white matter in a patient with acute MS. Neurology 57, 1248–1252.
Blesch, A., Tuszynski, M.H., 2003. Cellular GDNF delivery promotes growth of motor and
dorsal column sensory axons after partial and complete spinal cord transections and in-
duces remyelination. J. Comp. Neurol. 467, 403–417.
Blight, A.R., 1994. Effects of silica on the outcome from experimental spinal cord injury: im-
plication of macrophages in secondary tissue damage. Neuroscience 60, 263–273.
Bluechardt, M.H., Wiens, M., Thomas, S.G., Plyley, M.J., 1992. Repeated measurements of
pulmonary function following spinal cord injury. Paraplegia 30, 479–488.
Bocchiaro, C.M., Feldman, J.L., 2004. Synaptic activity-dependent persistent plasticity
in endogenously active mammalian motoneurons. Proc. Natl. Acad. Sci. U.S.A
101, 4292–4295.
Borisoff, J.F., Chan, C.C., Hiebert, G.W., Oschipok, L., Robertson, G.S., Zamboni, R.,
Steeves, J.D., Tetzlaff, W., 2003. Suppression of Rho kinase activity promotes axonal
growth on inhibitory CNS substrates. Mol. Cell. Neurosci. 22, 405–416.
200 CHAPTER 10 The challenges of respiratory motor system recovery

Bouhy, D., Malgrange, B., Multon, S., Poirrier, A.L., Scholtes, F., Schoenen, J., Franzen, R.,
2006. Delayed GM-CSF treatment stimulates axonal regeneration and functional recovery
in paraplegic rats via an increased BDNF expression by endogenous macrophages. FASEB
J. 20, 1239–1241.
Boulenguez, P., Gauthier, P., Kastner, A., 2007. Respiratory neuron subpopulations and path-
ways potentially involved in the reactiviation of phrenic motorneurons after C2 hemisec-
tion. Brain Res. 1148, 96–104.
Bradbury, E.J., Moon, L.D., Popat, R.J., King, V.R., Bennett, G.S., Patel, P.N., Fawcett, J.W.,
McMahon, S.B., 2002. Chondroitinase ABC promotes functional recovery after spinal
cord injury. Nature 416, 636–640.
Bretzner, F., Liu, J., Currie, E., Roskams, A.J., Tetzlaff, W., 2008. Undesired effects of a com-
binatorial treatment for spinal cord injury-transplantation of olfactory ensheathing cells
and BDNF infusion into the red nucleus. J. Neurosci. Res. 88, 2833–2846.
Bretzner, F., Plemel, J.R., Liu, J., Richter, M., Roskams, A.J., Tetzlaff, W., 2010. Combination
of olfactory ensheathing cells with local versus systemic cAMP treatment after a cervical
rubospinal tract injury. J. Neurosci. Res. 88, 2833–2846.
Brosamle, C., Huber, A.B., Fiedler, M., Skerra, A., Schwab, M.E., 2000. Regeneration of le-
sioned corticospinal tract fibers in the adult rat induced by a recombinant, humanized IN-1
antibody fragment. J. Neurosci. 20, 8061–8068.
Bundesen, L.Q., Scheel, T.A., Bregman, B.S., Kromer, L.F., 2003. Ephrin-B2 and EphB2 reg-
ulation of astrocyte-meningeal fibroblast interactions in response to spinal cord lesions in
adult rats. J. Neurosci. 23, 7789–7800.
Busch, S.A., Horn, K.P., Silver, D.J., Silver, J., 2009. Overcoming macrophage-mediated ax-
onal dieback following CNS injury. J. Neurosci. 29, 9967–9976.
Buss, A., Brook, G.A., Kakulas, B., Martin, D., Franzen, R., Schoenen, J., Noth, J., Schmitt, A.B.,
2004. Gradual loss of myelin and formation of an astrocytic scar during Wallerian
degeneration in the human spinal cord. Brain 127, 34–44.
Cafferty, W.B., Yang, S.H., Duffy, P.J., Li, S., Strittmatter, S.M., 2007. Functional axonal re-
generation through astrocytic scar genetically modified to digest chondroitin sulfate pro-
teoglycans. J. Neurosci. 27, 2176–2185.
Cafferty, W.B., Bradbury, E.J., Lidierth, M., Jones, M., Duffy, P.J., Pezet, S., McMahon, S.B.,
2008. Chondroitinase ABC-mediated plasticity of spinal sensory function. J. Neurosci.
28, 11998–12009.
Cai, D., Shen, Y., De Bellard, M., Tang, S., Filbin, M.T., 1999. Prior exposure to neurotrophins
blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent
mechanism. Neuron 22, 89–101.
Campbell, S.C., Krueger, R.C., Schwartz, N.B., 1990. Deglycosylation of chondroitin sulfate
proteoglycan and derived peptides. Biochemistry 29, 907–914.
Caroni, P., Schwab, M.E., 1988. Antibody against myelin-associated inhibitor of neurite
growth neutralizes nonpermissive substrate properties of CNS white matter. Neuron
1, 85–96.
Caroni, P., Schwab, M.E., 1989. Codistribution of neurite growth inhibitors and oligodendro-
cytes in rat CNS: appearance follows nerve fiber growth and precedes myelination. Dev.
Biol. 136, 287–295.
Carter, L.M., Starkey, M.L., Akrimi, S.F., Davies, M., McMahon, S.B., Bradbury, E.J., 2008.
The yellow fluorescent protein (YFP-H) mouse reveals neuroprotection as a novel mech-
anism underlying chondroitinase ABC-mediated repair after spinal cord injury.
J. Neurosci. 28, 14107–14120.
 References 201

Castellani, V., Falk, J., Rougon, G., 2004. Semaphorin3A-induced receptor endocytosis
during axon guidance responses is mediated by L1 CAM. Mol. Cell. Neurosci.
26, 89–100.
Chao, M.V., 2003. Neurotrophins and their receptors: a convergence point for many signalling
pathways. Nat. Rev. Neurosci. 4, 299–309.
Chatfield, P.O., Mead, S., 1948. Role of the vagi in the crossed phrenic phenomenon. Am. J.
Physiol. 54, 417–422.
Chau, C.H., Shum, D.K., Li, H., Pei, J., Lui, Y.Y., Wirthlin, L., Chan, Y.S., Xu, X.M., 2004.
Chondroitinase ABC enhances axonal regrowth through Schwann cell-seeded guidance
channels after spinal cord injury. FASEB J. 18, 194–196.
Chierzi, S., Ratto, G.M., Verma, P., Fawcett, J.W., 2005. The ability of axons to regenerate
their growth cones depends on axonal type and age, and is regulated by calcium, cAMP
and ERK. Eur. J. Neurosci. 21, 2051–2062.
Chitravanshi, V.C., Sapru, H.N., 1996. NMDA as well as non-NMDA receptors mediate the
neurotransmission of inspiratory drive to phrenic motoneurons in the adult rat. Brain Res.
715, 104–112.
Choi, H., Liao, W.-L., Newton, K.W., Onario, R.C., King, A.M., Desilets, F.C., Woodard, E.J.,
Eichler, M.E., Frontera, W.R., Subharwal, S., Teng, Y.D., 2005. Respiratory abnormalities
resulting from midcervical spinal cord injury and their reversal by serotonin 1A agonists in
conscious rats. J. Neurosci. 25, 4550–4559.
Coull, J., Beggs, S., Boudreau, D., Boivin, D., Tsuda, M., Inoue, K., Gravel, C., Salter, M., De
Koninck, Y., 2005. BDNF from microglia causes the shift in neuronal anion gradient un-
derlying neuropathic pain. Nature 438, 1017–1021.
Curinga, G.M., Snow, D.M., Mashburn, C., Kohler, K., Thobaben, R., Caggiano, A.O.,
Smith, G.M., 2007. Mammalian-produced chondroitinase AC mitigates axon inhibition
by chondroitin sulfate proteoglycans. J. Neurochem. 102, 275–288.
Dale, E.A., Mabrouk, R.B., Mitchell, G.S., 2014. Unexpected benefits of intermittent hypoxia:
enhanced respiratory and nonrespiratory motor function. Physiology 29, 39–48.
Dale, E.A., Mitchell, G.S., 2013. Spinal vascular endothelial growth factor (VEGF) and
erythropoietin (EPO) induced phrenic motor facilitation after repetitive acute intermitant
hypoxia. Respir. Physiol. Neurobiol. 185, 481–488.
Dale, E.A., Satriotomo, I., Mitchell, G.S., 2012. Cervical spinal erythropoietin induces phrenic
motor facilitation via ERK and Akt signaling? J. Neurosci. 32, 5973–5983.
Dale-Nagle, E.A., Satriotomo, I., Mitchell, G.S., 2011. Spinal vascular endothelial growth fac-
tor induces phrenic motor facilitation via ERK and Akt signaling. J. Neurosci.
31, 7682–7690.
Darlot, F., Cayetanot, F., Gauthier, P., Matarazzo, V., Kastner, A., 2012. Extensive respiratory
plasticity after cervical spinal cord injury in rats: axonal sprouting and rerouting of ven-
trolateral bulbospinal pathways. Exp. Neurol. 236, 88–102.
Dauchy, R.T., Dauchy, E.M., Tirrell, R.P., Hill, C.R., Davidson, L.K., Greene, M.W.,
Tirrell, P.C., Wu, J., Sauer, L.A., Blask, D.E., 2010. Dark-phase light contamination dis-
rupts circadian rhythms in plasma measures of endocrine physiology and metabolism in
rats. Comp. Med. 60, 348–356.
Davalos, D., Grutzendler, J., Yang, G., Kim, J.V., Zuo, Y., Jung, S., Littman, D.R., Dustin, M.L.,
Gan, W.B., 2005. ATP mediates rapid microglial response to local brain injury in vivo.
Nat. Neurosci. 8, 752–758.
David, S., Aguayo, A.J., 1981. Axonal elongation into peripheral nervous system bridges after
central nervous system injury in adult rats. Science 241, 931–933.
202 CHAPTER 10 The challenges of respiratory motor system recovery

Davies, J.G., Kirkwood, P.A., Sears, T.A., 1985. The distribution of monosynaptic connexions
from inspiratory bulbospinal neurons to inspiratory motorneurones in the cat. J. Physiol.
368, 63–87.
Davies, S.J., Fitch, M.T., Memberg, S.P., Hall, A.K., Raisman, G., Silver, J., 1997. Regener-
ation of adult axons in white matter tracts of the central nervous system. Nature
390, 680–683.
Davies, S.J., Goucher, D.R., Doller, C., Silver, J., 1999. Robust regeneration of adult sensory
axons in degenerating white matter of the adult rat spinal cord. J. Neurosci. 19, 5810–5822.
De Leon, R.D., Acosta, C.N., 2006. Effect of robotic-assisted treadmill training and chronic
quipazine treatment on hindlimb stepping in spinally transected rats. J. Neurotrauma
23, 1147–1163.
De Leon, R.D., Reinkensmeyer, D.J., Timoszyk, W.K., London, N.J., Roy, R.R., Edgerton, V.R.,
2002. Use of robotics in assessing the adaptive capacity of the rat lumbar spinal cord. Prog.
Brain Res. 137, 141–149.
De Winter, F., Oudega, M., Lankhorst, A.J., Hamers, F.P., Blits, B., Ruitenberg, M.J.,
Pasterkamp, R.J., Gispen, W.H., Verhaagen, J., 2002. Injury-induced class 3 semaphorin
expression in the rat spinal cord. Exp. Neurol. 175, 61–75.
Decherchi, P., Gauthier, P., 2002. Regeneration of acutely and chronically injured descending
respiratory pathways within post-traumatic nerve grafts. Neuroscience 112, 141–152.
Decherchi, P., Lammari-Barreault, N., Gauthier, P., 1996. Regeneration of respiratory path-
ways within spinal peripheral nerve grafts. Exp. Neurol. 137, 1–14.
Dergham, P., Ellezam, B., Essagian, C., Avedissian, H., Lubell, W.D., McKerracher, L., 2002.
Rho signaling pathway targeted to promote spinal cord repair. J. Neurosci. 22, 6570–6577.
Dietz, V., Colombo, G., Jensen, L., 1994. Locomotor activity in spinal man. Lancet
344, 1260–1263.
Dobbins, E.G., Feldman, J.L., 1994. Brainstem network controlling descending drive to
phrenic motorneurons in rat. J. Comp. Neurol. 347, 64–86.
Doperalski, N.J., Sandhu, M.S., Bavis, R.W., Reier, P.J., Fuller, D.D., 2008. Ventilation and
phrenic output following high cervical spinal hemisection in male vs. female rats. Respir.
Physiol. Neurobiol. 162 (2), 160–167.
Dou, C.L., Levine, J.M., 1994. Inhibition of neurite growth by the NG2 chondroitin sulphate
proteoglycan. J. Neurosci. 19, 8778–8788.
Dusart, I., Morel, M.P., Wehrle, R., Sotelo, C., 1999. Late axonal sprouting of injured Purkinje
cells and its temporal correlation with permissive changes in the glial scar. J. Comp. Neu-
rol. 408, 399–418.
Edgerton, V.R., McCall, G.E., Hodgson, J.A., Gotto, J., Goulet, C., Fleischmann, K., Roy, R.R.,
2001. Sensorimotor adaptations to microgravity in humans. J. Exp. Biol. 204, 3217–3224.
Ekström, P.A., Mayer, U., Panjwani, A., Pountney, D., Pizzey, J., Tonge, D.A., 2003. Involve-
ment of alpha7beta1 integrin in the conditioning-lesion effect on sensory axon regenera-
tion. Mol. Cell. Neurosci. 22, 383–395.
El Maarouf, A., Petridis, A.K., Rutishauser, U., 2006. Use of polysialic acid in repair of the
central nervous system. Proc. Natl. Acad. Sci. U.S.A 103, 16989–16994.
El-Bohy, A.A., Schrimsher, G.W., Reier, P.J., Goshgarian, H.G., 1998. Quantitative assess-
ment of respiratory function following contusion injury of the cervical spinal cord.
Exp. Neurol. 150, 143–152.
Ellenberger, H.H., Feldman, J.L., 1988. Monosynaptic transmission of respiratory drive to
phrenic motorneurons from brainstem bulbospinal neurons in rats. J. Comp. Neurol.
269, 47–57.
 References 203

Erickson, J.T., Millhorn, D.E., 1994. Hypoxia and electrical stimulation of the carotid sinus
nerve induce Fos-like immunoreactivity with catecholaminergic and serotonergic neurons
of the rat brainstem. J. Comp. Neurol. 348, 161–182.
Erickson, J.T., Conover, J.C., Borday, V., Champagnat, J., Barbacid, M., Yancopoulos, G.,
Katz, D.M., 1996. Mice lacking brain-derived neurotrophic factor exhibit visceral sensory
neuron losses distinct from mice lacking NT4 and display a severe developmental deficit
in control of breathing. J. Neurosci. 16, 5361–5371.
Erturk, A., Hellal, F., Enes, J., Bradke, F., 2007. Disorganized microtubules underlie the for-
mation of retraction bulbs and the failure of axonal regeneration. J. Neurosci.
27, 9169–9180.
Fabes, J., Anderson, P., Brennan, C., Bolsover, S., 2007. Regeneration enhancing effects of
EphA4 blocking peptide following corticospinal tract injury in adult rat spinal cord.
Eur. J. Neurosci. 26, 2496–2505.
Fehlings, M.G., Theodore, N., Harrop, J., Maurais, G., Kuntz, C., Shaffrey, C.I., Kwon, B.K.,
Chapman, J., Yee, A., Tighe, A., McKerracher, L., 2011. A phase I/IIa clinical trial of a
recombinant Rho protein antagonist in acute spinal cord injury. J. Neurotrauma
28, 787–796.
Fein, E.D., Grimm, D.R., Lesser, M., Bauman, W.A., Almenoff, P.L., 1998. The effects of
ipratropium bromide on histamine-induced bronchoconstriction in subjects with cervical
spinal cord injury. J. Asthma 35, 49–55.
Ferguson, A.R., Huie, J.R., Crown, E.D., Baumbauer, K.M., Hook, M.A., Garraway, S.M.,
Lee, K.H., Hoy, K.C., Grau, J.W., 2012. Maladaptive spinal plasticity opposes spinal
learning and recovery in spinal cord injury. Front. Physiol. 3, 1–17.
Fidler, P.S., Schuette, K., Asher, R.A., Dobbertin, A., Thornton, S.R., Calle-Patino, Y.,
Muir, E., Levine, J.M., Geller, H.M., Rogers, J.H., Faissner, A., Fawcett, J.W., 1999. Com-
paring astrocytic cell lines that are inhibitory or permissive for axon growth: the major
axon-inhibitory proteoglycan is NG2. J. Neurosci. 19, 877–8788.
Fleming, J.C., Bao, F., Chen, Y., Hamilton, E.F., Relton, J.K., Weaver, L.C., 2008. Alpha4-
beta1 integrin blockade after spinal cord injury decreases damage and improves neurolog-
ical function. Exp. Neurol. 214, 147–159.
Fleming, J.C., Bao, F., Chen, Y., Hamilton, E.F., Gonzalez-Lara, L.E., Foster, P.J., Weaver, L.C.,
2009. Timing and duration of anti-alpha4beta1 integrin treatment after spinal cord injury:
effect on therapeutic efficacy. J. Neurosurg. Spine 11, 575–587.
Fletcher, E.X., Lesske, J., Qian, W., Miller 3rd., C.C., Unger, T., 1992. Repetitive, episodic
hypoxia causes diurnal elevation of blood pressure in rats. Hypertension 19, 555–561.
Fouad, K., Pedersen, V., Schwab, M.E., Brosamle, C., 2001. Cervical sprouting of corticosp-
inal fibers after thoracic spinal cord injury accompanies shifts in evoked motor responses.
Curr. Biol. 11, 1766–1770.
Fouad, K., Klusman, I., Schwab, M.E., 2004. Regenerating corticospinal fibers in the Marmo-
set (Callitrix jacchus) after spinal cord lesion and treatment with the anti-Nogo-A antibody
IN-1. Eur. J. Neurosci. 20, 2479–2482.
Fouad, K., Schnell, L., Bunge, M.B., Schwab, M.E., Liebscher, T., Pearse, D.D., 2005. Com-
bining Schwann cell bridges and olfactory-ensheathing glia grafts with chondroitinase pro-
motes locomotor recovery after complete transection of the spinal cord. J. Neurosci.
25, 1169–1178.
Fouad, K., Pearse, D.D., Tetzlaff, W., Vavrek, R., 2009. Transplantation and repair: combined
cell implantation and chondroitinase delivery prevents deterioration of bladder function in
rats with complete spinal cord injury. Spinal Cord 47, 727–732.
204 CHAPTER 10 The challenges of respiratory motor system recovery

Fredholm, B.B., Hedquist, P., 1980. Modulation of neurotransmission by purine nucleotides

and nucleosides. Biochem. Pharmacol. 29, 1635–1643.
Freund, P., Schmidlin, E., Wannier, T., Bloch, J., Mir, A., Schwab, M.E., Rouiller, E.M., 2006.
Nogo-A-specific antibody treatment enhances sprouting and functional recovery after cer-
vical lesion in adult primates. Nat. Med. 12, 790–792.
Freund, P., Wannier, T., Schmidlin, E., Bloch, J., Mir, A., Schwab, M.E., Rouiller, E.M., 2007.
Anti-Nogo-Aantibody treatment enhances sprouting of corticospinal axons rostral to a uni-
lateral cervical spinal cord lesion in adult macaque monkey. J. Comp. Neurol.
502, 644–659.
Friedlander, D.R., Milev, P., Karthikeyan, L., Margolis, R.K., Margolis, R.U., Grumet, M.,
1994. The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell ad-
hesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and
neurite outgrowth. J. Cell Biol. 125, 669–680.
Fuller, D.D., Baker, T.L., Behan, M., Mitchell, G.S., 2001a. Expression of hypoglossal long-
term facilitation differs between substrains of Sprague-Dawley rat. Physiol. Genomics
4 (3), 175–181.
Fuller, D.D., Zabka, A.G., Baker, T.L., Mitchell, G.S., 2001b. Phrenic long-term facilitation
requires 5-HT receptor activation during but not following episodic hypoxia. J. Appl. Phy-
siol. 90, 2001–2006.
Fuller, D.D., Johnson, S.M., Johnson, R.A., Mitchell, G.S., 2002. Chronic cervical spinal sen-
sory denervation reveals ineffective spinal pathways to phrenic motor neurons in the rat.
Neurosci. Lett. 323, 25–28.
Fuller, D.D., Johnson, S.M., Olson Jr., E.B., Mitchell, G.S., 2003. Synaptic pathways to
phrenic motorneurons are enhanced by chronic intermittent hypoxia after cervical spinal
cord injury. J. Neurosci. 23, 2993–3000.
Fuller, D.D., Baker-Herman, T.L., Golder, F.J., Doperalski, N.J., Watters, J.J., Mitchell, G.S.,
2005. Cervical spinal cord injury upregulates ventral spinal 5-HT2A receptors.
J. Neurotrauma 22, 203–213.
Fuller, D.D., Golder, F.J., Olson, E.B., Mitchell, G.S., 2006. Recovery of phrenic activity and
ventilation after cervical spinal hemisection in rats. J. Appl. Physiol. 100, 800–806.
Fuller, D.D., Doperalki, N.J., Dougherty, B.J., Sandhu, M.S., Bolser, D.C., Reier, P.J., 2008.
Modest spontaneous recovery of ventilation following chronic high cervical hemisection
in rats. Exp. Neurol. 21, 97–106.
Fuller, D.D., Sandhu, M.S., Doperalski, N.J., Lane, M.A., White, T.E., Bishop, M.D., Reier, P.J.,
2009. Graded unilateral cervical spinal cord injury and respiratory motor recovery. Respir.
Physiol. Neurobiol. 165, 245–253.
Funakoshi, H., Frisen, J., Barbany, G., Timmusk, T., Zachrisson, O., Verge, V.M., Persson, H.,
1993. Differential expression of mRNAs for neurotrophins and their receptors after axot-
omy of the sciatic nerve. J. Cell Biol. 123, 455–465.
Gallo, G., Letourneau, P.C., 1998. Localized sources of neurotrophins initiate axon collateral
sprouting. J. Neurosci. 18, 5403–5414.
Gao, C., Che, L.W., Chen, J., Xu, X.J., Chi, Z.Q., 2003. Ohmefentanyl stereoisomers induce
changes of CREB phosphorylation in hippocampus of mice in conditioned place prefer-
ence paradigm. Cell Res. 13, 29–34.
Gao, Y., Deng, K., Hou, J., Bryson, J.B., Barco, A., Nikulina, E., Spencer, T., Mellado, W.,
Kandel, E.R., Filbin, M.T., 2004. Activated CREB is sufficient to overcome inhibitors in
myelin and promote spinal axon regeneration in vivo. Neuron 44, 609–621.
 References 205

Garcia-Alias, G., Barkhuysen, S., Buckle, M., Fawcett, J.W., 2009. Chondroitinase ABC treat-
ment opens a window of opportunity for task-specific rehabilitation. Nat. Neurosci.
12, 1145–1151.
Gaspar, P., Cases, O., Maroteaux, L., 2003. The developmental role of serotonin: news from
mouse molecular genetics. Nat. Rev. Neurosci. 4, 1002–1012.
Gauthier, P., Lammari-Barreault, N., 1992. Central respiratory neurons of the adult rat regrow
axons preferentially into peripheral nerve autografts implanted within ventral rather than
within dorsal parts of the medulla oblongata. Neurosci. Lett. 137, 33–36.
Gauthier, P., Rasminsky, M., 1988. Activity of medullary respiratory neurons regenerating
axons into peripheral nerve grafts in the adult rat. Brain Res. 438, 225–236.
Gauthier, P., Rega, P., Lammari-Barreault, N., Polentes, J., 2002. Functional reconnections
established by central respiratory neurons regenerating axons into a nerve graft bridging
the respiratory centers to the cervical spinal cord. J. Neurosci. Res. 70, 65–81.
Gauthier, P., Baussart, B., Stamegna, J.C., Tadie, M., Vinit, S., 2006. Diaphragm recovery by
laryngeal innervation after bilareral phrenicotomy or complete C2 spinal section in rats.
Neurobiol. Dis. 24, 53–66.
Gavazzi, I., Stonehouse, J., Sandvig, A., Reza, J.N., Appiah-Kubi, L.S., Keynes, R., Cohen, J.,
2000. Peripheral, but not central, axotomy induces neuropilin-1 mRNA expression in adult
large diameter primary sensory neurons. J. Comp. Neurol. 423, 492–499.
Ghosh, A., Haiss, F., Sydekum, E., Schneider, R., Gullo, M., Wyss, M.T., Mueggler, T.,
Baltes, C., Rudin, M., Weber, B., Schwab, M.E., 2010. Rewiring of hindlimb corticospinal
neurons after spinal cord injury. Nat. Neurosci. 13, 97–104.
Giger, R.J., Urquhart, E.R., Gillespie, S.K., Levengood, D.V., Ginty, D.D., Kolodkin, A.L.,
1998. Neuropilin-2 is a receptor for semaphorin IV: insight into the structural basis of re-
ceptor function and specificity. Neuron 21, 1079–1092.
Golan, M.H., Mane, R., Molczadzki, G., Zuckerman, M., Kaplan-Louson, V., Huleihel, M.,
Perez-Polo, J.R., 2009. Impaired migration signaling in the hippocampus following pre-
natal hypoxia. Neuropharmacology 57, 511–522.
Golder, F.J., Mitchell, G.S., 2005. Spinal synaptic enhancement with acute intermittent hyp-
oxia improves respiratory function after chronic cervical spinal cord injury. J. Neurosci.
25, 2925–2932.
Golder, F.J., Ranganathan, L., Satriotomo, I., Hoffman, M., Lovett-Barr, M.R., Watters, J.J.,
Baker-Herman, T.L., Mitchell, G.S., 2008. Spinal adenosine A2a receptor activation
elicits long-lasting phrenic motor facilitation. J. Neurosci. 28, 2033–2042.
Golder, F.J., Fuller, D.D., Lovett-Barr, M.R., Vinit, S., Resnick, D.K., Mitchell, G.S., 2011.
Breathing patterns after mid-cervical spinal contusion in rats. Exp. Neurol. 231, 97–103.
Goldshmit, Y., Galea, M.P., Wise, G., Bartlett, P.F., Turnley, A.M., 2004. Axonal regeneration
and lack of astrocytic gliosis in EphA4-deficient mice. J. Neurosci. 24, 10064–10073.
Gomez-Pinilla, F., Ying, Z., Roy, R.R., Molteni, R., Edgerton, V.R., 2002. Voluntary exercise
induces a BDNF-mediated mechanism that promotes neuroplasticity. J. Neurophysiol.
88, 2187–2195.
Goshgarian, H.G., 1981. The role of cervical afferent nerve fiber inhibition of the crossed
phrenic phenomenon. Exp. Neurol. 72, 211–225.
Goshgarian, H.G., Guth, L., 1977. Demonstration of functionally ineffective synapses in the
guinea pig spinal cord. Exp. Neurol. 57, 613–621.
Goshgarian, H.G., Rafols, J.A., 1984. The ultrastructure and synaptic architecture of phrenic
motor neurons in the spinal cord of the adult rat. J. Neurocytol. 13, 85–109.
206 CHAPTER 10 The challenges of respiratory motor system recovery

Goshgarian, H.G., Ellenberger, H.H., Feldman, J.L., 1991. Decussation of bulbospinal respi-
ratory axons at the phrenic level of the phrenic nuclei in adult rats: a possible substrate for
the crossed phrenic phenomenon. Exp. Neurol. 111, 135–139.
Gourine, A.V., Kasymov, V., Marina, N., Tang, F., Figueiredo, M.F., Lane, S.,
Teschemacher, A.G., Spyer, K.M., Deisseroth, K., Kasparov, S., 2010. Astrocytes control
breathing through pH-dependent release of ATP. Science 329, 571–575.
Gozal, D., Daniel, J.M., Dohanich, G.P., 2001. Behavioral and anatomical correlates for
chronic episodic hypoxia during sleep in the rat. J. Neurosci. 21, 2442–2450.
GrandPre, T., Li, S., Strittmatter, S.M., 2002. Nogo-66 receptor antagonist peptide promotes
axonal regeneration. Nature 417, 547–551.
Gray, P.A., Janczewski, W.A., Mellen, N., McCrimmon, D.R., Felman, J.L., 2001. Normal
breathing requires preBotzinger complex neurokinin-1 receptor-expressing neurons.
Nat. Neurosci. 4, 927–930.
Greer, J.J., Funk, G.D., Ballanyi, K., 2006. Preparing for the first breath: prenatal maturation of
respiratory neural control. J. Physiol. 570, 437–444.
Griesbeck, O., Parsadanian, A.S., Sendtner, M., Thoenen, H., 1995. Expression of neurotro-
phins in skeletal muscle: quantitative comparison and significance for motoneuron sur-
vival and maintenance of function. J. Neurosci. Res. 42, 21–33.
Grill, R., Murai, K., Blesch, A., Gage, F.H., Tuszynski, M.H., 1997. Cellular delivery of
neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after
spinal cord injury. J. Neurosci. 17, 5560–5572.
Grimm, D.R., Chandy, D., Almenoff, P.L., Schilero, G., Lesser, M., 2000. Airway hyper-
reactivity in subjects with tetraplegia is associated with reduced baseline airway caliber.
Chest 118, 1397–1404.
Gris, D., Marsh, D.R., Oatway, M.A., Chen, Y., Hamilton, E.F., Dekaban, G.A., Weaver, L.C.,
2004. Transient blockade of the CD11d/CD18 integrin reduces secondary damage after
spinal cord injury, improving sensory, autonomic, and motor function. J. Neurosci.
24, 4043–4051.
Gross, G.J., Hardman, H.F., Warltier, D.C., 1976. Adenosine on myocardinal oxygen con-
sumption. Br. J. Pharmacol. 57, 409–412.
Gruner, J.A., 1992. A monitored contusion model of spinal cord injury in the rat.
J. Neurotrauma 9, 123–126.
Guth, L., Zhang, Z., Steward, O., 1999. The unique histopathological responses of the injured
spinal cord. Implications for neuroprotective therapy. Ann. N. Y. Acad. Sci. 890, 366–384.
Gutierrez, D.V., Clark, M., Nawanna, O., Alilain, W.J., 2013. Intermittent hypoxia training
after C2 hemisection modifies the expression of PTEN and mTOR. Exp. Neurol.
248, 45–52.
Gwak, Y.S., Kong, J., Unabia, G.C., Hulsebosch, C.E., 2012. Spatial and temporal activation
of spinal glial cells: role of gliopathy in central neuropathic pain following spinal cord
injury in rats. Exp. Neurol. 234, 362–372.
Ha, Y., Kim, Y.S., Cho, J.M., Yoon, S.H., Park, S.R., Yoon, D.H., Kim, E.Y., Park, H.C., 2005.
Role of granulocyte-macrophage colony-stimulating factor in preventing apoptosis and
improving functional outcome in experimental spinal cord contusion injury.
J. Neurosurg. Spine 2, 55–61.
Hadley, S.D., Walker, P.D., Goshgarian, H.G., 1999a. Effects of serotonin inhibition on neu-
ronal and astrocyte plasticity in the phrenic nucleus 4h following C2 spinal cord hemisec-
tion. Exp. Neurol. 160, 433–445.
 References 207

Hadley, S.D., Walker, P.D., Goshgarian, H.G., 1999b. Effects of the serotonin synthesis inhib-
itor p-CPA on the expression of the crossed phrenic phenomenon 4h following C2 spinal
cord hemisection. Exp. Neurol. 160, 479–488.
Harris, N.G., Mironova, Y.A., Hovda, D.A., Sutton, R.L., 2010. Chondroitinase ABC en-
hances pericontusion axonal sprouting but does not confer robust improvements in behav-
ioral recovery. J. Neurotrauma 27, 1971–1982.
Hayashi, F., Coles, S.K., Bach, K.B., Mitchell, G.S., McCrimmon, D.R., 1993. Time-
dependent phrenic nerve responses to carotid afferent activation: intact vs. decerebellate
rats. Am. J. Physiol. 265, R811–R819.
Hayashi, F., Hinrichsen, C.F., McCrimmon, D.R., 2003. Short-term plasticity of descending
synaptic input to phrenic motorneurons in rats. J. Appl. Physiol. 94, 1421–1430.
Hayes, H.B., Jayaraman, A., Herrmann, M., Mitchell, G.S., Rymer, W.Z., Trumbower, R.D.,
2014. Daily intermittent hypoxia enhances walking after chronic spinal cord injury: a ran-
domized trial. Neurology 82, 104–113.
Hellal, F., Hurtado, A., Ruschel, J., Flynn, K.C., Laskowski, C.J., Umlauf, M., Kapitein, L.C.,
Strikis, D., Lemmon, V., Bixby, J., Hoogenraad, C.C., Bradke, F., 2011. Microtubule sta-
bilization reduces scarring and causes axon regeneration after spinal cord injury. Science
331, 928–931.
Hennigan, A., Trotter, C., Kelly, A.M., 2007. Lipopolysaccharide impairs long-term potenti-
ation and recognition memory and increases p75NTR expression in the rat dentate gyrus.
Brain Res. 1130, 158–166.
Herlenius, E., Lagercrantz, H., 2004. Development of neurotransmitter systems during critical
periods. Exp. Neurol. 190 (Suppl. 1), S8–S21.
Hoffman, M.S., Mitchell, G.S., 2011. Spinal 5-HT7 receptor activation induces long-lasting
phrenic motor facilitation. J. Physiol. 589, 1397–1407.
Hoffman, M.S., Mahamed, S., Golder, F.J., Mitchell, G.S., 2007. Adenosine A2A receptors
con-strain phrenic long term facilitation following acute intermittent hypoxia. FASEB
J. 918, 12.
Holstege, G., Blok, B.F., Ralston, D.D., 1988. Anatomical evidence for red nucleus projections
to motorneuronal cell groups in the spinal cord of the monkey. Neurosci. Lett. 95, 97–101.
Horn, K.P., Busch, S.A., Hawthorne, A.L., van Rooijen, N., Silver, J., 2008. Another barrier to
regeneration in the CNS: activated macrophages induce extensive retraction of dystrophi-
caxons through direct physical interactions. J. Neurosci. 28, 9330–9341.
Houle, J.D., Tom, V.J., Mayes, D., Wagoner, G., Phillips, N., Silver, J., 2006. Combining an
autologous peripheral nervous system "bridge" and matrix modification by chondroitinase
allows robust, functional regeneration beyond a hemisection lesion of the adult rat spinal
cord. J. Neurosci. 26, 7405–7415.
Howard, R.S., Wiles, C.M., Hirsch, N.P., Loh, L., Spencer, G.T., Newsom-Davis, J., 1992.
Respiratory involvement in multiple sclerosis. Brain 115, 479–494.
Huang, W., Matte, A., Suzuki, S., Sugiura, N., Miyazono, H., Cygler, M., 2000. Crystallization
and preliminary X-ray analysis of chondroitin sulphate ABC lyases I and II from Proteus
vulgaris. Acta Crystallogr. 56, 904–906.
Huang, W., Lunin, V.V., Li, Y., Suzuki, S., Sugiura, N., Miyazono, H., Cygler, M., 2003. Crys-
tal structure of Proteus vulgaris chondroitin sulfate ABC lyase 1 at 1.9A resolution. J. Mol.
Biol. 328, 623–634.
Huckstepp, R.T., id Bihi, R., Eason, R., Spyer, K.M., Dicke, N., Willecke, K., Marina, N.,
Gourine, A.V., Dale, N., 2010. Connexin hemichannel-mediated CO2-dependent release
208 CHAPTER 10 The challenges of respiratory motor system recovery

of ATP in the medulla oblongata contributes to central respiratory chemosensitivity.

J. Physiol. 588, 3901–3920.
Hulsebosch, C., 2008. Gliopathy ensures persistent inflammation and chronic pain after spinal
cord injury. Exp. Neurol. 214, 6–9.
Hurtado, A., Marcillo, A., Frydel, B., Bunge, M.B., Bramlett, H.M., Dietrich, W.D., 2012.
Anti-CD11d monoclonal antibody treatment for rat spinal cord compression injury.
Exp. Neurol. 233, 606–611.
Hutchinson, K.J., Gomez-Pinilla, F., Crowe, M.J., Ying, Z., Basso, D.M., 2004. Three exercise
paradigms differentially improve sensory recovery after spinal cord contusion in rats.
Brain 127, 1403–1414.
Huxtable, A.G., Vinit, S., Windelborn, J.A., Crader, S.M., Guenther, C.H., Watters, J.J.,
Mitchell, G.S., 2011. System inflammation improve respiratory chemoreflexes and plas-
ticity. Respir. Physiol. Neurobiol. 178, 482–489.
Iaci, J.F., Vecchione, A.M., Zimber, M.P., Caggiano, A.O., 2007. Chondroitin sulfate proteo-
glycans in spinal cord contusion injury and the effects of chondroitinase treatment.
J. Neurotrauma 24, 1743–1759.
Iarikov, D.E., Kim, B.G., Dai, H.N., McAtee, M., Kuhn, P.L., Bregman, B.S., 2007. Delayed
transplantation with exogenous neurotrophin administration enhances plasticity of corti-
cofugal projections after spinal cord injury. J. Neurotrauma 24, 690–702.
Ip, F.C., Cheung, J., Ip, N.Y., 2001. The expression profiles of neurotrophins and their recep-
tors in rat and chicken tissues during development. Neurosci. Lett. 301, 107–110.
Iseda, T., Okuda, T., Kane-Goldsmith, N., Mathew, M., Ahmed, S., Chang, Y.W., Young, W.,
Grumet, M., 2008. Single, high-dose intraspinal injection of chondroitinase reduces gly-
cosaminoglycans in injured spinal cord and promotes corticospinal axonal regrowth after
hemisection but not contusion. J. Neurotrauma 25, 334–349.
James, E., Nantwi, K.D., 2006. Involvement of peripheral adenosine A2 receptors in adenosine
A1 receptor-mediated recovery of respiratory motor function after upper cervical spinal
cord hemisection. J. Spinal Cord Med. 29, 57–66.
Jefferson, S.C., Tester, N.J., Howland, D.R., 2011. Chondroitinase ABC promotes recovery of
adaptive limb movements and enhances axonal growth caudal to a spinal hemisection.
J. Neurosci. 31, 5710–5720.
Jin, Y., Fischer, I., Tessler, A., Houle, J.D., 2002. Transplants of fibroblasts genetically mod-
ified to express BDNF promote axonal regeneration from supraspinal neurons following
chronic spinal cord injury. Exp. Neurol. 177, 265–275.
Jin, Y., Ketschek, A., Jiang, Z., Smith, G., Fischer, I., 2011. Chondroitinase activity
can be transduced by a lentiviral vector in vitro and in vivo. J. Neurosci. Methods
199, 208–213.
Johnson, R.A., Okragly, A.J., Haak-Frendscho, M., Mitchell, G.S., 2000. Cervical dorsal rhi-
zotomy increases brain-derived neurotrophic factor and neurotrophin-3 expression in the
ventral spinal cord. J. Neurosci. 20, RC77.
Jones, L.L., Margolis, R.U., Tuszynski, M.H., 2003. The chondroitin sulfate proteoglycans
neurocan, brevican, phosphacan, and versican are differentially regulated following spinal
cord injury. Exp. Neurol. 182, 399–411.
Juvin, L., Morin, D., 2005. Descending respiratory polysynaptic inputs to cervical and thoracic
motorneurons diminish during early postnatal maturation in rat spinal cord. Eur. J. Neu-
rosci. 21, 808–813.
Kajana, S., Goshgarian, H.G., 2008a. Administration of phosphodiesterase inhibitors and an
adenosine A1 receptor antagonist induces phrenic nerve recovery in high cervical spinal
cord injured rats. Exp. Neurol. 210, 671–680.
 References 209

Kajana, S., Goshgarian, H.G., 2008b. Spinal activation of the cAMP-PKA pathway induces
respiratory motor recovery following high cervical spinal cord injury. Brain Res.
1232, 206–213.
Karimi-Abdolrezaee, S., Eftekharpour, E., Wang, J., Schut, D., Fehlings, M.G., 2010. Syner-
gistic effects of transplanted adult neural stem/progrnitor cells, chondroitinase, and growth
factors promote functional repair and plasticity of the chronically injured spinal cord.
J. Neurosci. 30, 1657–1676.
Kaushal, V., Koeberle, P.D., Wang, Y., Schlichter, L.C., 2007. The Ca2+-activated K+
channel KCNN4/KCa3.1 contributes to microglia activation and nitric oxide-dependent
neurodegeneration. J. Neurosci. 27, 234–244.
Kerschensteiner, M., Gallmeier, E., Behrens, S., Leal, V.V., Misgeld, T., Klinkert, W.E.,
Kolbeck, R., Hoppe, E., Oropeza-Wekerle, R.L., Bartke, I., Stadelmann, C.,
Lassmann, H., Wekerle, H., Hohlfeld, R., 1999. Activated human T cells, B cells, and
monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain
lesions: a neuroprotective role of inflammation? J. Exp. Med. 189, 865–870.
Khristy, W., Ali, N.J., Bravo, A.B., de Leon, R., Roy, R.R., Zhong, H., London, N.J.,
Edgerton, V.R., Tillakaratne, N.J., 2009. Changes in GABA(A) receptor subunit gamma
2 in extensor and flexor motoneurons and astrocytes after spinal cord transection and mo-
tor training. Brain Res. 1273, 9–17.
Kigerl, K.A., Gensel, J.C., Ankeny, D.P., Alexander, J.K., Donnelly, D.J., Popovich, P.G.,
2009. Identification of two distinct macrophage subsets with divergent effects causing ei-
ther neurotoxicity or regeneration in the injured mouse spinal cord. J. Neurosci.
29, 13435–13444.
Kinkead, R., Mitchell, G.S., 1999. Time-dependent hypoxic ventilatory responses in rats: ef-
fects of ketanserin and 5-carboxamidotryptamine. Am. J. Physiol. 277, R658–R666.
Kirkwood, P.A., 1995. Synaptic excitation in the thoracic spinal cord from expiratory bulbosp-
inal neurones in the cat. J. Physiol. 484, 201–225.
Kishino, A., Nakayama, C., 2003. Enhancement of BDNF and activated-ERK immunoreac-
tivity in spinal motor neurons after peripheral administration of BDNF. Brain Res.
964, 56–66.
Kobayashi, N.R., Fan, D.P., Giehl, K.M., Bedard, A.M., Wiegand, S.J., Tetzlaff, W., 1997.
BDNF and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stim-
ulate GAP-43 and Talpha1-tubulin mRNA expression, and promote axonal regeneration.
J. Neurosci. 17, 9583–9595.
Kottis, V., Thibault, P., Mikol, D., Xiao, Z.C., Zhang, R., Dergham, P., Braun, P.E., 2002.
Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth.
J. Neurochem. 82, 1566–1569.
Kwo, S., Young, W., Decrescito, V., 1989. Spinal cord sodium, potassium, calcium, and
water concentration changes in rats after graded contusion injury. J. Neurotrauma
6, 13–24.
Kwon, C.H., Luikart, B.W., Powell, C.M., Zhou, J., Matheny, S.A., Zhang, W., Li, Y.,
Baker, S.J., Parada, L.F., 2006. Pten regulates neuronal arborization and social interaction
in mice. Neuron 50, 377–388.
Laabs, T.L., Wang, H., Katagiri, Y., McCann, T., Fawcett, J.W., Geller, H.M., 2007. Inhibiting
glycosaminoglycan chain polymerization decreases the inhibitory activity of astrocyte-
derived chondroitin sulfate proteoglycans. J. Neurosci. 27, 14494–14501.
Lammari-Barreault, N., Rega, P., Gauthier, P., 1991. Axonal regeneration from central respi-
ratory neurons of the adult rat into peripheral nerve autografts: effects of graft location
within the medulla. Neurosci. Lett. 125, 121–124.
210 CHAPTER 10 The challenges of respiratory motor system recovery

Lammari-Barreault, N., Rega, P., Gauthier, P., 1994. Central respiratory neuronal activity after
axonal regeneration within blind-ended peripheral nerve grafts: time course of recovery
and loss of functional neurons. Exp. Brain Res. 98, 238–244.
Lane, M.A., White, T.E., Coutts, M.A., Jones, A.L., Sandhu, M.S., Bloom, D.C., Bolser, D.C.,
Yates, B.J., Fuller, D.D., Reier, P.J., 2008. Cervical prephrenic interneurons in the normal
and lesioned spinal cord of the adult rat. J. Comp. Neurol. 511, 692–709.
Lane, M.A., Lee, K.-Z., Salazar, K., O’Steen, B.E., Bloom, D.C., Fuller, D.D., Reier, P.J.,
2012. Respiratory function following bilateral mid-cervical contusion injury in the adult
rat. Exp. Neurol. 235, 197–210.
Larsen, P.H., Wells, J.E., Stallcup, W.B., Opdenakker, G., Yong, V.W., 2003. Matrix
metalloproteinase-9 facilitates remyelination in part by processing the inhibitory NG2 pro-
teoglycan. J. Neurosci. 23, 11127–11135.
Lauder, J.M., 1993. Neurotransmitters as growth regulatory signals: role of receptors and sec-
ond messengers. Trends Neurosci. 16, 233–239.
Leblond, H., L’Esperance, M., Orsal, D., Rossignol, S., 2003. Treadmill locomotion in the
intact and spinal mouse. J. Neurosci. 23, 11411–11419.
Lee, J.K., Geoffroy, C.G., Chan, A.F., Tolentino, K.E., Crawford, M.J., Leal, M.A., Kang, B.,
Zheng, B., 2010. Assessing spinal axon regeneration and sprouting in Nogo-, MAG-, and
OMgp-deficient mice. Neuron 66, 663–670.
Lehmann, M., Fournier, A., Selles-Navarro, I., Dergham, P., Sebok, A., Leclerc, N., Tigyi, G.,
McKerracher, L., 1999. Inactivation of Rho signaling pathway promotes CNS axon regen-
eration. J. Neurosci. 19, 7537–7547.
Lemke, A.K., Sandy, J.D., Voigt, H., Dreier, R., Lee, J.H., Grodzinsky, A.J., Mentlein, R.,
Fay, J., Schunke, M., Kurz, B., 2010. Interleukin-1alpha treatment of meniscal explants
stimulates the production and release of aggrecanase-generated, GAG-substituted aggre-
can products and also the release of pre-formed, aggrecanase-generated G1 and mcalpain-
generated G1–G2. Cell Tissue Res. 340, 179–188.
Lemons, M.L., Howland, D.R., Anderson, D.K., 1999. Chondroitin sulfate proteoglycan im-
munoreactivity increases following spinal cord injury and transplantation. Exp. Neurol.
160, 51–65.
Levitt, P., Harvey, J.A., Friedman, E., Simansky, K., Murphy, E.H., 1997. New evidence for
neurotransmitter influences on brain development. Trends Neurosci. 20, 269–274.
Lewis, L.J., Brookheart, J.M., 1951. Significance of the crossed phrenic phenomenon. Am. J.
Physiol. 166, 241–254.
Li, W.W., Setzu, A., Zhao, C., Franklin, R.J., 2005. Minocycline-mediated inhibition of micro-
glia activation impairs oligodendrocyte progenitor cell responses and remyelination in a
non-immune model of demyelination. J. Neuroimmunol. 158, 58–66.
Ling, L., Bach, K.B., Mitchell, G.S., 1994. Serotonin reveals ineffective spinal pathways to
contralateral phrenic motor neurons in spinally hemisected rats. Exp. Brain Res.
101, 35–43.
Linn, W.S., Spungen, A.M., Gong Jr., H., Adkins, R.H., Bauman, W.A., Waters, R.L., 2001.
Forced vital capacity in two large outpatient populations with chronic spinal cord injury.
Spinal Cord 39, 263–268.
Lipski, J., Duffin, J., Kruszewska, B., Zhang, X., 1993. Upper cervical inspiratory neu-
rons in the rat: and electophysiological and morphological study. Exp. Brain Res.
95, 477–487.
Lipski, J.X., Duffin, J., Kruszewska, B., Kanjhan, R., 1994. Morphological study of long ax-
onal projections of ventral medullary inspiratory neurons in the rat. Brain Res.
640, 171–184.
 References 211

Lipton, S.A., Kater, S.B., 1989. Neurotransmitter regulation of neuronal outgrowth, plasticity
and survival. Trends Neurosci. 12, 265–270.
Liu, R.Y., Snider, W.D., 2001. Different signaling pathways mediate regenerative versus de-
velopmental sensory axon growth. J. Neurosci. 21, RC164.
Liu, G., Feldman, J.L., Smith, J.C., 1990. Excitatory amino acid-mediated transmission of
intraspiratory drive to phrenic motorneurons. J. Neurophysiol. 64, 423–436.
Liu, Y., Cox, S.R., Morita, T., Kourembanas, S., 1995. Hypoxia regulates vascular endothelial
factor gene expression in endothelial cells. Identification of a 50 enhancer. Circ. Res.
77, 638–643.
Liu, Y., Kim, D., Himes, B.T., Chow, S.Y., Schallert, T., Murray, M., Tessler, A., Fischer, I.,
1999. Transplants of fibroblasts genetically modified to express BDNF promote regener-
ation of adult rat rubrospinal axons and recovery of forelimb function. J. Neurosci.
19, 4370–4387.
Liu, K., Lu, Y., Lee, J.K., Samara, R., Willenberg, R., Sears-Kraxberger, I., Tedeschi, A.,
Park, K.K., Jin, D., Cai, B., Xu, B., Connolly, L., Steward, O., Zheng, B., He, Z., 2010.
PTEN deletion enhances the regenerative ability of adult corticospinal neurons. Nat. Neu-
rosci. 13, 1075–1081.
Lopez-Vales, R., Garcia-Alias, G., Fores, J., Udina, E., Gold, B.G., Navarro, X., Verdu, E.,
2005. FK 506 reduces tissue damage and prevents functional deficit after spinal cord injury
in the rat. J. Neurosci. Res. 81, 827–836.
Loveridge, B., Sanii, R., Dubo, H.I., 1992. Breathing pattern adjustments during the first year
following spinal cord injury. Paraplegia 30, 479–488.
Lovett-Barr, M.R., Satriotomo, I., Muir, G.D., Wilkerson, J.E.R., Hoffman, M.S., Vinit, S.,
Mitchell, G.S., 2012. Repetitive intermittent hypoxia induces respiratory and somatic mo-
tor recovery after chronic cervical spinal injury. J. Neurosci. 32, 3591–3600.
Lu, F., Qin, C., Foreman, R.D., Farber, J.P., 2004a. Chemical activation of C1-C2 spinal neu-
rons modulates intercostals and phrenic nerve activity in rats. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 286, R1069–R1076.
Lu, P., Yang, H., Jones, L.L., Filbin, M.T., Tuszynski, M.H., 2004b. Combinatorial therapy
with neurotrophins and cAMP promotes axonal regeneration beyond sites of spinal cord
injury. J. Neurosci. 24, 6402–6409.
Lu, P., Blesch, A., Graham, L., Wang, Y., Samara, R., Banos, K., Haringer, V., Havton, L.,
Weishaupt, N., Bennett, D., Faouad, K., Tuszynski, M.H., 2012. Motor axonal regenera-
tion after partial and complete spinal cord transection. J. Neurosci. 32, 8208–8218.
Lynskey, J.V., Sandhu, F.A., Dai, H.N., McAtee, M., Slotkin, J.R., MacArthur, L.,
Bregman, B.S., 2006. Delayed intervention with transplants and neurotrophic factors sup-
ports recovery of forelimb function after cervical spinal cord injury in adult rats.
J. Neurotrauma 23, 617–634.
MacFarlane, P.M., Mitchell, G.S., 2008. Respiratory long-term facilitation following intermit-
tent hypoxia requires reactive oxygen species formation. Neuroscience 152, 189–197.
MacFarlane, P.M., Mitchell, G.S., 2009. Episodic spinal serotonin receptor activation elicits
long-lasting phrenic motor facilitation by an NADPH oxidase-dependent mechanism.
J. Physiol. 587, 5469–5481.
MacFarlane, P.M., Wilkerson, J.E., Lovett-Barr, M.R., Mitchell, G.S., 2008. Reactive oxygen
species and respiratory plasticity following intermittent hypoxia. Respir. Physiol. Neuro-
biol. 164, 263–271.
MacFarlane, P.M., Satriotomo, I., Windelborn, J.A., Mitchell, G.S., 2009. NADPH oxidase
activity is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation.
J. Physiol. 587, 1931–1942.
212 CHAPTER 10 The challenges of respiratory motor system recovery

MacFarlane, P.M., Vinit, S., Mitchell, G.S., 2011. Serotonin 2A and 2B receptor-induced
phrenic motor facilitation: differential requirement for spinal NADPH oxidase activity.
Neuroscience 178, 45–55.
Macias, M., Dwornik, A., Ziemlinska, E., Fehr, S., Schachner, M., Czarkowska-Bauch, J.,
Skup, M., 2007. Locomotor exercise alters expression of pro-brain-derived neurotrophic
factor, brain-derived neurotrophic factor and its receptor TrkB in the spinal cord of adult
rats. Eur. J. Neurosci. 25, 2425–2444.
Macias, M., Nowicka, D., Czupryn, A., Sulejczak, D., Skup, M., Skangiel-Kramska, J.,
Czarkowska-Bauch, J., 2009. Exercise-induced motor improvement after complete spinal
cord transection and its relation to expression of brain-derived neurotrophic factor and pre-
synaptic markers. BMC Neurosci. 10, 144–169.
Maier, I.C., Ichiyama, R.M., Courtine, G., Schnell, L., Lavrov, I., Edgerton, V.R., Schwab, M.E.,
2009. Differential effects of anti-Nogo-A antibody treatment and treadmill training in rats
with incomplete spinal cord injury. Brain 132, 1426–1440.
Mantilla, C.B., Rowley, K.L., Zhan, W.Z., Fahim, M.A., Sieck, G.C., 2007. Synaptic vesicle
pools at diaphragm neuromuscular junctions vary with motoneuron soma, not axon termi-
nal inactivity. J. Neurosci. 146, 178–189.
Marino, P., Norreel, J.C., Schachner, M., Rougon, G., Amoureux, M.C., 2009. A polysialic
acid mimetic peptide promotes functional recovery in a mouse model of spinal cord injury.
Exp. Neurol. 219, 163–174.
Markus, A., Zhong, J., Snider, W.D., 2002. Raf and akt mediate distinct aspects of sensory
axon growth. Neuron 35, 65–76.
Martinez, F.O., Gordon, S., Locati, M., Mantovani, A., 2006. Transcriptional profiling of the
human monocyte-to-macrophage differentiation and polarization: new molecules and pat-
terns of gene expression. J. Immunol. 177, 7303–7311.
Massey, J.M., Hubscher, C.H., Wagoner, M.R., Decker, J.A., Amps, J., Silver, J., Onifer, S.M.,
2006. Chondroitinase ABC digestion of the perineuronal net promotes functional collat-
eral sprouting in the cuneate nucleus after cervical spinal cord injury. J. Neurosci.
26, 4406–4414.
McCrimmon, D.R., Smith, J.C., Feldman, J.L., 1989. Involvement of excitatory amino acids in
neurotransmission of inspiratory drive to spinal respiratory motorneurons. J. Neurosci.
9, 1910–1921.
McGuire, M., Zhang, Y., White, D.P., Ling, L., 2005. Phrenic long-term facilitation requires
NMDA receptors in the phrenic motornucleus in rats. J. Physiol. 576, 599–611.
McGuire, M., Liu, C., Cao, Y., Ling, L., 2008. Formation and maintenance of ventilatory long-
term facilitation require NMDA but not non-NMDA receptors in awake rats. J. Appl. Phy-
siol. 105, 942–950.
McKeon, R.J., Schreiber, R.C., Rudge, J.S., Silver, J., 1991. Reduction of neurite outgrowth in
a model of glial scarring following CNS injury is correlated with the expression of inhib-
itory molecules on reactive astrocytes. J. Neurosci. 11, 3398–3411.
McKeon, R.J., Hoke, A., Silver, J., 1995. Injury-induced proteoglycans inhibit the potential for
laminin-mediated axon growth on astrocytic scars. Exp. Neurol. 136, 32–43.
McKeon, R.J., Jurynec, M.J., Buck, C.R., 1999. The chondroitin sulfate proteoglycans neuro-
can and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar.
J. Neurosci. 19, 10778–10788.
McKerracher, L., David, S., Jackson, D.L., Kottis, V., Dunn, R.J., Braun, P.E., 1994. Identi-
fication of myelin associated glycoprotein as a major myelin-derived inhibitor of neurite
growth. Neuron 13, 805–811.
 References 213

Merrill, E.G., Lipski, J., 1987. Inputs to intercostal motoneurons from ventrolateral medullary
respiratory neurons in the cat. J. Neurophysiol. 57, 1837–1853.
Meyer-Franke, A., Wilkinson, G.A., Kruttgen, A., Hu, M., Munro, E., Hanson Jr., M.G.,
Reichardt, L.F., Barres, B.A., 1998. Depolarization and cAMP elevation rapidly recruit
TrkB to the plasma membrane of CNS neurons. Neuron 21, 681–693.
Millhorn, D.E., Eldridge, F.L., Waldrop, T.G., 1980a. Prolonged stimulation of respiration by
a new central neural mechanism. Respir. Physiol. 41, 87–103.
Millhorn, D.E., Eldridge, F.L., Waldrop, T.G., 1980b. Prolonged stimulation of respiration by
endogenous central serotonin. Respir. Physiol. 42, 171–188.
Minghetti, L., Greco, A., Potenza, R.L., Pezzola, A., Blum, D., Bantubungi, K., Popoli, P.,
2007. Effects of the adenosine A2A receptor antagonist SCH 58621 on
cyclooxygenase-2 expression, glial activation, and brain-derived neurotrophic factor
availability in a rat model of striatal neurodegeneration. J. Neuropathol. Exp. Neurol.
66, 363–371.
Minor, K.H., Akison, L.K., Goshgarian, H.G., Seeds, N.W., 2006. Spinal cord injury-
induced plasticity in the mouse—the crossed phrenic phenomenon. Exp. Neurol.
200, 486–495.
Minor, K.H., Bournat, J.C., Toscano, N., Giger, R.J., Davies, S.J., 2011. Decorin, erythroblas-
tic leukaemia viral oncogene homologue B4 and signal transducer and activator of tran-
scription 3 regulation of semaphorin 3A in central nervous system scar tissue. Brain
134, 1140–1155.
Miranda, J.D., White, L.A., Marcillo, A.E., Willson, C.A., Jaquid, J., Whittemore, S.R., 1999.
Induction of Eph B3 after spinal cord injury. Exp. Neurol. 156, 218–222.
Mire, E., Thomasset, N., Jakeman, L.B., Rougon, G., 2008. Modulating Sema3A signal with a
L1 mimetic peptide is not sufficient to promote motor recovery and axon regeneration after
spinal cord injury. Mol. Cell. Neurosci. 37, 222–235.
Miyata, H., Zhan, W.Z., Prakash, Y.S., Sieck, G.C., 1995. Myoneural interactions affect di-
aphragm muscle adaptations to inactivity. J. Appl. Physiol. 79, 1640–1649.
Mojsilovic-Petrovic, J., Jeong, G.B., Crocker, A., Arneja, A., David, S., Russell, D.S.,
Kalb, R.G., 2006. Protecting motor neurons from toxic insult by antagonism of adenosine
A2a and Trk receptors. J. Neurosci. 26, 9250–9263.
Moon, L.D., Asher, R.A., Rhodes, K.E., Fawcett, J.W., 2001. Regeneration of CNS axons back
to their target following treatment of adult rat brain with chondroitinase ABC. Nat. Neu-
rosci. 4, 465–466.
Moreno, D.E., Yu, X.J., Goshgarian, H.G., 1992. Identification of the axon pathways which
mediate functional recovery of a paralysed hemidiaphragm following spinal cord hemisec-
tion in the adult rat. Exp. Neurol. 182, 232–239.
Muir, E.M., Fyfe, I., Gardiner, S., Li, L., Warren, P., Fawcett, J.W., Keynes, R.J., Rogers, J.H.,
2010. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase
ABC from mammalian cells. J. Biotechnol. 145, 103–110.
Mukhopadhyay, G., Doherty, P., Walsh, F.S., Crocker, P.R., Filbin, M.T., 1994. A novel role
for myelin-associated glycoprotein as an inhibitor of axonal regeneration. Neuron
13, 757–767.
Nakamae, T., Tanaka, N., Nakanishi, K., Kamei, N., Sadaki, H., Hamasaki, T., Yamada, K.,
Yamamoto, R., Mochizuki, Y., Ochi, M., 2009. Chondroitinase ABC promotes corticosp-
inal axon growth in organotypic cocultures. Spinal Cord 47, 161–165.
Nandoe Tewarie, R.D., Hurtado, A., Bartels, R.H., Grotenhuis, J.A., Oudega, M., 2010.
A clinical perspective of spinal cord injury. NeuroRehabilitation 27 (2), 129–139.
214 CHAPTER 10 The challenges of respiratory motor system recovery

Nantwi, K.D., 2009. Recovery of respiratory activity after C2 hemisection (C2HS): involve-
ment of adenosinergic mechanisms. Respir. Physiol. Neurobiol. 169, 102–114.
Nantwi, K.D., Goshgarian, H.G., 1998. Theophylline-induced recovery in a hemidiaphragm par-
alyzed by hemisection in rats: contribution of adenosine receptors. Neuropharmacology
37, 113–121.
Nantwi, K.D., El-Bohy, A., Goshgarian, H.G., 1996. Actions of systemic theophylline on
hemidiaphragmatic recovery in rats following cervical spinal cord hemisection. Exp. Neu-
rol. 140, 53–59.
Nantwi, K.D., El-Bohy, A.A., Schrimsher, G.W., Reier, P.J., Goshgarian, H.G., 1999. Spon-
taneous functional recovery in a paralyzed hemidiaphragm following upper cervical spinal
cord injury in adult rats. Neurorehabil. Neural Repair 13, 225–234.
Nantwi, K.D., Basura, G.J., Goshgarian, H.G., 2003. Effects of long-term theophylline expo-
sure on recovery of respiratory function and expression of adenosine A1 mRNA in cervical
spinal cord hemisected adult rats. Exp. Neurol. 182, 232–239.
Nathan, P.W., Smith, M., Deacon, P., 1996. Vestibulospinal, reticulospinal and descending
propriospinal nerve fibres in man. Brain 119, 1809–1833.
Nicaise, C., Hala, T.J., Frank, D.M., Parker, J.L., Authelet, M., Leroy, K., Brion, J.-P.,
Wright, M.C., Lepore, A.C., 2012. Phrenic motor neuron degeneration compromises
phrenic axonal circultry and diaphragm activity in a unilateral cervical contusion model
of spinal cord injury. Exp. Neurol. 235, 539–552.
Nigro, J., Wang, A., Mukhopadhyay, D., Lauer, M., Midura, R.J., Sackstein, R., Hascall, V.C.,
2009. Regulation of heparin sulfate and chondroitin sulfate glycosaminoglycan biosynthe-
sis by 4-fluoroglucosamine in murine airway smooth muscle cells. J. Biol. Chem.
284, 16832–16839.
NSCISC, 2012. Spinal Cord Injury. Facts and Figures at a Glance. National Spinal Cord Injury
Statistical Center, Birmingham, University of Alabama at Birmingham, National Spinal
Cord Injury Statistical Center. Available at http://www.spinalcord.uab.edu. Assessed
November 2012.
O’Bryant, A.J., Allred, R.P., Maldonado, M.A., Cormack, L.K., Jones, T.A., 2011. Breeder
and batch-dependent variability in the acquisition and performance of a motor skill in adult
Long-Evans rats. Behav. Brain Res. 224, 112–120.
O’Hara Jr., T.E., Goshgarian, H.G., 1991. Quantitative assessment of phrenic nerve functional
recovery mediated by the crossed phrenic reflex at various time intervals after spinal cord
injury. Exp. Neurol. 111, 224–250.
Onai, T., Saji, M., Miura, M., 1987. Projections of supraspinal structures to the phrenic motor
nucleus in rats studied by horseradish peroxidase microinjection method. J. Auton. Nerv.
Syst. 21, 233–240.
Onimaru, H., Homma, I., 2003. A novel functional neuron group for respiratory rhythm gen-
eration in the ventral medulla. J. Neurosci. 23, 1478–1486.
Oo, T., Watt, J.W., Soni, B.M., Sett, P.K., 1999. Delayed diaphragm recovery in 12 patients
after high cervical spinal cord injury. A retrospective review of the diaphragm status of 107
patients ventilated after acute spinal cord injury. Spinal Cord 37, 117–122.
Park, S., Hong, Y.W., 2006. Transcriptional regulation of artemin is related to neurite out-
growth and actin polymerization in mature DRG neurons. Neurosci. Lett. 404, 61–66.
Park, K.K., Liu, K., Hu, Y., Smith, P.D., Wang, C., Cai, B., Xu, B., Connolly, L., Kramvis, I.,
Sahin, M., He, Z., 2008. Promoting axon regeneration in the adult CNS by modulation of
the PTEN/mTOR pathway. Science 322, 963–966.
Parsons, M.P., Hirasawa, M., 2010. ATP-sensitive potassium channel-mediated lactate effect on
orexin neurons: implications for brain energetics during arousal. J. Neurosci. 30, 8061–8070.
 References 215

Pedata, F., Corsi, C., Melani, A., Bordoni, F., Latini, S., 2001. Adenosine extracellular brain
concentrations and role of A2A receptors in ischemia. Ann. N. Y. Acad. Sci. 939, 74–84.
Petersen, J., Russell, L., Andrus, K., MacKinnon, M., Silver, J., Kliot, M., 1996. Reduction of
extra-neuronal scarring by ADCON-T/N after surgical intervention. Neurosurgery
38, 976–983.
Petruska, J.C., Ichiyama, R.M., Jindrich, D.L., Crown, E.D., Tansey, K.E., Roy, R.R.,
Edgerton, V.R., Mendell, L.M., 2007. Changes in motoneuron properties and synaptic in-
puts related to step training after spinal cord transection in rats. J. Neurosci.
27, 4460–4471.
Phillis, J.W., Delong, R.E., Towner, J.K., 1985. Adenosine and the regulation of cerebral blood
flow during anoxia. In: Stefanovich, V., Rudolphi, K., Scubert, P. (Eds.), Adenosine:
Receptors and Modulation of Cell Function. Oxford, United Kingdom, pp. 145–164.
Pindzola, R.R., Doller, C., Silver, J., 1993. Putative inhibitory extracellular matrix molecules
at the dorsal root entry zone of the spinal cord during development and after root and sci-
atic nerve lesions. Dev. Biol. 156, 34–48.
Pitts, R.F., 1940. The respiratory center and its descending pathways. J. Comp. Neurol.
72, 605–625.
Pizzorusso, T., Medini, P., Berardi, N., Chierzi, S., Fawcett, J.W., Maffei, L., 2002. Reactiva-
tion of ocular dominance plasticity in the adult visual cortex. Science 298, 1248–1251.
Pizzorusso, T., Medini, P., Landi, S., Baldini, S., Berardi, N., Maffei, L., 2006. Structural and
functional recovery from early monocular deprivation in adult rats. Proc. Natl. Acad. Sci.
U.S.A 103, 8517–8522.
Plant, G.W., Bates, M.L., Bunge, M.B., 2001. Inhibitory proteoglycan immunoreactivity is
higher at the caudal than the rostral Schwann cell graft-transected spinal cord interface.
Mol. Cell. Neurosci. 17, 471–487.
Poduslo, J.F., Curran, G.L., 1996. Permeability at the blood-brain and blood-nerve barriers of
the neurotrophic factors: NGF, CNTF, NT-3, BDNF. Brain Res. Mol. Brain Res.
36, 280–286.
Polentes, J., Stamegna, J.C., Nieto-Sampedro, M., Gauthier, P., 2004. Phrenic rehabilitation
and diaphragm recovery after cervical injury and transplantation of olfactory ensheathing
cells. Neurobiol. Dis. 16, 638–653.
Poon, P.C., Gupta, D., Shoichet, M.S., Tator, C.H., 2007. Clip compression model is
useful for thoracic spinal cord injuries: histologic and functional correlates. Spine
32, 2853–2859.
Popovich, P.G., Guan, Z., Wei, P., Huitinga, I., van Rooijen, N., Stokes, B.T., 1999. Depletion
of hematogenous macrophages promotes partial hindlimb recovery and neuroanatomical
repair after experimental spinal cord injury. Exp. Neurol. 158, 351–365.
Porter, W.T., 1895. The path of the respiratory impulse from the bulb to the phrenic neucli.
J. Physiol. Lond. 17, 455–485.
Powell, F.L., Milsom, W.K., Mitchell, G.S., 1998. Time domains of the hypoxic ventilatory
response. Respir. Physiol. 112, 123–134.
Prabhakar, V., Capila, I., Bosques, C.J., Pojasek, K., Sasisekharan, R., 2005. Chondroitinase
ABC 1 from Proteus vulgaris: cloning, recombinant expression and active site identifica-
tion. Biochem. J. 386, 103–112.
Prakash, Y.S., Miyata, H., Zhan, W.Z., Sieck, G.C., 1999. Inactivity-induced remodeling of
neuromuscular junctions in rat diaphragmatic muscle. Muscle Nerve 22, 307–319.
Qin, C., Chandler, M.J., Foreman, R.D., Farber, J.P., 2002. Upper thoracic respiratory inter-
neurons integrate noxious somatic and visceral information in rats. J. Neurophysiol.
88, 2215–2223.
216 CHAPTER 10 The challenges of respiratory motor system recovery

Qiu, J., Cai, D., Dai, H., McAtee, M., Hoffman, P.N., Bregman, B.S., Filbin, M.T., 2002a.
Spinal axon regeneration induced by elevation of cyclic AMP. Neuron 34, 895–903.
Qiu, J., Cai, D., Filbin, M.T., 2002b. A role for cAMP in regeneration during development and
after injury. Prog. Brain Res. 137, 381–387.
Raberger, G., Kraupp, O., Stuhlinger, W., Nell, G., Chirikdjian, J.J., 1970. The effects of an
intracoronary infusion of adenosine on cardiac performance, blood supply and on myocar-
dial metabolism in dogs. Pfluigers Arch. Gesamte. Physiol. 317, 20–34.
Rapalino, O., Lazarov-Spiegler, O., Agranov, E., Velan, G.J., Yoles, E., Fraidakis, M.,
Solomon, A., Gepstein, R., Katz, A., Belkin, M., Hadani, M., Schwartz, M., 1998. Implan-
tation of stimulated homologous macrophages results in partial recovery of paraplegic rats.
Nat. Med. 4, 814–821.
Richardson, P.M., McGuinness, U.M., Aguayo, A.J., 1980. Axons from CNS neurons regen-
erate into PNS grafts. Nature 284, 264–265.
Rolls, A., Avidan, H., Cahalon, L., Schori, H., Bakalash, S., Litvak, V., Lev, S., Lider, O.,
Schwartz, M., 2004. A disaccharide derived from chondroitin sulphate proteoglycan pro-
motes central nervous system repair in rats and mice. Eur. J. Neurosci. 20, 1973–1983.
Rolls, A., Shechter, R., Schwartz, M., 2009. The bright side of the glial scar in CNS repair. Nat.
Rev. Neurosci. 10, 235–241.
Rossignol, S., Barriere, G., Frigon, A., Barthelemy, D., Bouyer, L., Provencher, J.,
Leblond, H., Bernard, G., 2008. Plasticity of locomotor sensorimotor interactions after pe-
ripheral and/or spinal lesions. Brain Res. Rev. 57, 228–240.
Routal, R.V., Pal, G.P., 1999. Location of the phrenic nucleus in the human spinal cord.
J. Anat. 195, 617–621.
Rudge, J.S., Silver, J., 1990. Inhibition of neurite outgrowth on astroglial scars in vitro.
J. Neurosci. 10, 3594–3603.
Rudolphi, K.A., Schubert, P., Parkinson, F.E., Fredholm, B.B., 1992. Neuroprotective role of
adenosine in cerebral ischaemia. Trends Pharmacol. Sci. 13, 439–445.
Rutishauser, U., 2008. Polysialic acid in the plasticity of the developing and adult vertebrate
nervous system. Nat. Rev. Neurosci. 9, 26–35.
Sandhu, M.S., Dougherty, B.J., Lane, M.A., Bolser, D.C., Kirkwood, P.A., Reier, P.J.,
Fuller, D.D., 2009. Respiratory recovery following high cervical hemisection. Respir.
Physiol. Neurobiol. 169, 94–101.
Sandler, A.N., Tator, C.H., 1976. Effect of acute spinal cord compression injury on regional
spinal cord blood flow in primates. J. Neurosurg. 45, 660–676.
Sands, W.A., Palmer, T.M., 2008. Regulating gene transcription in response to cyclic AMP
elevation. Cell. Signal. 20, 460–466.
Satriotomo, I., Dale, E.A., Dahlberg, J.M., Mitchell, G.S., 2012. Repetitive actue intermittent
hypoxia increases expression of proteins associated with plasticity in the phrenic motor
nucleus. Exp. Neurol. 237, 103–115.
Schnell, L., Schwab, M.E., 1990. Axonal regeneration in the rat spinal cord produced by an
antibody against myelin-associated neurite growth inhibitors. Nature 343, 269–272.
Schnell, L., Schneider, R., Kolbeck, R., Barde, Y.A., Schwab, M.E., 1994. Neurotrophin-3
enhances sprouting of corticospinal tract during development and after adult spinal cord
lesion. Nature 367, 170–173.
Schucht, P., Raineteau, O., Schwab, M.E., Fouad, K., 2002. Anatomical correlates of locomo-
tor recovery following dorsal and ventral lesions of the rat spinal cord. Exp. Neurol.
176, 143–153.
 References 217

Schwartz, N.B., Galligani, L., Ho, P.L., Dorfman, A., 1974. Stimulation of synthesis of free
chondroitin sulfate chains by beta-D-xylosides in cultured cells. Proc. Natl. Acad. Sci. U.S.
A 71, 4047–4051.
Schwartz, M., Moalem, G., Leibowitz-Amit, R., Cohen, I.R., 1999. Innate and adaptive im-
mune responses can be beneficial for CNS repair. Trends Neurosci. 22, 295–299.
Seligman, A.M., Davis, W.A., 1941. The effects of some drugs on the crossed phrenic phe-
nomenon. Am. J. Physiol. 143, 102–106.
Sengottuvel, V., Leibinger, M., Pfreimer, M., Andreadaki, A., Fischer, D., 2011. Taxol facil-
itates axon regeneration in the mature CNS. J. Neurosci. 31, 2688–2699.
Shields, L.B., Zhang, Y.P., Burke, D.A., Gray, R., Shields, C.B., 2008. Benefit of chondroi-
tinase ABC on sensory axon regeneration in a laceration model of spinal cord injury in the
rat. Surg. Neurol. 69, 568–577.
Shimizu, H., Watanabe, E., Hiyama, T.Y., Nagakura, A., Fujikawa, A., Okado, H.,
Yanagawa, Y., Obata, K., Noda, M., 2007. Glial Nax channels control lactate signaling
to neurons for brain [Na +] sensing. Neuron 54, 59–72.
Singas, E., Grimm, D.R., Almenoff, P.L., Lesser, M., 1999. Inhibition of airway hyper-
reactivity by oxybutynin chloride in subjects with cervical spinal cord injury. Spinal Cord
37, 279–283.
Skup, M., Dwornik, A., Macias, M., Sulejczak, D., Wiater, M., Czarkowska-Bauch, J., 2002.
Long-term locotomotor training up-regulates TrkB(FL) receptor-like proteins,
brain-derived neurotrophic factor, and neurotrophin 4 with different topographies of ex-
pression in oligodendroglia and neurons in the spinal cord. Exp. Neurol. 176, 289–307.
Slack, S.E., Pezet, S., McMahon, S.B., Thompson, S.W., Malcangio, M., 2004. Brain derived
neurotrophic factor induces NMDA receptor subunit one phosphorylation via ERK and
PKC in the rat spinal cord. Eur. J. Neurosci. 20, 1769–1778.
Smith-Thomas, L.C., Fok-Seang, J., Stevens, J., Du, J.-S., Muir, E., Faissner, A., Geller, H.M.,
Rogers, J.H., Fawcett, J.W., 1994. An inhibitor of neurite outgrowth produced by astro-
cytes. J. Cell Sci. 107, 1687–1695.
Smith-Thomas, L., Stevens, J., Fok-Seang, J., Faissner, A., Rogers, J.H., Fawcett, J.W., 1995.
Increased axon regeneration in astrocytes grown in the presence of proteoglycan synthesis
inhibitors. J. Cell Sci. 108, 1307–1315.
Snow, D.M., Letourneau, P.C., 1992. Neurite outgrowth on a step gradient of chondroitin sul-
fate proteoglycan (CS-PG). J. Neurobiol. 23, 322–336.
Snow, D.M., Lemmon, V., Carrino, D.A., Caplan, A.I., Silver, J., 1990. Sulfated proteoglycans
in astroglial barriers inhibit neurite outgrowth in vitro. Exp. Neurol. 109, 111–130.
Snow, D.M., Smith, J.D., Gurwell, J.A., 2002. Binding characteristics of chondroitin sulfate
proteoglycans and laminin-1, and correlative neurite outgrowth behaviors in a standard
tissue culture choice assay. J. Neurobiol. 51, 285–301.
Stirling, D.P., Khodarahmi, K., Liu, J., McPhail, L.T., McBride, C.B., Steeves, J.D.,
Ramer, M.S., Tetzlaff, W., 2004. Minocycline treatment reduces delayed oligodendrocyte
death, attenuates axonal dieback, and improves functional outcome after spinal cord in-
jury. J. Neurosci. 24, 2182–2190.
Stohlman Jr., F., 1959. Observations on the physiology of erythropoietin and its role in the
regulation of red cell production. Ann. N. Y. Acad. Sci. 77, 710–724.
Tai, Q., Palazzolo, K.L., Goshgarian, H.G., 1997. Synaptic plasticity of
5-hydroxytryptamineimmunoreactive terminals in the phrenic nucleus following spinal
cord injury: a quantitative electron microscopic analysis. J. Comp. Neurol. 386, 613–624.
218 CHAPTER 10 The challenges of respiratory motor system recovery

Tan, W., Janczewski, W.A., Yang, P., Shao, X.M., Callaway, E.M., Feldman, J.L., 2008.
Silencing preBotzinger Complex somatostatin-expressing neurons induces persistent
apnea in awake rat. Nat. Neurosci. 11, 538–540.
Tang, X., Davies, J.E., Davies, S.J., 2003. Changes in distribution, cell associations, and pro-
tein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-
C during acute to chronic maturation of spinal cord scar tissue. J. Neurosci. Res.
71, 427–444.
Tian, G.F., Duffin, J., 1996. Connections from upper cervical inspiratory neurons to phrenic
and intercostals motoneurons studied with cross-correlation in the decerebrate rat. Exp.
Brain Res. 110, 196–204.
Tian, G.F., Duffin, J., 1998. The role of dorsal respiratory group neurons studied with cross-
correlation in the decerebrated rat. Exp. Brain Res. 121, 29–34.
Tian, G.F., Peever, J.H., Duffin, J., 1998. Botzinger-complex expiratory neurons monosynap-
tically inhibit phrenic motoneurons in the decerebrated rat. Exp. Brain Res. 122, 149–156.
Tillakaratne, N.J., Mouria, M., Ziv, N.B., Roy, R.R., Edgerton, V.R., Tobin, A.J., 2000. In-
creased expression of glutamate decarboxylase (GAD(67)) in feline lumbar spinal cord
after complete thoracic spinal cord transection. J. Neurosci. Res. 60, 219–230.
Tom, V.J., Houle, J.D., 2008. Intraspinal microinjection of chondroitinase ABC following
injury promotes axonal regeneration out of a peripheral nerve bridge. Exp. Neurol.
211, 315–319.
Tom, V.J., Steinmetz, M.P., Miller, J.H., Doller, C.M., Silver, J., 2004. Studies on the devel-
opment and behaviour of the dystrophic growth cone, the hallmark of regeneration failure,
in an in vitro model of the glial scar and after spinal cord injury. J. Neurosci.
24, 6531–6539.
Tom, V.J., Kadakia, R., Santi, L., Houle, J.D., 2009a. Administration of chondroitinase ABC
rostral or caudal to a spinal cord injury site promotes anatomical but not functional plas-
ticity. J. Neurotrauma 26, 2323–2333.
Tom, V.J., Sandrow-Feinberg, H.R., Miller, K., Santi, L., Connors, T., Lemay, M.A., Houle, J.D.,
2009b. Combining peripheral nerve grafts and chondroitinase promotes functional axonal
regeneration in the chronically injured spinal cord. J. Neurosci. 29, 14881–14890.
Tom, V.J., Sandrow-Feinberg, H.R., Miller, K., Domitrovich, C., Bouyer, J., Zhukareva, V.,
Klaw, M.C., Lemay, M.A., Houle, J.D., 2013. Exogenous BDNF enhances the integration
of chronically injured axons that regenerate through a peripheral nerve grafted into a
chondroitinase-treated spinal cord injury site. Exp. Neurol. 239, 91–100.
Totoiu, M.O., Keirstead, H.S., 2005. Spinal cord injury is accompanied by chronic progressive
demyelination. J. Comp. Neurol. 486, 373–383.
Tropea, D., Caleo, M., Maffel, L., 2003. Synergistic effects of brain derived neurotropic
factor and chondroitinase ABC on superior colliculus in adult rats. J. Neurosci.
23, 7034–7044.
Trumbower, R.D., Jayaraman, A., Mitchell, G.S., Rymer, W.Z., 2012. Exposure to acute in-
termittent hypoxia augments somatic motor function in humans with incomplete spinal
cord injury. Neurorehabil. Neural Repair 26, 163–172.
Vahidi, B., Park, J.W., Kim, H.J., Leon, N.L., 2008. Micro-fluidic-based strip assay for testing
the effects of various surface-bound inhibitors in spinal cord injury. J. Neurosci. Methods
170, 188–196.
Vavrek, R., Pearse, D.D., Fouad, K., 2007. Neuronal populations capable of regeneration fol-
lowing a combined treatment in rats with spinal cord transection. J. Neurotrauma
24, 1667–1673.
 References 219

Vinit, S., Gauthier, P., Stamegna, J.C., Kastner, A., 2006. High cervical lateral spinal cord in-
jury results in long-term ipsilateral hemidiaphragm paralysis. J. Neurotrauma
23, 1137–1146.
Vinit, S., Stamegna, J.C., Boulenguez, P., Gauthier, P., Kastner, A., 2007. Restorative respi-
ratory pathways after partial cervical spinal cord injury: role of ipsilateral phrenic affer-
ents. Eur. J. Neurosci. 25, 3551–3560.
Vinit, S., Darlot, F., Stamegna, J.C., Sanchez, P., Gauthier, P., Kastner, A., 2008. Longterm
respiratory pathways reorganization after partial cervical spinal cord injury. Eur. J. Neu-
rosci. 27, 897–908.
Vinit, S., Windelborn, J.A., Mitchell, G.S., 2011. Lipopolysaccharide attenuates phrenic long-
term facilitation following acute intermittent hypoxia. Respir. Physiol. Neurobiol.
176, 130–135.
Vorobyov, V., Kwok, J.C., Fawcett, J.W., Sengpiel, F., 2013. Effects of digesting chondroitin
sulphate proteoglycans on plasticity in cat primary visual cortex. J. Neurosci. 33, 234–243.
Wang, D., Ichiyama, R.M., Zhao, R., Andrews, M.R., Fawcett, J.W., 2011. Chondroitinase
combined with rehabilitation promotes recovery of forelimb function in rats with chronic
spinal cord injury. J. Neurosci. 31, 9332–9344.
Webb, A.A., Gowribai, K., Muir, G.D., 2003. Fischer (F-344) rats have different morphology,
sensorimotor and locomotor abilities compared to Lewis, Long-Evans, Sprague-Dawley
and Wistar rats. Behav. Brain Res. 144, 143–156.
Weishaupt, N., Li, S., DiPardo, A., Sipione, S., Fouad, K., 2013. Synergistic effects of BDNF
and rehabilitive training on recovery after cervical spinal cord injury. Behav. Brain Res.
239, 31–42.
White, T.E., Lane, M.A., Sandu, M.S., O’Steen, B.E., Fuller, D.D., Reier, P.J., 2010. Neuronal
progenitor transplantation and respiratory outcomes following upper cervical spinal cord
injury in adult rats. Exp. Neurol. 225, 231–236.
Wilkerson, J.E., Mitchell, G.S., 2009. Daily intermittent hypoxia augments spinal BDNF
levels. ERK phosphorylation and respiratory long-term facilitation. Exp. Neurol.
217, 116–123.
Wilkerson, J.E., MacFarlane, P.M., Hoffman, M.S., Mitchell, G.S., 2007. Respiratory plastic-
ity following intermittent hypoxia: roles of protein phosphatases and reactive oxygen spe-
cies. Biochem. Soc. Trans. 35, 1269–1272.
Wilkerson, J.E., Satriotomo, I., Baker-Herman, T.L., Watters, J.J., Mitchell, G.S., 2008. Oka-
daic acid-sensitive protein phosphatases constrain phrenic long-term facilitation after sus-
tained hypoxia. J. Neurosci. 28, 2949–2958.
Willson, C.A., Irizarry-Ramı́rez, M., Gaskins, H.E., Cruz-Orengo, L., Fiqueroa, J.D.,
Whittemore, S.R., Miranda, J.D., 2002. Upregulation of EphA receptor expression in
the injured adult rat spinal cord. Cell Transplant. 11, 229–239.
Willson, C.A., Miranda, J.D., Foster, R.D., Onifer, S.M., Whittemore, S.R., 2003. Transection
of the adult rat spinal cord up-regulates EphB3 receptor and ligand expression. Cell Trans-
plant. 12, 279–290.
Windelborn, J.A., Mitchell, G.S., 2012. Glial activation in the spinal ventral horn caudal to
cervical injury. Respir. Physiol. Neurobiol. 180, 61–68.
Windle, W.F., Clemente, C.D., Chambers, W.W., 1952. Inhibition of formation of a glial bar-
rier as a means of permitting a peripheral nerve to grow into the brain. J. Comp. Neurol.
96, 359–369.
Winn, H.R., Rubio, G.R., Berne, R.M., 1981a. The role of adenosine in the regulation of ce-
rebral blood flow. J. Cereb. Blood Flow Metab. 1, 239–244.
220 CHAPTER 10 The challenges of respiratory motor system recovery

Winn, H.R., Rubio, R., Berne, R.M., 1981b. Brain adenosine concentration during hypoxia in
rats. Am. J. Physiol. 241, H235–H242.
Wong, I., Liao, H., Bai, X., Zaknic, A., Zhong, J., Guan, Y., Li, H.Y., Wang, Y.J., Zhou, X.F.,
2010. ProBDNF inhibits infiltration of ED1 + macrophages after spinal cord injury. Brain
Behav. Immun. 24, 585–597.
Yamada, H., Ezure, K., Manabe, M., 1988. Efferent projections of inspiratory neurons of the
ventral respiratory group. A dual labeling study in the rat. Brain Res. 455, 283–294.
Yamagata, T., Saito, H., Habuchi, O., Suzuki, S., 1968. Purification and properties of bacterial
chondroitinases and chondrosulfatases. J. Biol. Chem. 243, 1523–1535.
Ying, Z., Roy, R.R., Edgerton, V.R., Gomez-Pinilla, F., 2005. Exercise restores levels of neu-
rotrophins and synaptic plasticity following spinal cord injury. Exp. Neurol. 193, 411–419.
Ying, Z., Roy, R.R., Zhong, H., Zdunowski, S., Edgerton, V.R., Gomez-Pinilla, F., 2008.
BDNF-exercise interactions in the recovery of symmetrical stepping after a cervical hemi-
section in rats. Neuroscience 155, 1070–1078.
Young, W., Koreh, I., 1986. Potassium and calcium changes in injured spinal cords. Brain Res.
365, 42–53.
Young, J.K., Baker, J.H., Montes, M.I., 2000. The brain response to 2-deoxy glucose is
blocked by a glial drug. Pharmacol. Biochem. Behav. 67, 233–239.
Zabka, A.G., Behan, M., Mitchell, G.S., 1985. Selected contribution: time-dependent hypoxic
respiratory responses in female rats are influenced by age and by the estrus cycle. J. Appl.
Physiol. 91 (6), 2831–2838.
Zabka, A.G., Behan, M., Mitchell, G.S., 2001. Long term facilitation of respiratory motor out-
put decreases with age in male rats. J. Physiol. 531, 509–514.
Zhang, Y., Zhang, X., Wu, D., Verhaagen, J., Richardson, P.M., Yeh, J., Bo, X., 2007.
Lentiviral-mediated expression of polysialic acid in spinal cord and conditioning lesion
promote regeneration of sensory axons into spinal cord. Mol. Ther. 15, 1796–1804.
Zhao, R.R., Muir, E.M., Alves, J.N., Rickman, H., Allan, A.Y., Kwok, J.C., Roet, K.C.,
Verhaagen, J., Schneider, B.L., Bensadoun, J.C., Ahmed, S.G., Yanez-Munoz, R.J.,
Keynes, R.J., Fawcett, J.W., Rogers, J.H., 2011. Lentiviral vectors express chondroitinase
ABC in cortical projections and promote sprouting of injured corticospinal axons.
J. Neurosci. Methods 201, 228–238.
Zheng, F., Wang, L., Brauner, M., Liewald, J.F., Kay, K., Watzke, N., Wood, P.G.,
Bamberg, E., Nagel, G., Gottschalk, A., Deisseroth, K., 2007. Multimodal fast optical in-
terrogation of neural circuitry. Nature 446, 633–639.
Zhou, S.Y., Goshgarian, H.G., 1999. Effects of serotonin on crossed phrenic nerve activity in
cervical spinal cord hemisected rats. Exp. Neurol. 160, 446–453.
Zhou, S.Y., Goshgarian, H.G., 2000. 5-Hydroxytryptophan-induced respiratory recovery after
cervical spinal cord hemisection in rats. J. Appl. Physiol. 89, 1528–1536.
Zhou, S.Y., Basura, G.J., Goshgarian, H.G., 2001. Serotonin(2) receptors mediate respiratory
recovery after cervical spinal cord hemisection in adult rats. J. Appl. Physiol.
91, 2665–2673.
Zimmer, M.B., Goshgarian, H.G., 2006. Spinal activation of serotonin 1A receptors enhances
latent respiratory activity after spinal cord injury. J. Spinal Cord Med. 29, 147–155.
Zimmer, M.B., Goshgarian, H.G., 2007. GABA, not glycine, mediates inhibition of latent re-
spiratory motor pathways after spinal cord injury. Exp. Neurol. 203, 493–501.
Zou, J., Neubauer, D., Dyess, K., Ferguson, T.A., Muir, D., 1998. Degradation of chondroitin
sulphate proteoglycan enhances the neurite-promoting potential of spinal cord tissue. Exp.
Neurol. 154, 654–666.