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REVIEW
Autonomic assessment of animals with spinal cord injury:
tools, techniques and translation
JA Inskip
1,2,4
, LM Ramer
1,2,4
, MS Ramer
1,2
and AV Krassioukov
1,3
1
International Collaboration on Repair Discoveries, University of British Columbia, Vancouver, British Columbia, Canada;
2
Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada and
3
Department of Medicine,
Division of Physical Medicine and Rehabilitation, University of British Columbia, Vancouver, British Columbia, Canada
Study design: Literature review.
Objectives: To present a comprehensive overview of autonomic assessment in experimental spinal
cord injury (SCI).
Methods: A systematic literature review was conducted using PubMed to extract studies that
incorporated functional motor, sensory or autonomic assessment after experimental SCI.
Results: While the total number of studies assessing functional outcomes of experimental SCI
increased dramatically over the past 27 years, studies with motor outcomes dramatically outnumber
those with autonomic outcomes. Within the areas of autonomic dysfunction (cardiovascular,
respiratory, gastrointestinal, lower urinary tract, sexual function and thermoregulation), not all aspects
have been characterized to the same extent. Studies focusing on bladder and cardiovascular function
greatly outnumber those on sexual function, gastrointestinal function and thermoregulation. This
review addresses the disparity between well-established motor-sensory testing presently used in
experimental animals and the lack of standardized autonomic testing following experimental SCI.
Throughout the review, we provide information on the correlation between existing experimental and
clinically used autonomic tests. Finally, the review contains a comprehensive set of tables and
illustrations to guide the reader through the complexity of autonomic assessment and dysfunctions
observed following SCI.
Conclusions: A wide variety of techniques exist to evaluate autonomic function in experimental
animals with SCI. The incorporation of autonomic assessment as outcome measures in experiments
testing treatments or interventions for SCI should be considered a high, clinically relevant priority.
Spinal Cord (2009) 47, 2–35; doi:10.1038/sc.2008.61; published online 10 June 2008
Keywords:
spinal cord injury; cardiovascular; respiratory; bladder; bowel; sexual
Introduction
Although it is generally appreciated that spinal cord injury
(SCI) disrupts all types of communication between the brain
and periphery below the lesion, the outcome of SCI is still
commonly described in terms of motor function. The
pervasive mental association between SCI and paralysis is
reflected in recent headlines announcing a study on
experimental SCI published in Nature Medicine.
1
The
headlines read ‘Scientists move toward helping paralysis
patients’
2
and ‘paralysis cure’.
3
In keeping with this perspective, an overwhelming
number of clinical studies have focused on the effects of
SCI on voluntary movement and the role of the somatic
nervous system. Despite the widespread effects of SCI on
autonomic control, it is only recently that autonomic
function following SCI has received significant attention in
clinical research.
4–6
The delay in addressing the autonomic
effects of SCI has not only limited their appreciation among
basic scientists and clinicians, but also efforts to develop new
treatments or rehabilitation strategies targeting autonomic
function following SCI. These shortcomings are not insig-
nificant, as autonomic dysfunctions represent the primary
causes of morbidity and mortality following SCI.
7,8
In
addition, individuals with SCI have identified recovery of
autonomic functions as a high priority for improving their
quality of life.
9
Recent data demonstrate that autonomic
function is not reliably predicted by the degree of residual
motor or sensory function.
10,11
Together these results suggest
that there is an imbalance between clinical priorities and the
general focus of SCI research.
A similar imbalance exists in animal research. This is
highlighted by a systematic review of the animal SCI
Received 20 February 2008; revised 5 May 2008; accepted 5 May 2008;
published online 10 June 2008
Correspondence: Dr AV Krassioukov, International Collaboration on Repair
Discoveries, University of British Columbia, 2469-6270 University Blvd.,
Vancouver, British Columbia, Canada V6T 1Z4.
E-mail: krassioukov@icord.org
4
These authors contributed equally to this work.
Spinal Cord
(2009) 47, 2–35
&
2009 International Spinal Cord Society All rights reserved 1362-4393/09
$
32.00
www.nature.com/sc
literature (Figure 1; see ‘Methods’). We reviewed published
studies of animals with experimental SCI, and identified
studies with functional outcomes designed to evaluate
motor, sensory or autonomic function. While the total
number of studies increased dramatically over the past 27
years, published studies with motor outcomes dramatically
outnumber those with sensory or autonomic outcomes. The
disproportionate number of studies focusing on motor
recovery after experimental SCI represents a significant
mismatch between the clinical priorities of improved
autonomic function and the direction of SCI research in
animals.
When the functional autonomic outcomes are separated
according to organ system dysfunction, it is immediately
apparent that not all aspects of SCI-induced autonomic
dysfunction have been examined to the same extent. The
available data characterize mainly bladder and cardiovascu-
lar functions in SCI animals, whereas sexual function,
gastrointestinal (GI) function and thermoregulation remain
essentially uninvestigated. These findings are particularly
alarming, as recovery of sexual function in particular has
been identified by both paraplegics and quadriplegics as an
urgent priority.
9
There are several likely reasons for the paucity of research
addressing autonomic dysfunctions following SCI. The
complex organization of the autonomic nervous system,
and its involvement in the control of almost every system in
the body, makes it difficult to select appropriate functional
tests. There is also some confusion surrounding the opera-
tional definitions of autonomic dysfunctions that are
present in animals following SCI. Finally, there may be a
lack of agreement on (and awareness of) well-designed,
clinically relevant tests that have been validated to evaluate
autonomic functions in animals with SCI.
The main goal of this review is to provide the scientific
community with an overview of the current methods used to
assess the autonomic function of animals with SCI. For six
aspects of autonomic functionFcardiovascular function,
respiratory function, GI function, lower urinary tract (LUT)
function, sexual function and thermoregulationFwe review
the innervation of the system in humans and rats, the
clinical implications of dysfunction following SCI and the
tests or techniques that are currently available to evaluate
function after experimental SCI. We also include functional
tests that have been developed in other animal models, but
that appear to be applicable to animals with SCI. Each test is
reviewed in terms of its methodology, the type of informa-
tion that it provides and the available data in animals with
SCI. We hope to improve the understanding of functional
tests used to evaluate autonomic function and to increase
their incorporation in SCI experiments, particularly those
studies testing potential therapeutic agents (see Table 9). We
also hope that this review will be useful to veterinarians in
clinical practice, to add to their arsenal of assessments of
animals with naturally occurring SCI. Throughout the review
we also highlight clinical assessments that are similar to the
experimental methods we describe. The use of similar tests in
the clinic and the laboratory is valuable because it facilitates
Figure 1 Autonomic dysfunction remains underrepresented in experimental spinal cord injury (SCI). (a) The total number of publications
reporting the functional outcome in animal models of SCI has increased steadily since 1980. However, the rate of increase is dramatically
different between studies characterizing function/dysfunction of different divisions of the nervous system. At every time period examined,
published studies with motor outcomes far outnumber published studies investigating autonomic or sensory function. The disparity is
particularly pronounced in the past 7 years, when studies incorporating a motor outcome outnumber publications with any autonomic
outcome by more than four times. (b) When published data on autonomic function are categorized according to organ system dysfunction
after SCI, it is clear that not every area is equally represented. Lower urinary tract (LUT) function/dysfunction is the best-characterized
component of experimental SCI, whereas experiments studying sexual, gastrointestinal and thermoregulatory function remain comparatively
scant. However, when we compare the number of published studies characterizing motor outcome in the past 7 years (554) with the number
of published studies characterizing LUT function in the same period (60), it is obvious that every aspect of SCI-related autonomic dysfunction
should be considered a priority in animal research.
Autonomic assessment in experimental SCI
JA Inskip et al
3
Spinal Cord
the translation of our knowledge about autonomic function
and dysfunction following SCI between the bench and the
bedside.
Methods: literature review
A systematic literature review was conducted using PubMed
to identify studies that incorporated functional assessment
after experimental SCI. Each search was limited to animal
studies published in English, and to specified publication
date ranges. Search terms were always used in combination
with ‘spinal cord injury’. A separate search was performed
using each of the terms listed in Table 1. The abstracts
returned by each search were reviewed to identify studies
with relevant functional outcomes; inclusion and exclusion
criteria are listed in Table 2. If the outcome measure(s) used
in the study were not evident from reading the abstract, the
‘Methods’ section of the paper was reviewed.
Publications were not counted twice in the same section
(that is, no paper was counted as both locomotor and
movement), but some were considered as spanning two
sections (that is, some papers contained both motor and
sensory functional assessments). All studies incorporating
the functional outcome of interest were included, regardless
of the objective of the study. For example, studies aimed at
characterizing the effects of a treatment on pain after SCI
that also included motor testing were counted as both motor
and sensory. Finally, publications were categorized according
to the intent of the outcome measure. For example, if
authors used a ladder-walking test to assess locomotor
function, the study was counted as motor (even though
there are presumably proprioceptive components to the
performance).
Cardiovascular function
Autonomic innervation of the cardiovascular system
In humans and animals with an intact neuraxis, both tonic
neurogenic and reflex autonomic control of the cardiovas-
cular system ensure adequate regional blood supply under a
wide range of physiological conditions. The autonomic
innervation of the cardiovascular system has important
ramifications for the pattern of cardiovascular dysfunction
that emerges after SCI. Here we review the most relevant
features of cardiovascular innervation (Figure 2), the details
of which have been extensively reviewed elsewhere.
12,13
Clinical implications of cardiovascular dysfunction following SCI
Unlike dysfunction of the LUT, which can be described in
general terms for suprasacral SCI, cardiovascular dysfunction
varies dramatically with level of injury,
14,15
its severity
determined by the relative loss of supraspinal control over
spinal sympathetic outflow.
16
Cervical SCI disrupts suprasp-
inal connections to preganglionic sympathetic innervation
of the heart and blood vessels, eliminating tonic excitatory
input to these organs. If the injury occurs at T5 or above,
Table 1 Search terms used to identify studies of motor, sensory and autonomic outcome of experimental SCI
Motor Sensory Autonomic
Autonomic; sympathetic; parasympathetic
Lower urinary tract Cardiovascular Respiratory Sexual Gastrointestinal Thermoregulation
Motor Sensory Urinary Cardiovascular Respiratory Sexual Gastrointestinal Thermoregulation
Movement Sensation Bladder Blood pressure Breathing Uterus Bowel Sweating
Walking Touch Micturition Hypertension Lung Vagina Colon Piloerection
Stepping Proprioception Renal Hypotension Menstruation
Locomotor Pain Dysreflexia Menstrual
Locomotion Allodynia Heart Penis
Postural Hyperalgesia Arrhythmia Erection
Spasticity Dysaesthesia Ejaculation
Paresthesia
Each search term was used in combination with ‘spinal cord injury’. Searches were limited to animal studies published in English within specified dateranges(Figure1).
Abstracts and manuscripts were reviewed to obtain the final publication count in each area: see Methods for details and Table 2 for inclusion/exclusion criteria.
Table 2 Criteria used to identify studies with relevant functional
assessment after experimental SCI
Included Excluded
All experimental models of SCI
(for example, traumatic,
ischemic) in all species
K Clinical (veterinary) SCI
K In vitro and ex vivo preparations
K Review articles
Studies incorporating functional
assessments of SCI, including
those that:
K Characterize an outcome
measure
K Characterize function/
dysfunction
K Use functional outcome to
assess manipulation or
treatment
K Studies detected by the search
criteria that contained no
functional assessments (For
example, motor neuron atrophy
after SCI)
K Studies that only monitored
vital signs during surgery or
treatment
K Electrophysiological studies of
individual neuron properties
(versus neurological function)
K Studies of spinal cord blood
flow/lesion perfusion or
microperfusion (versus systemic
cardiovascular function)
K Studies testing the effects of
vasopressor therapy as a
treatment for SCI
Abstracts returned by literature searches were reviewed; if necessary, manu-
scripts were also reviewed to determine what outcome measures were included
in the study. Studies were included/excluded according to the listed criteria.
Autonomic assessment in experimental SCI
JA Inskip et al
4
Spinal Cord
supraspinal control over sympathetic innervation of the
splanchnic vascular bed is lost, jeopardizing blood pressure
control. For this reason, injuries at and above T5–T6 are most
likely to precipitate severe cardiovascular dysfunction.
14,17
The acute phase of clinical SCI is marked by neurogenic
shock, a period of profoundly altered cardiovascular con-
trol.
18
This phenomenon is particularly pronounced follow-
ing cervical SCI, which frequently induces severe
hypotension and bradycardia.
14,17
After neurogenic shock
resolves, basal cardiovascular parameters remain altered in
people with cervical and high-thoracic SCI.
19
People with
mid-thoracic SCI typically have elevated heart rates,
20
whereas people with higher injuries often present with low
heart rate and resting blood pressure.
21
Cervical SCI also
abolishes circadian blood pressure rhythms
22–24
and blunts
the cardiovascular responses to exercise.
25
In addition to low
resting blood pressure, many people with high SCI experi-
ence orthostatic hypotension (OH), a decrease in blood
pressure that occurs with assumption of a sitting position.
26
These individuals are also prone to autonomic dysreflexia
(AD), episodes of paroxysmal hypertension, often accom-
panied by baroreflex-mediated bradycardia, induced by
sensory stimulation below the level of the injury.
17
As a
group, cardiovascular disorders are the most common
underlying or contributing causes of death in people
with SCI.
8,27
Figure 2 Autonomic innervation of the cardiovascular system. The major organs of the cardiovascular system are the heart and the blood
vessels. The heart receives both parasympathetic and sympathetic innervation. Parasympathetic efferents travel to the heart in the vagus nerve,
which exits the central nervous system (CNS) at the level of the medulla. The vagus nerve innervates the atria, nodes and Purkinje fibers via
local cardiac ganglia, and vagal activity decreases heart rate, contractility and conduction velocity. Sympathetic activity has an opposite,
stimulatory effect on the heart. All tissues of the heart receive sympathetic input from the upper thoracic (T1–T5) cord. Blood vessels are under
sympathetic control, and vessels supplying the splanchnic regionFthe liver, spleen and intestinesFare most important in cardiovascular
control. The splanchnic bed is densely innervated, highly compliant and contains approximately one-fourth of the total blood volume in
humans at rest.
403
As such, it is the primary capacitance bed in the body. Sympathetic outflow to the splanchnic bed exits the thoracolumbar
cord (T5–L2) and provides tonic vasoconstriction. The relative amount of sympathetic and parasympathetic activity governing cardiovascular
control is determined (in part) by information from two types of afferents: baroreceptors and chemoreceptors. Baroreceptors in the aortic arch,
carotid sinus and coronary arteries detect changes in arterial pressure, and chemoreceptors in the carotid bodies respond to changes in partial
pressures of oxygen and carbon dioxide in the blood. Baroreceptor activity is the primary drive for rapid blood pressure adjustment.
Baroreceptor afferents travel primarily in the vagus nerve and the glossopharyngeal nerve to reach the medulla. Abbreviations: AR, adrenergic
receptors; CVLM, caudal ventrolateral medulla; DMNX, dorsal vagal motor nerve; g, ganglion; mAChR, muscarinic cholinergic receptors; NA,
nucleus ambiguous; n, nerve; NTS, nucleus of the solitary tract; P2X, purinergic receptors; RVLM, rostral ventrolateral medulla; ( þ ) denotes
excitatory synapses; () denotes inhibitory synapses.
Autonomic assessment in experimental SCI
JA Inskip et al
5
Spinal Cord
Assessment of cardiovascular function following experimental SCI
SCI alters the connectivity of autonomic circuits between the
brainstem and spinal cord that governs cardiovascular
control and modifies sympathetic intraspinal and neurovas-
cular transmission resulting in altered vascular smooth
muscle responses.
28–34
These phenomena (as well as others)
are likely to contribute to SCI-induced cardiovascular
dysfunction. Here we focus on the systemic cardiovascular
outcome of experimental SCI. Several aspects of SCI-induced
cardiovascular dysfunction have been successfully modeled
in experimental animals. Cardiovascular parameters of
animals with SCI are monitored from direct arterial blood
pressure measurements: indirect measurements of blood
pressure (collected using a tail cuff, for example) have never
been used in animals with SCI. Arterial blood pressure is
taken either directly from a fluid-filled cannula or via
radiotelemetric monitoring of animals with chronically
implanted cannulae.
Short-term cardiovascular assessment using fluid-filled cannulae
The bulk of available cardiovascular data from animals with
SCI has been collected using arterial cannulae implanted
acutely prior to data collection. Typically, the cannula is
placed in one carotid or femoral artery hours or days before
data collection, although it is possible to maintain cannulae
for at least 1 week with daily flushing. Cannulae are filled
with heparin solution to preserve patency and tunneled
subcutaneously for externalization. After arterial cannulae
are implanted, animals are housed singly to protect cannulae
from cage-mate chewing. Cardiovascular parameters can be
monitored directly in conscious animals in their home cage
by connecting the cannulae to a pressure transducer: this
method has been used frequently to study many types of
cardiovascular dysfunction in animals with SCI.
The aspect of cardiovascular dysfunction that has been
best characterized in rodent models of SCI is AD, episodic
hypertension that has been described in both rats and mice,
and is induced by visceral and somatic stimuli (see Table 3
for detailed references). The nature of rodent AD and its
inciting stimuli is very similar to that described in humans.
Rats, like humans, typically experience AD when SCI occurs
at or above T5–T6. In one study, bladder filling induced AD
in rats with severe contusion SCI at T4, but not in rats with
severe T10 contusion.
68
The most common experimental
stimulus for AD is colorectal distension (CRD), in which a
balloon catheter is inserted into the colon through the anus
in the conscious animal.
28
Air is infused into the balloon to
mimic the pressure of several fecal boli in the colon.
Distension is typically maintained for 1 min, and arterial
pressure is monitored before, during and after CRD.
Formerly considered to be associated with chronic SCI, AD
may also emerge during the acute stages in both animals and
humans.
74
Rats with complete transection of the spinal cord
at T5 develop AD in response to CRD as early as 24 h after
injury.
28
The severity of AD subsequently decreases during
the first post-injury week, and AD is relatively mild at 7 days
following T5 SCI.
28,38
Thereafter, the severity of AD in-
creases. Although the actual rise in blood pressure varies
with the level and severity of injury, as well as the
experimental conditions, the majority of experiments are
conducted at least 2 weeks after SCI when AD is pronounced.
This well-characterized model has been used extensively to
identify mechanisms contributing to the development of
AD; still, only a few studies have examined the severity of AD
as an outcome measure when testing therapies for SCI
(Table 9).
Compared to its hypertensive counterpart, OH has
received little attention in experimental SCI. Some clues to
mechanisms of OH are provided by animal models of
microgravity-induced cardiovascular deconditioning, in
which microgravity is simulated by hindlimb unloading
(HU).
75–77
Animals exposed to HU experience a pronounced
drop in blood pressure when subjected to head-up tilt.
78
Considering this, we are currently developing an animal
model of OH after SCI. We have recently observed OH in rats
with complete transection injury at T3–T4 (JAI, LMR and
AVK, unpublished observations). When these unanesthe-
tized rats are subjected to passive head-up tilt 1 month after
injury, they experience a pronounced drop in blood pressure
(Figure 3); uninjured rats subjected to the same maneuver
exhibit either a rise in blood pressure or very little change.
Blood pressure data collected directly from arterial cannu-
lae have also been used to monitor animals in the early
phases of recovery from SCI. Neurogenic shock is generally
considered to be less pronounced in experimental animals
than in human SCI. This notion might stem from the fact
that the overwhelming majority of experiments are per-
formed in animals with thoracic SCI. Animals with cervical
SCI may experience neurogenic shock that is more compar-
able to the clinical situation. Rats with complete transection
between C7 and T1 developed both hypotension and
bradycardia.
79
Mean arterial pressure fell precipitously (by
approximately 30 mm Hg) in the first 24 h after injury, and
slowly recovered to preinjury levels (around 100 mm Hg) by
9 days after injury. Heart rate also decreased dramatically
(from about 360 b.p.m. to about 300 b.p.m.) and remained
significantly lower than preinjury heart rate for 9 days (the
duration of the study). Owing to the challenges involved in
animal care, there are very few data on animals with high,
severe SCI. These animal models are currently being devel-
oped (for example Pearse et al.
80
) and should facilitate
our understanding of cardiovascular control following
cervical SCI.
Cardiovascular data collected from animals with acutely
implanted arterial cannulae have also been subjected to
spectral analysis. This type of analysis exploits the relation-
ship between frequency of spontaneous fluctuations in
cardiovascular parameters and the activity of systems (local,
sympathetic and parasympathetic) governing cardiovascular
control. Spectral analysis has been validated in humans,
81,82
dogs
82–84
and rats,
85
and provides indices of autonomic
nervous system activity from relatively short segments of
continuous recordings of blood pressure (and in humans,
electrocardiogram signals). Spectral analysis has been ap-
plied to examine blood pressure variation in rats with acute
SCI.
70
At 1 and 6 days after complete transection at T4–T5,
rats exhibited reduced power in the low-frequency range,
Autonomic assessment in experimental SCI
JA Inskip et al
6
Spinal Cord
indicative of reduced sympathetic tone (or increased para-
sympathetic tone
85
) that affects blood pressure control.
There are currently no data applying spectral analysis to
more chronic experimental SCI. However, as this analysis is
related to clinical measures of autonomic control following
human SCI,
86
it represents a readily translatable tool for
autonomic assessment in experimental animals and humans
with SCI.
Advantages/disadvantages. Blood pressure measurements
collected directly from fluid-filled cannulae provide high-
resolution cardiovascular data with a relatively modest
investment in equipment. However, preserving catheter
patency for repeated measurements in the same animals
can present a challenge. For this reason, this method
typically only provides a snapshot of information about
cardiovascular function in each animal (that is, taken at a
single time point). The presence of investigators at the time
of data collection, as well as possible effects of recent surgery
for cannula implantation, may contribute to stress in the
animals during data collection.
Telemetric monitoring of cardiovascular parameters
Telemetric monitoring provides continuous physiological
information about the cardiovascular function of conscious,
freely moving animals over a long time course. This
technique was first applied to animals with SCI in our
laboratory (Figure 4),
38
and allowed us to characterize
changes in resting blood pressure and heart rate in rats
during recovery from high-thoracic SCI. Rats were instru-
mented with a telemetric transducer (catheter implanted
into the descending aorta) 1 month prior to SCI, and arterial
pressure data were transmitted as a radiofrequency signal to a
receiver under the cage. The recovery time after transducer
implantation was critical, as the effects of simultaneous
Table 3 Overview of studies characterizing or targeting cardiovascular dysfunction after experimental SCI
Technique used Species Injury model Time range post-injury References
Acute arterial cannulation
Basal cardiovascular parameters Rat T1–T4 clip compression o4h
35–37
1.5 months
38
C6/7 Tx; T2 Tx; T9 Tx 4 h
38
p1 week
39
T4–T7 Tx 0 h–6 weeks
40
p1 week
41
T5–T8 contusion o1h
42–44
T9 clip compression 1, 2 h
45
Rat, cat C7 Tx (ligature crush) o1h
46
Cat T6 contusion p24 h
47
Dog C4 Tx o1h
48
Monkey T5 balloon compression
49
Autonomic dysreflexia
Colorectal distension Mouse T2 clip compression 2 weeks
50
T2 Tx 2 weeks
50–52
Rat C7–T1; C8 Tx 3 days
53,54
2–3 months
55
T4–T5 clip compression p1 week
38,56
2–4 weeks
38,57–59
4–6 weeks
38,59
T3–T5 Tx p24 h
40,60,61
p1 week
28,40,60
2–4 weeks
28,34,40,60–65
2–3 months
55
Bladder distension Rat C7–T1 Tx 24 h
66
p1 week
67
T4 contusion 4 weeks
68
T5 Tx p1 week
28
Somatic stim. Mouse T2 clip compression 2 weeks
50
T2 Tx
50–52
Rat T4 clip compression 2–6 weeks
59
Vag.-cerv. stim. Rat T7 Tx 1–3 weeks
69
Spectral analysis Rat T1–T5 Tx p1 week
70,71
Telemetric monitoring Rat C7–T1 Tx 2–3 months
55
T4–5 Tx p24 h, p1 week
40
4–6 weeks
40,72
2–3 months
73
T4 clip compression 1.5 months
38
Abbreviations: stim., stimulation; Tx, transection; vag.-cerv., vaginocervical.
Studies have been classified by the technique used to assess cardiovascular function. Studies employing a combination of techniques are referenced under both
headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.
Autonomic assessment in experimental SCI
JA Inskip et al
7
Spinal Cord
transducer implantation and SCI can impede blood supply to
the hindlimbs.
38
Telemetric monitoring of animals with SCI suggests that
SCI-induced alterations in basal cardiovascular control are
similar between rats and humans. Rats with T5 clip
compression SCI experience transient hypotension and
persistent tachycardia following injury.
38,87
Telemetric mon-
itoring revealed that blood pressure recovered to preinjury
levels by 48 h after SCI, and remained equivalent to preinjury
levels for at least 6 weeks following injury. Heart rate was
increased relative to preinjury levels by 3 days after SCI, and
remained elevated for at least 6 weeks. Diurnal fluctuations
in blood pressure and heart rate returned approximately 5
days after injury.
38
Interestingly, recent telemetric data
demonstrate that rats with more rostral injuries (complete
T4 transection) experience persistent hypotension and
tachycardia, with altered basal blood pressure and heart rate
for at least 6 weeks following SCI.
40
Thus, it seems that in
rats, as in humans, severity of cardiovascular dysfunction
varies with both level and severity of SCI. Radiotelemetry has
also been used to investigate AD induced by CRD, which is
similar in progression between rats with T5 clip compression
and rats with T4 complete transection (Figure 4).
38,40
Advantages/disadvantages. As data collection is continuous,
telemetric monitoring is extremely informative. Since it
avoids the potential stressors of intra-arterial catheterization,
catheter maintenance, or restraint for catheter connection,
telemetry can be considered the gold standard for cardio-
vascular assessment in conscious experimental animals. It
does not eliminate stress due to handling when animals
must be loosely restrained for cardiovascular measurements
(such as during CRD to induce AD), so this remains a
consideration in telemetric studies. Most significantly,
adopting telemetric monitoring entails considerable invest-
ment in equipment. Perhaps for this reason, data in animals
with SCI remain scant (Table 3).
Respiratory function
Innervation of the respiratory system
Coordinated activity of somatic (diaphragm and accessory
respiratory skeletal muscles) and autonomic (smooth mus-
cles of the bronchial tree) nervous systems is crucial for
normal respiration. Here we review efferent and afferent
innervation as it pertains to respiratory dysfunction that
emerges following SCI (Figure 5). For simplicity, we omit
many important aspects, and the interested reader is directed
to comprehensive reviews on the subject (for example, see
Brading
12
and Canning and Fischer
88
).
Clinical implications of respiratory dysfunction following SCI
Respiratory dysfunction after SCI is determined by relative
loss of descending autonomic innervation to the respiratory
Figure 3 Representative tracings using fluid-filled cannulae to assess blood pressure during head-up tilt. One month after complete T3–T4
transection injury, conscious rats with a left carotid artery cannulation subjected to a passive 901 head-up tilt experienced a pronounced drop in
blood pressure. Uninjured rats subjected to the same maneuver exhibited either a rise in blood pressure or very little change (JAI, LMR and AVK,
unpublished observations).
Autonomic assessment in experimental SCI
JA Inskip et al
8
Spinal Cord
muscles. Injuries above C3 paralyze the diaphragm, and
ventilatory support is typically required to sustain life. After
C3–C5 SCI, the diaphragm is partially denervated, and
inspiration is compromised; still, most individuals with
C4–C5 SCI do not require artificial ventilation (or can be
weaned during recovery
89
). In lower cervical SCI, innerva-
tion of the primary (and some accessory) inspiratory muscles
is preserved, but ventilation is still impaired, because
denervated intercostal muscles do not coordinate chest
expansion with diaphragm descent.
90
Finally, any SCI above
L1–L2 denervates abdominal muscles, reducing the effec-
tiveness of coughing.
The clinical ramifications of respiratory dysfunctions
following SCI are severe, and encompass pneumonia,
atelectasis, bronchitis, reduced lung volumes and compli-
ance, sleep apnea and respiratory insufficiency or dyspnea,
particularly during exercise.
91–95
The incidence and severity
of respiratory dysfunction increases with the level of SCI.
Respiratory complications are the leading cause of death in
acute SCI.
96
In chronic SCI, respiratory dysfunction con-
tributes significantly to mortality,
8
and is associated with
reduced quality of life.
97
Assessment of respiratory function following experimental SCI
The most common experimental model used to study SCI-
induced respiratory dysfunction is rat cervical hemisection.
This injury reliably disrupts innervation to one-half of the
diaphragm. Recently, cervical hemicontusion models have
been developed that also induce respiratory deficits due to
hemidiaphragm paralysis.
98,99
Data from both injury models
have increased our understanding of endogenous plastic
processes that might improve respiratory function following
SCI (Table 4; reviewed in Zimmer et al.
134
). A few studies have
also examined the potential for regenerative therapies to
restore diaphragm function (Table 9). The obvious limitation
of these models is that they are incomplete injuries, and thus
do not model all aspects of clinical SCI. As more models of
severe cervical SCI are developed, our understanding of
respiratory dysfunction after SCI is likely to improve.
Fortunately, outcome measures used to assess respiratory
function are similar between experimental and clinical SCI,
facilitating translation of these data.
Phrenic nerve conduction
Phrenic nerve conduction testing is used clinically for
planning phrenic nerve or diaphragmatic pacing for assisted
activation of the diaphragm.
135–137
In animals, phrenic
nerve responses during spontaneous breathing or spinal
cord stimulation are examined in anesthetized, paralyzed
(mechanically ventilated) animals.
101–103,121,122
These data
have been used extensively to characterize respiratory
Figure 4 Use of telemetric monitoring to assess basal cardiovascular parameters (a, b) and autonomic dysreflexia (c, d) after experimental
spinal cord injury (SCI). Rats were instrumented with radiotelemetric pressure transducers and cannulae were implanted into the descending
aorta 1 month prior to T5 clip compression SCI. (a, b) Telemetric monitoring permitted data collection in freely moving rats for 3 days prior to
SCI (to establish reliable uninjured baseline responses) and for the first 10 days following SCI. Mean values (
±
s.e.m.) of 3 h intervals for five rats
are shown, and light and dark periods are indicated on the x axes. (c, d) Autonomic dysreflexia (AD) was examined at 7 and 28 days following
SCI. Average responses (
±
s.e.m.) to colorectal distension in five rats are shown. This figure is reprinted from Mayorov et al.
38
Autonomic assessment in experimental SCI
JA Inskip et al
9
Spinal Cord
plasticity after SCI, particularly during the crossed phrenic
phenomenon (CPP).
134,138
The CPP relies on a functionally latent bulbospinal
pathway that innervates the diaphragm bilaterally. When
the hemidiaphragm is paralyzed by C2 hemisection, hypoxia
induced by contralateral phrenic nerve section activates the
latent pathway to restore diaphragmatic function. This
pathway also becomes spontaneously active over time
following SCI, but this spontaneous return of activity may
not be sufficient to restore diaphragmatic function.
104
A
series of phrenic nerve function studies has revealed
that systemic delivery of xanthines (adenosine receptor
Figure 5 Innervation of the respiratory system. The main respiratory muscles are the diaphragm, intercostals and abdominals. The diaphragm
is the major inspiratory muscle and is innervated by phrenic motor neurons that lie in the cervical spinal cord (C3–C5 in humans; C3–C6 in
rats
404
). Innervation of respiratory intercostal and abdominal muscles exits the thoracolumbar spinal cord, from T1–T11 and T7–L2,
respectively. Activity of these muscles (as well as that of accessory muscles) is modulated by autonomic premotor neurons in the VLM, which
project to motor neurons in the spinal cord. The airways receive both parasympathetic and sympathetic inputs. The parasympathetic nervous
system provides the most important innervation to the smooth muscle of the airways, and is thus most important in controlling its diameter.
Preganglionic parasympathetic neurons originate in the NA, and innervate the trachea and the bronchi via the laryngeal and vagus nerves
(respectively). Parasympathetic innervation is predominately cholinergic and its action is excitatory, reducing airway diameter (via mAChR).
Sympathetic innervation of smooth muscle is comparatively scant. Preganglionic sympathetic axons exit at T4 and travel to paravertebral
ganglia, and post-ganglionic adrenergic fibers elicit bronchodilation, acting through b-AR. The airways also have extensive afferent innervation.
The most important afferents regulating respiration are vagal Ad, with cell bodies in the nodose ganglia and central axons projecting to the
NTS. Abbreviations:Ad, mechanoreceptors; b-AR, b-adrenergic receptors; C, cervical spinal cord; g, ganglion; L, lumbar spinal cord; mAChR,
muscarinic cholinergic receptors; NA, nucleus ambiguous; nAChR, nicotinic cholinergic receptors; NE, norepinephrine; n, nerve; NTS, nucleus
of the solitary tract; T, thoracic spinal cord; VLM, ventrolateral medulla; ( þ ) denotes excitatory synapses; () denotes inhibitory synapses.
Autonomic assessment in experimental SCI
JA Inskip et al
10
Spinal Cord
antagonists) can activate this pathway without phrenic
nerve section, to restore respiratory drive to phrenic motor
neurons following SCI.
105–107,123,126
These data, and pre-
liminary clinical experience, suggest that xanthine treat-
ment may represent a viable therapeutic option in weaning
individuals with SCI from ventilatory support.
139
Phrenic nerve recording has also been used to test the
efficacy of olfactory ensheathing cell (OEC) transplantation
in improving the respiratory outcome of experimental
SCI
140,141
(Table 9). In these experiments, rats with high
cervical hemisection (that abolished ipsilateral phrenic nerve
activity) received OEC transplantation at the time and site of
injury. In one study, rats that received OECs recovered
spontaneous respiratory rhythm in the ipsilateral phrenic
nerve by 2 months after SCI.
140
In another set of experi-
ments, ipsilateral phrenic nerve activity recovered to
approximately 80% of contralateral nerve activity by 3–6
months post-SCI.
141
In the latter set of experiments, the
authors transected the contralateral spinal cord to demon-
strate that a significant proportion of this recovery was due
to ipsilateral projections. However, the underlying mechan-
ism of recovery is not known.
Pneumotachometry
Pneumotachometry is the evaluation of the respiratory
volumes and rate that can be readily applied in conjunction
with phrenic nerve recording. This type of assessment was
important in identifying altered breathing patterns in rats
with unilateral cervical SCI.
127
Rats with C2 hemisection
exhibit a reduced expiratory volume and an increased
respiratory rate to preserve total (minute) ventilation.
127
Pneumotachography was also applied to verify the func-
tional significance of crossed phrenic pathways.
128
Diaphragmatic electromyography
Similar to clinical practice, electromyography (EMG) of the
diaphragm can be performed in conjunction with phrenic
nerve conduction studies in animals with SCI. For example,
rats that received OECs at the time of C2 hemisection
recovered both ipsilateral phrenic nerve activity and ipsilat-
eral diaphragm activity 3–6 months after treatment and
injury.
141
A recent study used diaphragmatic EMG to test the
effect of administering a N-methyl-D-aspartic acid (NMDA)
receptor antagonist (MK-801) in acute cervical SCI.
131
In
these experiments, i.p. MK-801 administration after C2
hemisection was associated with both recovery of ipsilateral
diaphragm function and upregulation of NMDA receptor
subunit NR2A.
Advantages/disadvantages (phrenic nerve conduction, pneumota-
chometry and diaphragmatic EMG). Although diaphragmatic
EMG, phrenic nerve recordings and pneumotachometric
evaluations have clinical correlates, they are much more
invasive procedures in the experimental laboratory than in
the clinical setting. These experiments are technically
demanding and terminal preparations. Although they
provide a quantitative and informative index of diaphrag-
matic function, other outcome measures may be more
suitable when respiratory function is not the sole or primary
focus of an experiment.
Table 4 Overview of studies characterizing or targeting respiratory dysfunction after experimental SCI
Technique used Species Injury model Time range post-injury References
Phrenic nerve conduction Rat C2 hemicontusion 1 week
99
4–6 weeks
99,100
C2 hemisection p24 h
101–118
p1 week
104,116,117,119,120
2–4 weeks
103,119,121–124
4–8 weeks
121,125
2–4 months
116,117,126
C4/5 contusion 2–11 weeks
100
Pneumotachometry Rat C2 hemisection 1–2 months
127,128
Dog T2, T4, T8 (seg. epid.) Immediate
129
Turtle D8–D10 Tx 4, 8 weeks
130
Diaphragm EMG Rat C2 hemisection p24 h
105,117,131
p1 week
119
2–4 weeks
119
90 days
117
C2 hemicontusion 7 days, 1 month
99
Dog T2, T4, T8 (seg. epid.) Immediate
129
Mouse C2 hemisection 1–48 h
132
Plethysmography Rat C2 hemisection 2, 3, 5 weeks
122
C4/5 hemicontusion 24 h, 1, 2, 4, 6 weeks
98
T8 contusion 24 h, 7 days
133
Abbreviations: EMG, electromyography; seg. epi., segmental epidural; Tx, transection.
Studies have been classified by the technique used to assess respiratory function. Studies employing a combination of techniques are referenced under both
headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.
Autonomic assessment in experimental SCI
JA Inskip et al
11
Spinal Cord
Plethysmography
An alternative outcome measure for assessing respiratory
function in experimental SCI is whole-body plethysmogra-
phy (WBP), in which respiratory function is determined in
conscious animals, without invasive instrumentation. Prior
to experimentation, rats are typically trained to become
acclimated to the plethysmography chamber to prevent
confounds of stress. In a recent study, WBP was used to
characterize respiratory function in rats with a C5 hemi-
contusion SCI.
98
These rats exhibited respiratory deficits that
are reminiscent of clinical SCI:
142
specifically, their ability to
augment tidal volume in response to hypercapnic challenge
was impaired 4 weeks after severe SCI. This technique has
also been used to verify the altered breathing pattern
induced by C2 hemisection previously reported in anesthe-
tized rats.
122
Although spirometry is more commonly used in
the clinic, WBP has recently been validated for respiratory
evaluation of people with SCI.
143,144
Advantages/disadvantages. The most obvious advantage of
WBP is that it is not a terminal experiment. Rather,
respiratory data can be collected in the same animals at
different time points following SCI. It does not provide a
direct index of diaphragm function, but does provide
clinically relevant indices of respiratory function in experi-
mental animals. As it also requires less technical expertise
than other methods, WBP may represent an attractive
method for many laboratories investigating respiratory
dysfunction after experimental SCI.
Gastrointestinal function
Innervation of the gastrointestinal system
The GI system control involves a complex interaction
between the somatic nervous system (anal sphincters), both
divisions of the autonomic nervous system, and the unique
intrinsic enteric nervous system (ENS). The ENS controls the
secretion, motility, blood flow, storage and evacuation of the
GI tract, and its basic organization and function is similar
across species. The autonomic nervous system modulates the
intrinsic activity of the ENS, and is especially important at
the proximal and distal ends of the GI tract (Figure 6). More
detailed information about the interactions between the
autonomic nervous system and ENS can be found in recent
reviews.
145–147
Clinical impact of gastrointestinal dysfunctions following SCI
Although the ENS does have some intrinsic functional
capacity, this is significantly impaired when it loses central
coordination following SCI. The disruption of autonomic
innervation to the GI tract is primarily revealed by abnormal
motor function as opposed to changes in secretory or
absorptive function.
148
GI problems are prevalent in both
the acute and chronic periods following SCI, and are a
significant cause of rehospitalization and morbidity.
149–153
Although there is some controversy over the effect of SCI on
the upper GI tract, there is evidence of gastric dilation,
delayed gastric emptying (GE), gastric ulceration and
prolonged orocecal transit time (OCTT) following
SCI.
154–157
The most common complications are lower GI
tract dysfunctions and most research has focused on their
identification and resolution. These typically present as
constipation, compaction and fecal incontinence, and can
trigger both physical and psychological problems that
restrict lifestyle choices and disrupt rehabilitation and
overall quality of life of individuals with SCI.
158,159
The term ‘neurogenic bowel’ describes the loss of neuronal
control to the colon, and resulting dysfunction.
160
The
neurogenic bowel can be divided into two main types, each
with characteristic colonic dysfunctions; supraconal lesions
result in damage to descending supraspinal pathways (upper
motor neuron bowel syndrome) and infraconal (cauda
equina or pelvic nerve lesions) damage to motor and
parasympathetic innervation to the colon (lower motor
neuron bowel syndrome).
160
The upper motor neuron
bowel, or hyperreflexive bowel, is associated with spastic
activity in the colon and external anal sphincter (EAS),
which interferes with the voluntary ability to defecate but
leaves the bowel reflex intactFthe basis for bowel emptying
using chemical or mechanical stimuli. The lower motor
neuron, or areflexive, bowel is associated with a relaxed
colon, perturbed peristalsis, slow stool propulsion and
constipation.
160
Assessment of gastrointestinal function following experimental
SCI
Given that SCI primarily affects GI motor control, the
majority of GI assessments following SCI consist of tests of
functional motility or contractile activity. This section
focuses on functional GI assessments used in experimental
animal models (Table 5), some of which have been used as
outcome measures for assessing autonomic function of the
GI tract in humans following SCI and for testing clinically
relevant therapies for restoring GI function.
Gastrointestinal transit with oral markers
GI transit can be assessed in experimental animals by
segmental dye recovery along the GI tract following oral
marker delivery.
170,171
In the experimental animal, a non-
toxic, nonabsorbable dye is administered by gavage feeding
to the stomach. After the desired time interval, animals are
euthanized, clamps are secured between each GI segment
and marker recovery is detected by spectrophotometry to
quantify the amount of dye in each segment, an indicator of
movement through the GI tract.
170
Dye recovery has been used as an outcome measure in rats
with SCI at various levels.
161,172
Rats with cervical or thoracic
spinal cord transection showed increased dye recovery in the
stomach and decreased recovery in the small intestine
throughout the first week after injury, indicating decreased
GE and overall GI transit.
161
However, after 10 days post-SCI,
there were no differences in dye recovery between the
stomach and intestine, indicating that there is a recovery of
GE and overall GI transit.
164
Unlike humans, these rats also
showed concurrent spontaneous recovery of bowel func-
tion.
172
A follow-up study revealed that large bowel empty-
Autonomic assessment in experimental SCI
JA Inskip et al
12
Spinal Cord
Figure 6 Innervation of the distal gastrointestinal (GI) tract. The ENS is composed of two main ganglionated plexuses that lie between the
longitudinal and circular muscle layers (myenteric or Auerbach’s plexus) and within the submucosal layer of the GI tract (submucosal or
Meissner’s plexus). The myenteric plexus is continuous throughout the entire length of the GI tract, and is primarily involved in the
coordination of smooth muscle activity, whereas the submucosal plexus is present primarily in the small and large intestines, where it controls
secretion and absorption.
145
Autonomic innervation of the GI tract is required to modulate the intrinsic activity of the enteric nervous system
(ENS). This modulation is especially important in the distal GI tract (illustrated here), where the ANS coordinates storage and evacuation by
regulating colon motility and resting anal sphincter tone.
405,406
Parasympathetic innervation of the distal colon and rectum originates in the
sacral cord (S2–S4), whereas the upper GI tract (to the level of the splenic flexure) is innervated by the vagus nerve (not illustrated here).
Preganglionic parasympathetic neurons synapse directly on Auerbach’s plexus that enhances smooth muscle activity (via mAChR).
Sympathetic innervation is mainly postganglionic, and arises from paravertebral and prevertebral ganglia of the abdominal and pelvic
cavitiesFceliac, superior and inferior mesenteric and pelvic ganglia. Sympathetic neurons in prevertebral ganglia inhibit muscle and secretory
activity indirectly, by noradrenergic modulation of activity in both the Meissner and Auerbach’s plexuses. The IAS receives both sympathetic
and parasympathetic innervation, whereas the EAS is innervated by somatic fibers traveling in the pudendal nerve (S2–S4 in humans, L6–S1 in
rats). Afferent information from this area travels in both the pelvic and pudendal nerves. Abbreviations:Ad, C, mechanosensitive primary
afferents; aAR, a-adrenergic receptors; DR g, dorsal root ganglion; EAS, external anal sphincter; g, ganglion; IAS, internal anal sphincter; IM g,
inferior mesenteric ganglion; mAChR, muscarinic cholinergic receptors; n, nerve; nAChR, nicotinic cholinergic receptors; NO, nitric oxide; ( þ )
denotes excitatory synapses; () denotes inhibitory synapses.
Autonomic assessment in experimental SCI
JA Inskip et al
13
Spinal Cord
ing prevented the development of delayed GE and GI transit
following SCI.
162
Although the effects of SCI on the upper GI
tract are clinically still somewhat controversial, with some
groups reporting GE delays and others demonstrating
normal GE,
155–157,173
these results suggest that delayed GE
is in fact secondary to lower GI delayed motility.
162
This
corroborates previous research showing that the GE reflex is
mediated by vagovagal reflexes and is not disrupted by
sympathetic denervation of the upper GI tract.
174,175
Visualization techniques, similar to orally administered
radionucleotides used to assess GI motility clini-
cally,
156,176,177
can also be applied in the experimental
setting. Video fluoroscopy has been recently used to assess
the functional benefits of colonic electrical stimulation as a
treatment to improve colonic transit in rats
178,179
and SCI
cats.
165
These results suggest that colonic transit times are
improved with the use of colonic electrical stimulation.
180
Magnetic resonance imaging (MRI) has also been used to
visualize GI motility by detection of solid food labeled with
trace amounts of nontoxic iron oxide particles in rats.
181
Advantages/disadvantages. Oral marker delivery for evalua-
tion of GI transit can require training and animal habitua-
tion, particularly if gavage feeding is required. The detection
of the markers can be performed as a terminal preparation, or
by indirect visualization. The latter type of detection
techniques (MRI or X ray) are attractive as they could be
easily combined with sensory or motor outcomes; however,
these techniques are more expensive and technically
demanding.
Electromyography
EMG recording of EAS activity has been used as a tool to
investigate the physiology and pathophysiology of the EAS
in rats with SCI.
168
This preparation allows for the measure-
ment of both baseline EMG activity in the EAS and the
contractions stimulated by EAS distension.
168
Animals are
restrained in a supine position using a loose-fitting cylinder,
or masking tape, to secure their torso, hind limbs and
tail.
168,169
Temporary bipolar EMG electrodes are implanted
in the EAS, with the external wire attached to the tail and
connected to a preamplifier. After recording baseline EAS
EMG activity, EAS distension is initiated with a plastic probe
(used to mimic fecal bolus), and the resultant EAS contrac-
tions are recorded.
168
Anesthetics are not normally used in
this assessment, as they significantly attenuate EAS hyper-
reflexia.
This technique has also been used to assess functional
recovery of autonomic reflexes after the period of spinal
shock, and the development of hyperreactive autonomic
reflexes.
169
EAS hyperreflexia, reflected in prolonged burst
duration of EAS activity, developed 2 days post-contusion at
T9–T10 and resolved to preoperative levels, not significantly
different from controls, by 6 weeks post-injury. In contrast,
spinally transected animals developed EAS hyperreflexia 7
days post-injury and did not demonstrate any EAS reflex
recovery.
169
This research demonstrates the sensitivity of EAS
EMG recording and its potential as an objective assessment
of pelvic autonomic reflexes.
EMG recording from the jejunum has been recently
adapted for use with telemetry, allowing for recording in
awake and mobile rats.
182
In this technique, an EMG
transmitter is implanted dorsally between the shoulder
blades and connected to the electrodes in the jejunum.
182
Advantages/disadvantages. EMG is technically demanding,
usually terminal, and requires both training and specialized
equipment. However, it represents the most sensitive and
direct evaluation of the activity in the GI tract.
Manometry
Manometry, the measurement of pressure changes within
different parts of the GI tract, has also been used to assess
colonic motility in rats following SCI.
166
Similar to the
clinical technique
183–185
a fluid-filled catheter is inserted
into the colon and is attached to recording probes at
different regions along the colon; however, in the rat these
probes are secured with ligatures to allow for chronic
Table 5 Overview of studies characterizing or targeting gastrointestinal dysfunction after experimental SCI
Technique used Species Injury model Time range post-injury References
GI transit with oral markers Rat T4–T5 Tx 30 min–7 days
161
1day
162,163
1–30 days
164
C7–T1 Tx 30 min; 6 h; 1, 3, 7 days
161
Cat T4 hemostat clamp 1–2 weeks
165
Manometry Rat T4 Tx 1 day–2 weeks
166
Cat T4 hemostat clamp 1–2 weeks
165
Dog T10 Tx 2–6 weeks
167
EMG Rat T9–T10 Tx p24 h
168
2 days–6 weeks
168,169
T9–T10 contusion 2 days–6 weeks
169
Abbreviations: EMG, electromyography; GI, gastrointestinal; Tx, transection.
Studies have been classified by the technique used to assess gastrointestinal function. Studies employing a combination of techniques are referenced under both
headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.
Autonomic assessment in experimental SCI
JA Inskip et al
14
Spinal Cord
recording.
166
To quantify motor activity, a motility index can
be calculated that incorporates the amplitude, duration,
frequency and number of contractions. Using this techni-
que, spinally transected rats showed a reduction of distal
colonic motility acutely after injury, which returned to
preoperative values after 7 days.
186
Unfortunately, the
combined motility index does not provide an indication of
the functional motility in the colonFan important con-
sideration given that one of the main problems following
SCI is the lack of coordinated peristalsis rather than lack of
overall contractile activity.
155
However, when used in
combination with oral marker delivery, the effects of reduced
motility can be more clearly assessed.
165,179,187
Strain gauge transducers
Strain gauge transducers have also been used to continuously
record gastric, small intestinal and colonic motility in awake
rats.
188
In contrast to manometry, this technique measures
the pressure changes on the extraluminal surface of the GI
tract.
189–191
Although each transducer has only uniaxial
sensitivity, by placing transducers at right angles to each
other in the same segment, both longitudinal and circular
contractile muscle activities can be recordedFa good
indicator of coordinated motility.
189
This technique has
been used to model postoperative GI tract paresis,
188
assess
neurochemical effects on GI motility
192
and examine the
functional effects of altered pacemaker activity in the gut.
193
Although this technique has not yet been used in experi-
mental SCI, its relative noninvasiveness and capacity for
chronic use makes it a promising way to measure functional
GI motility.
Advantages/disadvantages (manometry and strain gauge
transducers). Both manometry and strain gauge techniques
require a fairly significant investment in training and
equipment. Manometry is ideal for acute measurements,
but its use is restricted to the colon. The use of strain
gauges is surgically demanding, but does not interfere with
normal GI motility, and is therefore preferable for chronic
recording.
194
Hydrogen breath test
Hydrogen breath tests are commonly used to assess the
pathophysiology of clinical GI disorders. As there is no
bodily source of hydrogen other than that produced by
bacterial metabolism in the cecum, an increase in hydrogen
expiration following carbohydrate administration indicates
the arrival of the nutrient bolus in the cecum, and can be
used as a measure of OCTT.
195–197
Using this test, individuals
with SCI show significantly delayed OCTT compared to
controlsFan effect that is more pronounced in quadriplegic
than paraplegic patients.
198
Although this technique has also
been validated in feline, canine and rodent models,
195,199,200
it has not yet been used in experimental SCI. However, it
may prove to be a useful outcome measure to assess GI
function following SCIFat both the bench and the bedside.
Advantages/disadvantages. The hydrogen breath test is mini-
mally invasive and could easily be used in conjunction with
other assessments in SCI animals. It can be used at repeated
intervals in the same animals. However, specialized equip-
ment is necessary for detection of hydrogen in the breath.
Urinary bladder function
Innervation of the lower urinary tract
Although interspecies differences exist, the basic organiza-
tion and innervation of the LUT is similar among common
experimental species and humans (Figure 7). In general
terms, storage of urine is sympathetically mediated, whereas
micturition is elicited by parasympathetic activation: how-
ever, normal LUT function requires the coordinated activity
of the sympathetic, parasympathetic and somatic nervous
systems. Here we provide only the basic scheme of LUT
innervation, as neural control of the LUT and associated
reflexes are comprehensively reviewed elsewhere.
201–207
Clinical implications of lower urinary tract dysfunction
following SCI
The manifestation of LUT dysfunction after SCI is
similar in experimental animals,
67,208
(Table 6) and
humans,
205,206,268–270
and is broadly termed neurogenic
bladder. The initial period following SCI is marked by
bladder areflexia and urinary retention. When SCI occurs
at or below the sacral level (that is, infraconal; a lower
motoneuron injury) bladder areflexia persists. If the lesion
extends rostrally to the thoracolumbar region to involve
sympathetic preganglionic neurons, the bladder neck may
also become hypoactive. Suprasacral (supraconal) SCI (an
upper motor neuron lesion) typically produces hyperreflexia
of the smooth (detrusor) muscle of the bladder and tonic
activation of the striated urethral sphincter. Therefore,
sphincter contractions are dyssynergic with detrusor con-
tractions. Detrusor hyperreflexia and detrusor–sphincter
dyssynergia result from both a loss of tonic supraspinal
inhibition and the emergence of aberrant spinal reflexes. The
clinical profile of LUT dysfunction is highly variable between
individuals with SCI, but can be generally described in terms
of impaired continence (most common in supraconal SCI),
impaired emptying (most severe in infraconal SCI) and
impaired sensation of bladder filling (a component of
dysfunction for most people with SCI). Clinically, complica-
tions of LUT dysfunction remain the leading cause of
rehospitalization among people with SCI.
271
Assessment of lower urinary tract function following
experimental SCI
SCI induces profound changes in bladder innervation, parti-
cularly afferent, circuitry,
206,217,272,273
morphology
209,213,274
and structure,
275–278
all of which likely contribute to neuro-
genic bladder. We limit this discussion to functional assess-
ments only, with an emphasis on tests used in clinical SCI.
Although an animal model of cauda equina/conus medullaris
injury (lumbosacral ventral root avulsion in rat) has been
recently developed,
279,280
available experimental data describe
Autonomic assessment in experimental SCI
JA Inskip et al
15
Spinal Cord
bladder dysfunction following supraconal (not infraconal) SCI.
Although SCI-induced LUT dysfunction has been well-char-
acterized in experimental animals, few studies have included
LUT assessment as an outcome measure in testing therapies for
SCI (Table 9).
Cystometric urodynamic analysis
The most common method for assessing LUT function in
experimental animals is cystometric urodynamic analysis.
Cystometry can be performed using a urethral or transvesical
catheter implanted into the bladder dome: although the
latter method is more invasive, it does not partially obstruct
the urethra and permits EMG of the external urethral
sphincter (see ‘External urinary sphincter electromyogra-
phy’) to be performed concurrently. Cystometry is routinely
conducted in both conscious and anesthetized animals, with
the caveat that anesthesia does affect micturition reflexes,
281
particularly in animals with chronic SCI.
235,236,257
This
undesirable effect may be partially addressed by reducing
the dose of anesthetic.
236
Whether transvesical or transure-
thral, conscious or unconscious, cystometry in experimental
animals is essentially similar to clinical cystometry: the
bladder is filled with saline while recording intravesical
pressure to examine the relationship between bladder
volume and pressure during filling and micturition.
Detrusor hyperreflexia and detrusor–sphincter dyssynergia
create a similar urodynamic pattern in rats
209,237,218
and
humans
206,269,282,283
with suprasacral SCI. Post-injury cysto-
metrograms are characterized by increases in volume thresh-
old for inducing micturition, pressure during micturition,
volume expelled during micturition and residual volume
after micturition. In addition, spinal-cord-injured rats and
humans often exhibit nonvoiding detrusor contractions
during bladder filling, a hallmark of detrusor hyperreflexia.
Rats assessed via conscious transvesical cystometry 2–3
weeks after complete thoracic (T8–T10) spinal transection
had larger volume thresholds for micturition (1.43 versus
0.34 ml), larger micturition pressures (48 versus 26 mm Hg),
increased voided volumes (0.72 versus 0.31 ml) and in-
creased residual volumes (0.71 versus 0.03 ml) compared to
uninjured controls.
218
Figure 7 Innervation of the lower urinary tract (LUT). The LUT is comprised of the bladder, urethral sphincter and urethra. The LUT receives
the bulk of its innervation from three nerves. The hypogastric nerve carries sympathetic innervation to the LUT; contributing spinal nerves exit
the spinal cord (SC) between L1 and L2. Muscle activity for storage is mediated by a-AR expressed in the trigone, bladder neck and urethra
(excitatory), and by b-AR expressed in the bladder dome (inhibitory). The pelvic nerve contains parasympathetic input originating in the sacral
cord (L6–S1 in rat
407
) and controls micturition via cholinergic muscarinic receptors (mAChR) expressed throughout the LUT. The human
pudendal nerve exits the sacral SC, and provides somatic innervation to the striated muscles of the external urethral sphincter; in rats, the
pudendal nerve originates in the L6–S1 cord. In addition to their efferent function, each of these nerves carries afferent input from the LUT.
Information about bladder distension is carried by mechanosensitive afferents (Ad, C) found primarily in the pelvic nerve. These afferents signal
the coordinated switch between storage and micturition. The pudendal and hypogastric nerves mostly contain nociceptive afferents, which are
not depicted here. Abbreviations: AR, adrenergic receptors; DR g, dorsal root ganglion; EUS, external urinary sphincter; g, ganglion; IM g,
inferior mesenteric ganglion; L, lumbar spinal cord; mAChR, muscarinic cholinergic receptors; n, nerve; nAChR, nicotinic cholinergic receptors;
NO, nitric oxide; P2X, purinergic receptor; S, sacral spinal cord; ( þ ) denotes excitatory synapses; () denotes inhibitory synapses.
Autonomic assessment in experimental SCI
JA Inskip et al
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Spinal Cord
Preclinical urodynamic analysis has been used extensively
to characterize mechanisms of and identify therapeutic
candidates for bladder dysfunction following SCI (Table 6).
Such experiments continue to be informative even after
treatments enter clinical practice: for example, rats were
recently treated with botulinum-A to examine the effects of
acute versus delayed therapy following SCI.
238
To date, only
a few experimental studies testing regenerative, plasticity-
promoting or protective treatments for SCI have incorpo-
rated urodynamic analysis as an outcome measure (Table 9).
In one such study, rats that received transplants of
immortalized neural stem cells at the site of thoracic SCI
(T8 contusion) exhibited reduced micturition pressure and
reduced residual urine compared to untreated controls.
284
Table 6 Overview of studies characterizing or targeting lower urinary tract dysfunction after experimental SCI
Technique used Species Injury model Time range post-injury References
Cystometry Rat T8–T10 contusion p1 week
209–211
2–4 weeks
209,211,212
8 weeks
210,213
T8–T11 Tx Immediate
214
24 h
215,216
p1 week
217
2–4 weeks
209,214,216–234
4–8 weeks
225,231,235–245
8–12 weeks
232,246,247
T12 heat injury 1 month
248
L3/4; L6/S1 Tx Immediate; 2–5 weeks
214
Rabbit T10 Tx 1, 2, 7–21 days
249
Cat C6–T1 Tx 10 weeks
250
T1 clip compression 2–3, 5–7 weeks
251
T8 Tx; T8 contusion 3 weeks
252
T10 d. fun. Tx Immediate
253
T10–T12 Tx p24 h
254
2–4 weeks
255,256
4–8 weeks
255–261
6–12 months
257,260
Dog T8–T11 Tx 1–8 weeks
262
1–8 months
263,264
External urinary sphincter EMG Rat T8 contusion p1 week
209,213
2 weeks
209
6–8 weeks
210,213
T8–T9 Tx Immediate
214
2–4 weeks
209,214
4–6 weeks
214,241,243
T8–T11 Tx 2–4 weeks
219
4–8 weeks
236,237,239,240
8–12 weeks
246
L3/4; L6/S1 Tx Immediate; 2–5 weeks
214
Rabbit T10 Tx 1, 2, 7–21 days
249
Cat C6–T1 Tx 10 weeks
250
T1 clip compression 2–3, 5–7 weeks
251
T9–T12 Tx 2–4 weeks
256
6–8 weeks
253,258,261
Dog T8–T10 Tx 1–8 weeks
251
1–8 months
264
Bladder volume Rat T8; T9/10 contusion p1 week
213,265,266
2–4 weeks
265–267
4–6 weeks
265,266
T10 Tx 0–20 days
217
Cat T8 Tx; T8 contusion 2 weeks
252
Videofluoroscopy Cat C6–T1 Tx 10 weeks
250
T1 clip compression 2–7 weeks
251
Dog T8–T9 Tx 1–8 weeks
262
Pressure recording (c. spongiosus) Rat T9/10 contusion p1 week; 2–4 weeks
266
Abbreviations: compr., compression; c. spongiosus, corpus spongiosus; d. fun., dorsolateral funiculus; EMG, electromyography; Tx, transection.
Studies have been classified by the technique used to assess lower urinary tract (LUT) function. Studies employing a combination of techniques are referenced
under both headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.
Autonomic assessment in experimental SCI
JA Inskip et al
17
Spinal Cord
Two subsequent studies in the same injury model demon-
strated that transplantation of neural precursor cells
285
or
genetically modified fibroblasts
286
accelerated recovery from
bladder areflexia and reduced micturition pressure and the
number of nonvoiding detrusor contractions.
External urinary sphincter electromyography
In both clinical and experimental assessment of SCI, external
urethral sphincter EMG (EUS EMG) can be performed in
conjunction with cystometry to provide a direct measure-
ment of detrusor–sphincter dyssynergia. Wire electrodes are
inserted into the muscle of the EUS, and EUS activity during
bladder filling and micturition is recorded. The data are
typically expressed as mean EMG activity, mean EMG power
frequency, mean EMG-spiking activity (ESA) and durations
of activity or contractions.
213,236
Also, the relationship
between EUS EMG amplitude and detrusor contractions is
examined to determine the extent of coordination between
detrusor and sphincter activities. Changes in EUS EMG
throughout the micturition cycle can be examined by power
spectrum analysis using the fast Fourier transform algo-
rithm.
213
In rats
209,213,237
and humans
206,269,287,288
with suprasacral
SCI, EUS EMG reveals sphincter activity that is not associated
with detrusor contraction or micturition. In rats, the
micturition-induced increase in external sphincter activity
(dESA) is lost following thoracic SCI: as dESA is negatively
correlated with SCI severity, it can be used as an index of
detrusor–sphincter dyssynergia.
213
Other intriguing indices
of bladder-sphincter synergy have been identified by analyz-
ing fractal dimensions and power spectra of EUS EMG and
cystometrograms.
239,240
Akin to urodynamic analysis, EUS
EMG has been used as an outcome measure in mechanistic
studies of LUT dysfunction in experimental SCI (Table 6).
However, this technique is rarely included in preclinical
studies that assess the functional benefits of therapies for SCI
(Table 9).
Advantages/disadvantages (Cystometric urodynamic analysis and
External urinary sphincter electromyography). Cystometry and
EUS EMG applied in combination definitely represent the
most informative and clinically relevant assessment of LUT
function following SCI. However, these are invasive, techni-
cally demanding procedures that can only be performed at a
single time point (that is, at the end of the experiment).
Laboratories that lack expertise in these techniques may
wish to adopt another method of monitoring bladder
function after SCI.
Functional bladder volume
In rats, increases in bladder volume are proportional to
severity of SCI.
213,274
Bladder volume is estimated by
measuring the volume of urine expressed at micturition,
either by using a metabolic cage
267,284–286
or by measuring
the volume of urine that can be manually expressed.
289
Bladder volume has been used as an outcome measure in
experiments testing chondroitinase (a bacterial enzyme) as a
regenerative therapy for SCI.
289
In these experiments,
intrathecal chondroitinase treatment reduced the volume
of urine that could be manually expressed in rats with
moderate thoracic SCI.
An attractive alternative to estimating bladder function by
expressed urine is transabdominal ultrasound, which has
been recently used in rat SCI.
265
In this study, a handheld
digital ultrasound imaging system was used to measure
bladder volume following severe thoracic (T10) contusion.
Bladder volume was 3.51
±
0.47 ml (compared to
0.089
±
0.04 ml in uninjured rats) on day 4 post-injury and
decreased to 1.83
±
0.50 ml by day 8; it did not change
significantly for the duration of the experiment (46 days).
The authors found that ultrasonic measurements of bladder
volume were comparable to estimates based on manual
expression of urine.
Advantages/disadvantages. Measurements of volume of ur-
ine per micturition represent a cost-saving alternative for
monitoring bladder function, but are tedious and lack
precision. Transabdominal ultrasound is noninvasive, less
stressful than other methods, does not disrupt concomitant
motor and sensory testing and permits assessment in the
same animals throughout the recovery period. As this is a
common clinically used test, it could be recommended for
more frequent use in animal experiments. This method will
require some investment in technology and training.
Videofluoroscopy
In videofluoroscopy, the bladder is filled with radio-opaque
medium and imaged by X-ray video recording. This
approach is commonly used to evaluate a variety of organ
functions in the clinical setting. Fluoroscopy has been used
in preclinical investigations of electrical stimulation to
improve bladder function following SCI (Table 6). These
studies characterize the effects of sacral spinal stimula-
tion,
262
direct bladder stimulation,
251,290
and more recently,
neuroprosthetic microstimulators targeting the bladder wall
and plexus
291,292
in dogs and cats with SCI.
Advantages/disadvantages. Videofluoroscopy is a clinically
relevant technique and provides useful information for
specific applications (those mentioned above) in larger
species; however, it has never been used in rodent SCI. It
also requires specialized equipment and expertise that is not
likely to be available to most investigators.
Corpus spongiosum pressure recording
Another method of examining LUT function that has been
recently developed involves telemetric monitoring of pres-
sure within the corpus spongiosum of the penis (CSP).
266,293
Traditionally used to study sexual function in experimental
animals
294–296
(see ‘Sexual function’), CSP pressure has been
recently validated for assessing LUT function in spinal-cord-
injured rats.
293
In this study, the authors found that volume
of urine expelled per micturition was highly correlated with
micturition duration recorded by CSP pressure: thus, tele-
metric CSP pressure monitoring can provide information
about voiding volume and frequency of micturition in freely
Autonomic assessment in experimental SCI
JA Inskip et al
18
Spinal Cord
moving, conscious animals throughout their recovery from
SCI. At 21 days after thoracic (T10) contusion, rats with
telemetric pressure monitoring had micturition CSP pressure
waveforms that were increased in duration, mean pressure
and peak frequency compared to those observed before
SCI.
266
Advantages/disadvantages. Pressure recording from erectile
tissue is technically challenging, but has the benefits of
being able to be used chronically and in unanesthetized,
freely moving animals. It is of particular interest for outcome
assessment post-SCI because it allows for simultaneous
recording of micturition and erection eventsFboth of which
are perturbed following SCI.
Sexual function
Innervation of the pelvic organs and genitalia
The autonomic innervation of the pelvic organs is essentially
similar across mammalian species, and generally similar
between males and females.
297
Here we review only the most
pertinent features of tissue innervation in the pelvic organs
and genitalia (Figure 8). More detailed reviews can be found
elsewhere.
298–300
The role of the autonomic nervous system in sexual function
Normal sexual function requires a combination of local
spinal reflexes and descending cortical control.
300,301
Genital
arousal is a combined neurovascular and neuromuscular
response that can be initiated reflexively or psychogenically.
In both sexes, reflex sexual arousal results from increased
parasympathetic activity, which causes nitric-oxide-
mediated vasodilation,
302–304
accompanied by inhibition of
sympathetic adrenergic activity. Together these two changes
lead to an increase in genital blood flow, engorgement of
erectile tissues and intracavernous pressure increase and,
in women, lubrication of the vaginal surface. Unlike
reflex arousal, psychogenic arousal appears to be facilitated
by the sympathetic nervous system.
305–308
In the male
rat, and probably also in humans, the contraction of the
somatic striated perineal muscles (bulbospogiosus and
ichiocavernosus) also contributes to penile rigidity during
erection.
309–312
Sexual climax appears to be mediated by a spinal reflex:
313
in response to genital stimulation, anesthetized, acutely
spinalized rats show a urethrogenital response similar to that
seen in human sexual climax.
307,314–316
This reflex includes
clonic contractions of the perineal muscles, rhythmic firing
of the cavernous nerve, penile erections and ejaculation in
the male, and rhythmic vaginal and uterine contractions in
the female.
316
This response is mediated by efferent output
from hypogastric and pelvic nerves, suggesting that both
parasympathetic and sympathetic nervous systems are
involved.
317
With regards to the autonomic nervous system,
ejaculation itself is considered to be a sympathetically
mediated event;
318
however, normal anterograde ejaculation
requires the coordination of both the somatic and sympa-
thetic nervous systems.
The autonomic nervous system also has a role in the
maintenance of reproductive capacity, although there is
comparatively little research in this area.
319
The activity of
the epidydimis, whose main functions include sperm
transport
320,321
and fluid resorption,
319
is primarily regulated
by the sympathetic nervous system, with particularly
important innervation arising from the inferior mesenteric
ganglion.
320,321
Clinical impact of sexual dysfunctions following SCI
Low sexual satisfaction and sexual dysfunction are both well
documented after SCI, and the resolution of these problems
has been identified as a high priority.
9,322
The disruption to
autonomic circuits following SCI can result in a number of
different sexual changes and the sexual function subgroup of
the ASIA/ISCOS working group is currently developing
international autonomic standards for documenting these
changes.
323
Here we focus primarily on changes in sexual
function rather than reproductive function. The effect of SCI
on sexual function is highly dependent on injury level and
the most commonly affected sexual responses are arousal
and orgasm.
Individuals with upper motor neuron lesions, and pre-
served S2–S5 roots, generally have preserved reflex genital
arousal, as the reflexes mediating erection are located in the
spinal cord. However, these individuals generally are unable
to initiate genital arousal psychogenically. On the other
hand, lower level SCI (infraconal or cauda equina) tends to
disrupt reflex vasocongestion, but can leave sympathetically
mediated psychogenic arousal intact.
306,307
These psycho-
genic erections are comparable in duration, rigidity and
tumescence to the preserved reflex erections in men with
higher lesions.
306
Many women with SCI maintain the ability to reach
orgasm. However, men often have difficulty maintaining
erection, ejaculating and sensing orgasm. As a result, male
reproductive function is significantly affected by SCI. Penile
vibratory stimulation and electroejaculation have been used
to successfully manage infertility in many cases.
324–326
Most
women maintain reproductive capacity, and are able to
become pregnant and undergo normal pregnancy after a
short period of amenorrhea acutely after SCI.
327,328
AD must also be included in the discussion of sexual
function, as it can be triggered by sexual activity and sperm
retrieval in those with injuries above T5.
329–334
During
periods of AD, blood pressure can reach levels that are
potentially life threatening. However, despite these extreme
blood pressure levels, the symptoms of AD are not always
subjectively detected by the individual (termed silent AD).
335
Therefore, it is imperative that blood pressure be assessed in
both private sexual activity and during sperm retrieval
procedures to assure that patients are not at risk.
335
Interestingly, although AD interferes with sexual activity
for some individuals, the symptoms of AD during
sexual activity can also be perceived as pleasurable or
arousing.
333,336
Autonomic assessment in experimental SCI
JA Inskip et al
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Spinal Cord
Assessment of sexual response and reproductive function following
experimental SCI
Although the subjective responses from human studies are
indispensable to fully elucidate sexual functioning following
SCI,
337
physiological data can be obtained from experimen-
tal animal models regarding normal sexual function
338
and
recovery of autonomic circuits after injury.
266
Spinal transec-
tion models have been used to study the neurophysiology of
spinal sex reflexes in the absence of supraspinal control,
316
and to study infertility post-SCI.
339–341
However, sexual
function has been rarely used as an outcome measure in
testing therapies for SCI (Table 7). Like human research,
Figure 8 Innervation of the pelvic organs and genitalia. In the pelvic organs and genitalia there are three main tissue types: secretory, erectile
and striated muscle. The majority of the autonomic innervation to these tissues comes from the bilateral pelvic ganglia (PG), which contains
both sympathetic and parasympathetic neurons. Parasympathetic preganglionic neurons originate in the sacral cord (S2–S4 in humans; L6–S1
in rodents) and travel in the pelvic nerve to the PG. Sympathetic innervation originates in the lumbar cord (L1–L2) and travels via the
hypogastic nerves to innervate the PGFin rodents, sympathetic nerves also travel to the PG via the pelvic nerve, which is mixed sympathetic
and parasympathetic. Unlike human PG, which form a diffuse plexus on either side of the prostate (men) or cervix (women), rat PG are more
condensed, and form two true ganglia. In both sexes, the largest nerve exiting from the PG is the cavernous nerve (also called penile nerve in
males). In humans, somatic innervation of the striated perineal muscles, which include the ischiocavernosus, bulbocavernosus and levator ani,
originates in the sacral cord (S2–4), whereas in the rats, this is shifted rostrally (L6–S1). Afferent information from the pelvic organs is relayed to
the spinal cord via the ‘genitospinal’ nerves (pelvic, hypogastric and pudendal; for simplicity, only the pelvic nerve is illustrated here) and
sensory pathways ascend bilaterally in the dorsal quadrant of the spinal cord.
408
Abbreviations: AChR, cholinergic receptors; DR g, dorsal root
ganglion; g, ganglion; IM g, inferior mesenteric ganglion; n, nerve; nAChR, nicotinic cholinergic receptors; NE, norepinephrine; NO, nitric
oxide; NPY, neuropeptide Y; ( þ ) denotes excitatory synapses; () denotes inhibitory synapses.
Autonomic assessment in experimental SCI
JA Inskip et al
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Spinal Cord
experimental animal research has been dominated by the
study of male sexual function, and there are comparably few
validated experimental animal models of female sexual
function.
348
This section focuses on functional assessments
of autonomically mediated components of sexual and
reproductive function, with a focus on those that have been
used in experimental SCI.
Ex copula visual scoring
Visual scoring has long been used to assess sexual function in
experimental animals. This semi-quantitative technique
includes observation, grading and quantification of sexual
arousal and copulatory events (mounts, intromissions and
ejaculations in the male; lordosis in the female animals).
349
Ex copula tests have been primarily used in experimental SCI
models, as functional copulatory behavior is unrealistic
given the associated motor deficits. Animals are generally
tested in a supine position with their legs and torso
restrained by straps.
350
To test reflex sexual function, penile reflexes are elicited by
the retraction of the penile sheath and light pressure on the
base of the penis,
351
mechanical stimulation of the ure-
thra
316,352
or by electrical stimulation of the dorsal penile
nerve.
343
The stereotyped responses occur in clusters and
include erections (reddening of the glans), cups (flaring of
the glans into a trumpet shape) and flips (anteroflexions of
the glans of varying lengths).
351,353,344
After SCI, these reflex
erections occur more quickly and more frequently than in
uninjured animals.
342
Visual scoring can also be used to investigate psychogenic
erectile function. Penile reflexes triggered by central stimula-
tion of the medial preoptic area (a brain region with a well-
established role in the facilitation of sexual behavior)
revealed that the capacity for centrally mediated erections
is preserved in a rat model of cauda equina injury.
350
As
erectile function has been traditionally associated with
parasympathetic activation, this research revealed that the
presence of thoracolumbar sympathetic activity is sufficient
to mediate erection, and likely forms a component of normal
erectile function.
350
Similar results have also been found
clinically.
354
Like many of the other assessment methods described
herein, visual scoring is most valuable when used in
conjunction with other physiological measures, such as
EMG recording. Together, these two techniques have been
recently used as outcome measures for assessing the effects of
pharmacological manipulation to facilitate penile reflexes
and ejaculation after experimental SCI.
344
Similarly, the
combination of visual scoring and blood pressure recording
following penile reflex stimulation in a rodent model of SCI
could be used to address the important clinical issue of
vibrostimulation-triggered AD in men undergoing fertility
treatment (JAI, LMR and AVK, unpublished observations).
Corpus spongiosum and cavernosus pressure recording
During erection, dilation of arteries and erectile tissue
relaxation increase blood flow and result in increased
intrapenile pressure. Pressure recording of the corpus
cavernosum (CC) or corpus spongiosum (CSP) has been used
to study sexual function and erectile function in intact
rats,
294,355,356
and is the most common assessment of erectile
function used in preclinical trials.
356
In this technique, the
CC or CSP is catheterized using a polyethylene tubing, or a
hypodermic needle attached to tubing, connected to a
pressure transducer.
345
The parameters that are commonly
measured include the baseline, peak and plateau pressures,
total area under the curve, as well as erection latency and
total number of erections.
356
These outcome measures have
been used to characterize the sexual response changes that
occur after SCI and the effectiveness of drugs in restoring
normal responses.
Recent validation in rats with SCI showed that CSP
pressure recording is a reliable method to evaluate erectile
function over extended periods of time in conscious and
freely moving animals as well as in restrained animals.
266,293
Akin to previous reports using reflex erection tests on SCI
rats,
311,351
SCI rats exhibited shorter time to first observed
erectile event compared to baseline values.
266
CSP pressure
recording was sensitive to early changes in SCI rats: the
number of CSP pressure peaks increased in SCI rats as early as
1 day post-injury, even though at this time the total number
of erectile events was not different from baseline values.
266
Furthermore, SCI rats had many more CSP pressure peaks per
erectile event, revealing that although erections may be
qualitatively similar after SCI, their physiology may be
significantly altered.
266
Pressure recording has been recently
Table 7 Overview of studies characterizing or targeting sexual dysfunction after experimental SCI
Technique used Species Sex Injury model Time range post-injury References
Behavioral scoring Rat M Mid-T Tx 28–52 days
342
T8–T9 Tx 1–7 days
343
T6 Tx 3, 4 weeks
344
Pressure recording
c. cavernosus M T8–T9 Tx 2 weeks
345
c. spongiosus M T10 contusion p1 week, 2–4 weeks
266
Doppler flowmetry F T10 Tx 6–8 weeks
346
Epididymal sperm transport M T9 contusion 10 days
347
Abbreviations: c. cavernosus, corpus cavernosus; c. spongiosus, corpus spongiosus; Tx, transection.
Studies have been classified by the technique used to assess sexual function. Studies using similar experimental animals, injury models and time range of study
post-injury are grouped together.
Autonomic assessment in experimental SCI
JA Inskip et al
21
Spinal Cord
used as an outcome measure for evaluating the effectiveness
of pharmacological manipulation to restore erectile capacity
after SCI.
345
Advantages/disadvantages. Described above; see Urinary
bladder function (‘Assessment of lower urinary tract function
following experimental SCI’ and ‘Corpus spongiosum pres-
sure recording’).
Perineal muscle electromyography
Physiological responses and the role of perineal muscles can
be measured during sexual behavior using EMG recording.
This technique was used in the discovery of the urethrogen-
ital reflex, a model used to study human sexual climax.
316
In
the men, wire electrodes are placed in the bulbospongiosus
muscle, which is attached to the CSP; in women, this
recording is usually taken from the smooth muscle of the
vagina.
316
In both sexes, mechanical stimulation of the
urethra elicits clonic contractions of the perineal muscles
and rhythmic cavernous muscle activity, with expulsion of
the urethral contents in the men.
316
This technique has been
validated in uninjured rats as well as spinalized, anesthetized
animals.
316,357
Advantages/disadvantages. Like EMG in other systems,
perineal EMG is generally a terminal preparation and is
conducted on anesthetized animals. However, it offers a
readily quantifiable measure of the sexual response.
Flowmetry
As signs of female genital arousal are quite subtle, experi-
mental research in this area relies heavily on physiological
recording. Vaginal photoplethysmography offers a reliable
clinical measurement of vaginal blood volume and pulse
amplitude.
307,358–360
Experimentally, vaginal blood flow
recording by laser Doppler flowmetry has been recently
established as a method to investigate sexual arousal in
rats.
348,361,362
In this technique, capillary blood flow is
measured by a surface probe, placed on the ventral surface
of the vagina or clitoral glans, which is connected to a
flowmeter.
346,348,361
Using a bipolar hook electrode, electro-
stimulation is applied to the clitoral nerve or pelvic plexus to
elicit a vascular response.
361
After suprasacral SCI, unlike male rats, female rats
exhibited decreased response to electrostimulation; only
50% of animals showed increased clitoral blood flow, and
even then, the increase was weak and nonsustained.
346
In
contrast, uninjured female rats showed immediate and
sustained increases in both clitoral and vaginal capillary
blood flow.
361
Recently, this technique has been used as a
functional outcome to evaluate the effectiveness of pharma-
cological agents to restore sexual arousal following experi-
mental SCI.
346
Advantages/disadvantages. This technique offers a new way
to investigate the underrepresented issue of female sexual
dysfunction following SCI. Flowmetry itself is noninvasive,
but it is often used in combination with nerve stimulation,
in invasive and terminal preparations. It is conducted in
anesthetized animals, as the recording is very sensitive to
movement.
Epididymal sperm transport assay
Unlike the techniques described above, which assess sexual
function, this assay is utilized to assess reproductive function
following SCI. Epididymal sperm transport is assessed by
labeling of spermatozoa followed by quantification of
labeled sperm throughout the length of the epididymis.
347
This technique offers an indirect way to assess the preserva-
tion of sympathetic innervation to the epididymis, as sperm
transport through the epididymis is sympathetically
mediated.
320,321
Following SCI, rats show decreased epididy-
mal sperm transport.
347
Advantages/disadvantages. This assay is a terminal prepara-
tion that demands some level of technical skillFfor both the
injection of the label and the epididymal dissection.
Thermoregulatory function
Autonomic control of thermoregulatory effectors
The thermoregulatory role of the autonomic nervous system
is primarily mediated by the sympathetic nervous system
and its vasomotor, sudomotor and pilomotor effectors.
Although each of these effectors can also be activated by
emotional stimuli, for the purposes of this review, we focus
only on their thermoregulatory function. The preganglionic
neurons of these effectors are cholinergic, and lie in the
thoracolumbar cord (T1–L2); the ganglionic neurons are also
cholinergic, and lie in the paravertebral ganglia.
Vasomotor efferents that innervate cutaneous vascular
beds regulate the amount of heat that is dissipated to the
surrounding environment. Unlike the visceral vasoconstric-
tors, cutaneous vasoconstrictors are not under strong
baroreflex control, but are strongly affected by core tem-
perature changes. Some areas of the skin are more specialized
to perform thermoregulatory roles; areas with arteriovenous
anastamoses allow for quick transfer of blood from the
arteries to veins. Although the location of anastamoses varies
from species to species, their function is similar.
In contrast to most sympathetic efferents, sudomotor
efferents are cholinergic. When activated, these neurons act
via muscarinic receptors to increase sweat gland secretion. In
humans, there are two types of sudomotor neurons, apocrine
or eccrine. Eccrine glands (present over the entire skin
surface, with especially high levels on the palms, soles, face
and axillae) are primarily used for diffuse thermoregulatory
sweating; apocrine glands are associated with hair follicles,
and are present in the genital, axillary and mammary
regions. Unlike humans, rats only have apocrine glands,
present on the plantar and palmar paw surfaces. These
glands are activated by psychogenic rather than thermal
stimuli, and do not have a role in thermoregulation.
363
Pilomotor neurons are noradrenergic, and innervate
piloerector muscles, which are found throughout the hairy
skin of experimental animals and skin of humans. When
Autonomic assessment in experimental SCI
JA Inskip et al
22
Spinal Cord
these muscles contract, they raise their associated hairs and
ultimately augment insulation. The rostrocaudal innerva-
tion pattern of piloerector muscles is similar to that of the
sensory dermatomes.
Clinical implications of thermoregulatory dysfunction after SCI
SCI can disrupt the descending sympathetic input, leaving
large areas of skin and associated blood vessels, sweat glands
and hair follicles disconnected from supraspinal sympathetic
control.
364
As a result, sympathetic mechanisms do not
respond adequately to adjust core temperature and maintain
equilibrium. The resulting thermoregulatory instability in
both the acute and chronic periods can be severely
debilitating.
Clinically, there are three main phenomena associated
with temperature dysregulation reported following SCI:
poikilothermia, acute hyperthermia and exercise-induced
hyperthermia.
5
Poikilothermia is also called ‘environmental
fever’, but can refer to both the hypo- and hyperthermia
experienced by individuals with SCI as a result of the
ambient environment that they are exposed to. ‘Quad fever’
is used to refer to hyperthermia present in the first few weeks
or months after SCI that is unrelated to infection or other
identifiable causes.
In general, individuals with high injuries are predisposed
to more severe temperature dysregulation than those with
lower injuries.
365,366
However, even individuals with low-
level SCI can exhibit up to a 50% reduction in whole-body
sweating capacity compared to able-bodied controlsFa
reduction that correlates with their reduced skin surface
area for sweating.
367,368
Although there is some evidence of
increased productivity of sweat glands above the injury as a
mechanism of compensation for decreased sweating below
the injury,
369
individuals with SCI remain at a significant risk
for heat illness due to increased heat storage.
370
Heat storage
is particularly problematic in wheelchair athletes,
371
but
recent evidence suggests that precooling or cooling during
exercise can reduce the development of a high core
temperature.
372,373
As well as being at increased risk for thermoregulatory
dysfunction, individuals with high-thoracic and cervical
injuries are also at risk for AD. During periods of AD, when
reflex sympathetic adrenergic vasoconstriction below the
level of injury elevates systemic blood pressure, reflex
sympathetic cholinergic activity can also cause excessive
sweating. In combination with thermoregulatory dysfunc-
tion, profuse sweating can contribute to a lowering of body
temperature, which can result in hypothermia.
374
Assessment of thermoregulatory function following
experimental SCI
In this section we focus primarily on functional tests of
thermoregulatory control that are currently employed in
experimental SCI research. These studies most commonly
aim to characterize the thermoregulatory changes that occur
after SCI, and a small number of them test approaches to
mitigate these dysfunctions (Table 8). Molecular approaches
to thermoregulation are beyond the scope of this review and
have been recently reviewed elsewhere.
377,378
Core temperature recording
Core temperature recording provides a rough indication of
the thermoregulatory capacity. Clinically, this type of
information is easy to obtain, as it is part of routine care of
individuals following SCI. In animals, core temperature is
also quite easily obtained using a rectal thermometer.
Acutely after cervical and high-thoracic SCI, rat core
temperatures decline significantly and are vulnerable to
changes in ambient environmental temperatures.
40,379
Although the use of heating mats and raised environmental
temperatures can be used to maintain body temperature in
the acute setting,
379
at 6 weeks post-injury core temperatures
remained significantly lower in SCI rats compared to
preoperative levels.
40
Advantages/disadvantages. Core temperature recording is
minimally stressful for SCI animals with limited colorectal
sensation. The tools are easily available and the technique
itself requires minimal training. However, investigators must
be aware that rectal probe insertion could induce AD, which
can alter core temperature.
40,374
Cutaneous temperature recording
Cutaneous temperature recording provides a simple indirect
way to investigate cutaneous vasoconstrictor activity. De-
spite the fact that skin blood flow is important in heat loss
during exercise in individuals with SCI,
380
there has been
little research investigating cutaneous blood flow after
experimental animal SCI.
40
The skin of the tail is the most
important thermoregulatory organ in the rodent, and has
been targeted in many investigations of thermoregulatory
function.
381,382
Infrared thermography can be used to record
the surface temperature of the rat tail and hindlimb.
40,383,384
Table 8 Overview of studies characterizing or targeting thermoregulatory dysfunction after experimental SCI
Technique used Species Injury model Time range post-injury References
Core temperature recording Rats C6/7 Tx 6 h
375
Infrared thermography T4 Tx 1 day–6 weeks
40
Flowmetry T2; T9 Tx 1 h–7 days
39
Microneurography T12–T13 Tx 0–72 h
376
Abbreviation: Tx, transection.
Studies have been classified by the technique used to assess thermoregulatory function. Studies employing a combination of techniques are referenced under both
headings. Studies using similar experimental animals, injury models and time range of study post-injury are grouped together.
Autonomic assessment in experimental SCI
JA Inskip et al
23
Spinal Cord
Following SCI, the skin temperature of the mid-tail and the
hindpaw increased. These changes correlated with the
decrease in core temperature, suggesting that the two are
potentially related.
40
Advantages/disadvantages. Cutaneous temperature record-
ing is noninvasive, causes no stress to the animals and can
be used chronically. However, specialized equipment is
required for both the data acquisition and analysis.
Flowmetry
Blood flow recording provides a noninvasive way to assess
sympathetic vasomotor pathways following SCI. Clinically,
this method has been used to show that individuals with
high SCI have reduced skin vasoconstriction in response to
cutaneous cold compared to controls, and reduced vasodila-
tion in response to local heating below the injury.
385,386
This
method is also used in experimental animal research, where
ultrasound flow probes are surgically implanted around the
artery, tissue or organ of interest, and flow is recorded using a
flowmeter.
39,382
Hong and colleagues have recently looked at
organ system microcirculation, including the skin in the
forepaw and hindpaw, after acute SCI at high and low
thoracic levels.
39
In the acute period, there was a significant
decrease in forepaw blood flow, which may be related to
changes in regional peripheral vascular resistance.
39
Advantages/disadvantages. Flowmetry can be minimally in-
vasive (when recording from the skin surface), but requires a
significant investment in equipment. Although the equip-
ment is quite sensitive to movements, caused by breathing
for example, there are ways to reduce these artifacts.
39
Microneurography
In vivo microneurography allows for the direct intraneural
recording of sympathetic neuronal activity.
387
Changes in
sudomotor and vasoconstrictor activity can be recorded in
response to thermoregulatory stimuli, or nerve activity can
be evoked to investigate the function of the peripheral
effectors. Microneurography has been recently used to assess
whether therapeutic electrical stimulation can facilitate skin
sympathetic nerve activity following SCI in rats.
376
Advantages/disadvantages. Microneurography is both inva-
sive and technically challenging. However, it provides a
sensitive and direct measurement of sympathetic nerve
activity.
Sympathetic skin response
Clinical sudomotor tests have proven successful in the
identification and localization of autonomic nervous system
lesions.
388
The sympathetic skin response (SSR) is a widely
used clinical technique that assesses the integrity of
sympathetic cholinergic pathways. In humans, it is typically
recorded from the palmar and plantar surfaces, areas rich in
eccrine glands, and measures changes in skin conductance in
response to electrical nerve stimulation.
389
SSR has been
suggested as one way to assess autonomic completeness of
SCI clinically, as it requires supraspinal input.
390
However,
no animal studies of SCI have included assessment of
sudomotor function as an outcome measure of autonomic
nervous system function.
Advantages/disadvantages. SSR is a noninvasive technique,
but it has not yet been developed for use in animals.
Visualization of sudomotor function
The visualization of sudomotor function is used in both
clinical and in experimental research. Thermoregulatory
sweat testing with alizarin red has been used to define the
extent of peripheral neuropathies.
391,392
Preliminary results
show that this technique can also be used as a tool to map
the preserved function of cholinergic sympathetic pathways
in individuals with SCI.
393
The starch iodine technique is an
analogous assessment that is used in rats to visualize sweat
gland function.
394,395
Advantages/disadvantages. This technique is minimally in-
vasive and the results could be easily translated between the
clinic and the experimental laboratory. However, in rodents,
it can only inform about the presence or absence of
sympathetic innervation of the palmar and plantar surfaces.
Furthermore, it does not reveal any information about the
thermoregulatory response.
Conclusion
Historically, SCI was synonymous with paralysis, and the
ultimate goal of therapy was recovery of movement. Today,
SCI is more correctly recognized as a potential disruption of
all nervous system functions, including motor, sensory and
autonomic. Although motor impairments are the most
obvious manifestation of disability, they may not be the
most catastrophic: injury to the spinal autonomic pathways
results in functional deficits of the major organ systems,
manifesting as bladder, gastrointestinal and sexual dysfunc-
tions, and disordered cardiovascular function, thermoregu-
lation and respiration. These aspects of autonomic
dysfunction are fast emerging as priorities in clinical
management of SCI.
5,323
To address these priorities, discovery scientists must
incorporate autonomic assessments as outcome measures
in their experimental SCI research. Here we have reviewed a
wide variety of techniques that are available to evaluate
autonomic function in experimental animals, including
some that have not yet been used in experimental SCI, but
appear to be feasible. For some laboratories, incorporating
autonomic assessment may involve adding new techniques
to their existing battery of outcome measures. For others,
collaboration may be required to attain the necessary
expertise. In some cases, it might be necessary to add
additional groups of animals to maintain scientific rigor.
Whatever the practical considerations of incorporating
autonomic assessment, the gainFan improved understand-
ing of autonomic dysfunction after SCIFis indisputably
worth the effort. There is an urgent need to include
Autonomic assessment in experimental SCI
JA Inskip et al
24
Spinal Cord
autonomic evaluations in experiments testing therapeutic
(that is, regenerative or protective) agents after SCI, as there
are very few experiments currently doing so (see Table 9).
At present, treatments are moving to clinical trial without
any animal data that might predict their effects on
autonomic function.
402
If we continue to neglect autonomic
function after SCI in the laboratory, the consequences for
people with SCI could be disastrous.
Acknowledgements
We gratefully acknowledge the Heart and Stroke Foundation
of BC and the Yukon (AVK and MSR), the Christopher Reeve
Paralysis Foundation (AVK), the Rick Hansen Foundation
(AVK and MSR), the Michael Smith Foundation for Health
Research (JAI, LMR and MSR), the Canadian Institutes of
Health Research (JAI and MSR) and the National Science and
Engineering Research Council (LMR) for their support. We
thank our colleagues at ICORD (International Collaboration
on Repair Discoveries) for providing a supportive environ-
ment for our research.
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5 weeks Anti-CD11d antibody
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