ABSTRACT: Cerebral palsy (CP) is the most prevalent neurologic disease
in children and a leading cause of severe physical disability. Research and
clinical experience indicate that children with CP have abnormal neuromuscular junctions (NMJs), and we present evidence that nonapposition of
neuromuscular junction components is associated with the severity of motor
system deficit in CP. Leg muscle biopsies collected from ambulatory (n ⫽
21) or nonambulatory (n ⫽ 38) CP patients were stained in order to detect
acetylcholine receptor (AChR) and acetylcholine esterase (AChE). Image
analysis was used to calculate the extra-AChE spread (EAS) of AChR
staining to estimate the amount of AChR occurring outside the functional,
AChE-delimited NMJ. Nonambulatory children exhibited higher average
EAS (P ⫽ 0.025) and had a greater proportion of their NMJs with significantly elevated EAS (P ⫽ 0.023) than ambulatory children. These results
indicate that physical disability in children with CP is associated with structurally dysmorphic NMJs, which has important implications for the management of CP patients, especially during surgery and anesthesia.
Muscle Nerve 32: 626 – 632, 2005
DYSMORPHIC NEUROMUSCULAR JUNCTIONS
ASSOCIATED WITH MOTOR ABILITY
IN CEREBRAL PALSY
MARY C. THEROUX, MD,1,2 KARYN G. OBERMAN, BA,1,3 JUSTINE LAHAYE, MS,1,4
BOBBIE A. BOYCE,1 DAVID DUHADAWAY, BS,1
FREEMAN MILLER, MD,5 and ROBERT E. AKINS, PhD1
1
Nemours Biomedical Research, A. I. duPont Hospital for Children, 1600 Rockland Road,
Wilmington, Delaware 19803, USA
2
Department of Anesthesiology and Critical Care, A. I. duPont Hospital for Children,
Wilmington, Delaware, USA
3
Department of Biological Sciences, University of Delaware, Newark, Delaware, USA
4
Physiology and Informatics Program, University of Poitiers, Poitiers, France
5
Department of Orthopaedics, A. I. duPont Hospital for Children, Wilmington,
Delaware, USA
Accepted 25 May 2005
Previous studies suggest that children with cerebral
palsy (CP) have abnormal neuromuscular junctions
(NMJs). Distribution of acetylcholine receptors
(AChRs) at abnormal NMJs is clinically important as
the depolarization–repolarization characteristics of
the muscle membrane may be affected by extrajunctional AChR. This is especially significant when neuromuscular blocking agents (NMBAs) are used during anesthesia, as dangerous quantities of potassium
may be released into the extracellular fluid.9,10,29,30 It
Abbreviations: AChE, acetylcholine esterase; AChR, acetylcholine receptor;
BTX, bungarotoxin; CP, cerebral palsy; EAS, extra AChE spread; GMFM,
gross motor function measure; MAS, modified Ashworth score; NMBA, neuromuscular blocking agent; NMJ, neuromuscular junction; PBS, phosphatebuffered saline.
Key words: acetylcholine esterase; acetylcholine receptor; gross motor
function measure; hyperkalemia; neuromuscular blocking agents; spasticity
Correspondence to: R. E. Akins; e-mail: rakins@nemours.org
© 2005 Wiley Periodicals, Inc.
Published online 15 July 2005 in Wiley InterScience (www.interscience.wiley.
com). DOI 10.1002/mus.20401
626
Neuromuscular Junctions in CP
has been shown that patients with CP have increased
sensitivity to the depolarizing NMBA succinylcholine27 and increased resistance to the nondepolarizing NMBA vecuronium,11 but these results provide
only indirect support for the existence of abnormal
NMJs. More recently, laboratory-based assessments
of NMJs in erector spinae collected from children
during spinal fusion surgery gave direct evidence
that children with CP have dysmorphic NMJs.28 In
particular, bungarotoxin (BTX)–labeled AChR
staining extended significantly beyond the functional NMJ as delimited by junctional acetylcholine
esterase (AChE) immunoreactivity in CP patients,
whereas AChE completely overlapped AChR staining in samples from children with idiopathic scoliosis.28
The presence of disorganized NMJs in children
with nonprogressive central nervous system lesions
like those found in CP is unexpected. The establishment and maintenance of NMJ organization has
MUSCLE & NERVE
November 2005
been well studied, and the colocalization of junctional components in NMJs is highly controlled.13,15
The nonapposition of AChR and AChE in CP therefore suggests a fundamental dysregulation of nerve–
muscle interactions or a lack of NMJ maturation
associated with the disease. An improved understanding of AChR localization and the organization
of NMJ components in CP is needed to assure safe
and adequate anesthesia, to define further the manifestations of CP, and to improve understanding of
potential alterations in nerve–muscle interactions associated with disease state. Of interest in this regard
is whether the nonapposition of NMJ components is
functionally related to the level of motor system
impairment.
In this study, we examined the relationship between the degree of motor system involvement in CP
and the nonapposition of AChR and AChE. We
tested the hypothesis that the degree of AChR expansion beyond the limits of the functional NMJ
increases with disease severity. Motor system function of CP children was estimated based on the
ability to ambulate without assistance and on Gross
Motor Function Measure (GMFM). A digital imaging
algorithm was used to quantify the degree of AChR–
AChE nonapposition.
MATERIALS AND METHODS
Patient Selection and Sample Acquisition. We enrolled 59 CP patients in the study after obtaining
institutional review board approval and signed informed consent. Signed assents were obtained where
appropriate. Patients were 2–20 years of age, had
spastic quadriplegic CP, and were scheduled to undergo muscle and tendon release surgery. During
surgery, muscle biopsies were obtained from gracilis,
vastus lateralis, or gastrocnemius, depending on the
exposure of muscles during the procedure. Samples
were immediately snap frozen in N2(l)-chilled isopentane.
Preoperative Assessment of Patient. Prior to surgery, patients were assessed for their ability to bear
their own weight in a standing position without assistance. Experienced physiotherapists performed
this evaluation during a routine appointment before
the day of surgery, and the results of the evaluation
were collected from patient charts. Those patients
that could bear their own weight were classified as
ambulatory (n ⫽ 21) and those that could not were
classified as nonambulatory (n ⫽ 38). Patients who
did not have their ambulatory status evaluated prior
to surgery were excluded from the study. In addi-
Neuromuscular Junctions in CP
tion, GMFM Section D scores were determined for
51 patients (15 ambulatory and 36 nonambulatory)
in a separate visit to our institutional Gait Laboratory. The GMFM Section D score is a quantitative
measure of motor ability associated with standing. It
includes 13 separate assessments graded from 0 (no
part of the task was accomplished) to 3 (the task was
completed); the total score is used to assess the level
of motor function. The GMFM has been validated.21,25 It is predictive of the ambulatory status of
children with CP 3,20 and correlates with CP severity.19,21 Patients were also graded using the modified
Ashworth scale (MAS) to estimate spasticity.2 MAS
was determined for 66 muscle groups (24 ankle plantar flexion, 22 hip adduction, 10 knee extension) in
39 patients (15 ambulatory, 24 nonambulatory). The
MAS ranges from 0 to 5 and was assigned by a
physiotherapist based on the resistance to passive
stretch within a particular muscle group.
To evaluate the
presence of AChRs outside functional NMJs, a previously developed double-stain method to visualize
AChR and AChE was employed.28 This allowed the
direct determination of AChR–AChE nonapposition
within histologic sections. AChE was chosen because
of its well-characterized distribution pattern and its
localization to the functional NMJ.13,16,23,24
Slides containing 8-␮m-thick cryosections were
fixed at room temperature for 5 min using 10%
neutral-buffered formalin (Fisher, Fairlawn, New Jersey) and blocked at room temperature for 30 min
using 3% bovine serum albumin (Sigma, St. Louis,
Missouri). Sections were rinsed with Dulbecco’s
phosphate-buffered saline (PBS; Invitrogen, Carlsbad, California) and stained in a solution containing 0.16 ␮g/ml tetramethylrhodamine-conjugated
␣-BTX (Molecular Probes, Eugene, Oregon). After
1-h incubation at room temperature, the sections
were rinsed with PBS and stained using a monoclonal antibody to AChE (AE-2; Biogenesis, Sansdown,
New Hampshire) diluted 1:150 in PBS, followed by a
fluorescein-conjugated secondary antibody (Jackson
Labs, Westchester, Pennsylvania). Samples were
viewed and digitally photographed on an Olympus
BX-60 fluorescence microscope (Olympus, Tokyo,
Japan) equipped with a Spot RT-Slider digital camera (Diagnostic Instruments, Sterling Heights, Michigan). Corrected lenses were used to minimize
chromatic aberration, and the registration of fluorescence images was routinely verified using test images. The distribution of AChR relative to AChE was
quantified using Image Pro software (Media Cybernetics, Silver Spring, Maryland) and a customized
Histological Assessment of Samples.
MUSCLE & NERVE
November 2005
627
software macro. The value extra-AChE spread (EAS)
was calculated as the fraction of pixels in digitized
images exhibiting AChR but no AChE staining.
The inter-rater reliability of the
digital image analysis was determined using parallel
determinations carried out by blinded research assistants. To test whether EAS was related to ambulatory status, a median value was determined for each
child and ambulatory and nonambulatory groups
were compared using a Mann–Whitney test. Effect
size was estimated using Cohen’s d calculation: effect
size ⫽ (mean difference)/(pooled SD).5 The relationship between GMFM score and EAS was evaluated using Pearson’s correlation. The relationship
between MAS and EAS values was evaluated using
Spearman’s correlation. All statistical analyses were
carried out using SPSS for Windows (SPSS, Chicago,
Illinois).
Statistical Analysis.
RESULTS
Validation of Methodology and Determination of Normal
Image analysis was applied to determine
the area of AChR staining outside the limits of AChE
immunoreactivity. As illustrated in Figure 1, staining
patterns for AChR and AChE were nondiffuse and
readily visualized. To validate our approach to NMJ
assessment, three different investigators performed
independent determinations of EAS for 100 identical NMJs from children with CP. An inter-rater reliability test yielded an alpha value of 0.8 for absolute
agreement among the three measurements. Subsequent comparisons were based on values determined
by a single investigator.
The normal value and range of EAS were determined in control NMJs. Since all children enrolled
in the current study had a primary diagnosis of CP,
we used muscle samples from a previous study.28 EAS
Values.
FIGURE 1. Calculation of EAS values. Biopsy samples were stained for AChR and AChE. Images were digitally acquired for each label
and analyzed to determine which pixels were illuminated by each fluorophore. (A) Sample images for a single NMJ from the
gastrocnemius of a child with CP showing the distribution of AChR (red), AChE (green), and the combined staining. (B) Topographic
representations of AChR and AChE intensities from images shown in A; distinct boundaries were easily identifiable relative to
background, allowing discrimination of staining pattern edges. (C) The corresponding threshold images for AChR, AChE, and the
combined information showing which pixels have AChR alone (red ⫽ 445 pixels) and which have both (yellow ⫽ 1,543 pixels). In this
example, the EAS value ⫽ R/(R⫹Y) ⫽ 0.224.
628
Neuromuscular Junctions in CP
MUSCLE & NERVE
November 2005
was significantly lower in the erector spinae of children with idiopathic scoliosis (0.07 ⫾ 0.04; n ⫽ 26)
than in children with CP (0.28 ⫾ 0.04; n ⫽ 7).
Assessment of Variability between Groups. Patients
in the ambulatory and nonambulatory groups were
matched for age (8.1 ⫾ 5.2 and 8.2 ⫾ 4.4 years,
respectively) and weight (24.7 ⫾ 14.0 and 19.7 ⫾ 9.1
kg). There were no significant differences in these
parameters (P ⬎ 0.1; Student’s t-test). In addition,
there was no correlation between EAS and age or
weight (P ⬎ 0.8; Spearman’s correlation) for either
group.
Abnormal NMJs coexisted in the same histological sections as normal
NMJs (Fig. 2) in both ambulatory and nonambulatory groups. Comparison of EAS values between the
three muscle types collected indicated no significant
difference in either patient group (P ⬎ 0.5 by
Kruskal–Wallis test), and the data obtained from all
muscles collected from an individual child were
pooled for analysis. Descriptive statistics are summarized in Table 1.
EAS differed between the ambulatory and
nonambulatory groups (P ⫽ 0.025 by Mann–WhitAnalysis of NMJ Components.
FIGURE 2. Nonapposed NMJs occur in the same region as apposed NMJs. Biopsy samples were stained for AChR and AChE, and
images were acquired separately for each label. Images were analyzed to determine which pixels were illuminated for each label. (A)
Cross section of gracilis from a child with CP showing normal and abnormal NMJ distributions in the same anatomic location and in the
same histologic section. Examples of normal and abnormal NMJs are denoted by a square and a circle. (B) Higher magnification view
of normal NMJ from A, showing that AChE signal overlapped the AChR signal. (C) Higher magnification view of abnormal NMJ from A,
showing AChR–AChE nonapposition.
Neuromuscular Junctions in CP
MUSCLE & NERVE
November 2005
629
Table 1. Descriptive statistics for ambulatory and nonambulatory patients.*
Parameter
No. of patients
Mean GMFM Section D score†
Mean EAS†‡
Percentage of NMJs with EAS ⬎ normal†
No. of NMJs assessed
No. of pixels per NMJ image†
Mean EAS of NMJs in vastus§
Mean EAS of NMJs in gracilis§
Mean EAS of NMJs in gastrocnemius§
Ambulatory
Nonambulatory
21
23.0 ⫾ 13.1 (15)
0.16 ⫾ 0.08 (21)
34
853
1,142 ⫾ 657 (853)
0.17 ⫾ 0.22 (45)
0.17 ⫾ 0.21 (319)
0.15 ⫾ 0.18 (489)
38
2.2 ⫾ 7.3 (36)
0.23 ⫾ 0.14 (38)
45
2,060
1,026 ⫾ 626 (2,060)
0.22 ⫾ 0.24 (352)
0.20 ⫾ 0.22 (1,120)
0.22 ⫾ 0.25 (588)
EAS, extra acetylcholine esterase spread; GMFM, gross motor function measure; NMJ, neuromuscular junction.
*Data presented as mean value ⫾ SD; numbers in parentheses indicate the number of determinations for each measurement.
†
Statistically significant comparisons (P ⬍ 0.05). Statistical comparisons between groups were carried out using parametric or nonparametric approaches as
appropriate for the type of data (see text).
‡
Each child received an EAS score equivalent to the arithmetic average for all junctions assessed within that child. The mean and standard deviation of these
scores are reported.
§
EAS values did not vary by muscle type.
ney test), with nonambulatory patients having
higher EAS values. The mean EAS values for ambulatory (0.16 ⫾ 0.08) and nonambulatory (0.23 ⫾
0.14) patients were each more than two standard
deviations above the mean EAS of normal NMJs
(0.07 ⫾ 0.04) with the difference between groups
indicating a medium effect size (d ⫽ 0.54). There
was also a significant correlation (R ⫽ ⫺0.056; P ⫽
0.005) between EAS values and GMFM Section D
score. There was no correlation between EAS and
MAS (R ⫽ .028; P ⫽ 0.8; n ⫽ 66).
The prevalence of abnormal NMJs versus normal
NMJs was also determined. On average, nonambulatory CP patients had 45% of their NMJs with EAS
exceeding 2 standard deviations above normal (i.e.,
EAS ⬎ 0.15) compared to ambulatory patients who
had 34% of their NMJs with EAS values that high.
This was statistically significant (P ⫽ 0.023) by
Mann–Whitney test. The post-junctional area of
NMJs, as estimated by the number of pixels present
in BTX-stained images, was found to be about 10%
smaller in nonambulatory than ambulatory children
(P ⬍ 0.001).
DISCUSSION
We found that differences in NMJ structure exist in
CP patients such that there is a greater degree of
NMJ nonapposition present in more highly affected
patients. The analytical approach to estimate NMJ
nonapposition was based on the detection of AChR
in the absence of corresponding AChE within digitized images. This approach may underestimate true
EAS since each digital image element comprised
optical information from a full thickness (8 ␮m) of
the histological section examined. Thus, AChR–
630
Neuromuscular Junctions in CP
AChE mismatches along the z-axis would have been
overlooked. Confocal microscopy or image deconvolution may have provided more accurate estimates of
EAS, but the reported approach allowed us to evaluate a much larger number of tissue sections than
would have been possible otherwise. The underlying
differences between ambulatory and nonambulatory
patients were substantial, and significant differences
were found without the application of more involved
techniques.
Patients with CP exhibit poor voluntary muscle
control and decreased functional motor capability.
These were estimated using the GMFM. GMFM Section D scores range from 0 to 39 and were considered continuous variables. Pearson’s correlation was
applied to compare EAS with motor function. Although there was a strong correlation between
GMFM and EAS, there was little predictive value to
the relationship. GMFM scores tended to cluster into
two groups (Table 1) with a large number of zero
values present in the more highly affected patients.
These GMFM clusters matched the independently
determined values for ambulatory status. Ambulatory status was used as the principal grouping for
analysis. Our results indicate an association between
AChR–AChE nonapposition and the degree of voluntary motor function, with nonambulators having
higher EAS values and a greater proportion of highly
abnormal NMJs than ambulators.
Spasticity is another characteristic feature of CP
in which hyperexcitability of the stretch reflex results
in increased motor neuron activity.12 Since motor
neuron activity has been associated with AChR expression and NMJ size,17,31 we suspected that CPassociated differences in NMJs were related to the
MUSCLE & NERVE
November 2005
degree of spasticity. Surprisingly, no correlation was
found between MAS and EAS, indicating that nonapposition of NMJ components may result from
mechanisms that are independent of reflex muscle
control. The MAS, however, does not distinguish
effectively heightened stretch reflex from increased
intrinsic stiffness,7 and in some cases it may not
accurately indicate spasticity per se, especially in the
lower extremities.1,6 Thus, although it would be intriguing to speculate that NMJ distortion in CP is
associated with voluntary muscle control but independent of reflex muscle control, such a conclusion
cannot be made without improved means to assess
spasticity.
The two groups of children in our study had
different levels of mobility, and certain types of immobilization can lead to NMJ disruption. Generally,
immobilization implies decreased NMJ activation
due to disuse. Evidence suggests that gravitational
unloading increases the average size of motor endplates in adult rats8 and that casting results in increased AChR expression.32 Other disease states associated with decreased NMJ activation, e.g.,
denervation, spinal cord injury, or burn, can also
lead to expansion of AChR staining and upregulation of AChR subunit expression.4,9,14,18,22,29 Increased muscle impulse activity, by contrast, is associated with decreased NMJ size,17 and sustained
activity inhibits ␥-AChR subunit expression in denervated muscle.31 Interestingly, because their lack of
mobility is largely due to weakness, spasticity, and
loss of motor control, immobilization in CP patients
occurs in conjunction with continuing NMJ activation as opposed to the reduced NMJ activation commonly associated with other conditions. Consistent
with this, nonambulatory (or less mobile) patients
had slightly smaller NMJs, perhaps associated with
greater spasticity, and in a previous study, the NMJs
of CP patients appeared smaller than those of controls with no evidence for ␥-AChR expression.28
Thus, the pathways contributing to NMJ deformation in CP may be unrelated to patient mobility and
distinct from those previously described for other
diseases associated with disuse.
Our study has implications for the management
of CP patients, especially during surgery and anesthesia. The increased oral secretions and gastroesophageal reflux in children with CP26 often render
the use of a rapidly acting NMBA, such as succinylcholine, attractive in securing the airway during anesthetic management. Given the NMJ abnormalities
and known sensitivities of CP patients, however, anesthesiologists should exercise caution in the use of
succinylcholine in patients with CP. Conversely, it is
Neuromuscular Junctions in CP
important to be aware of the decreased potency of
nondepolarizing NMBAs in patients with disrupted
NMJs because inadequate relaxation could result in
procedural difficulties during surgery. Our study
also suggests that treatments, therapies, and diagnostics aimed at NMJs may be of benefit to CP patients,
but further elucidation of the mechanisms and
pathophysiology associated with NMJ disruption and
AChR–AChE nonapposition is needed.
This research was presented in part to the Pediatric Academic
Society, May 2004, San Francisco, California, and to the Society
for Neuroscience, October 2004, San Diego, California. This work
was supported in part by NAG9-1339 from the Office of Biological
and Physical Research at NASA, by 1P20-RR020173-01 from the
National Center for Research Resources at NIH, and by AI2003026 from the Nemours Foundation. The authors thank Kelly
Quaile, Nancy Lennon, Lauren Kirstetter, Anusha Gopalrathnam,
and Funbi Fagbami for their assistance with the preoperative
evaluation of patients and the validation of the image analysis
method.
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