Figure 1.
Determination of appositional score.
Sample images are shown for a single NMJ from a spinalis biopsy of a child with CP. The distribution of AChR (Panel A), AChE (B), and the composite image of both AChR and AChE (C) are shown. Each image was thresholded using the standard algorithm provided in the Image Pro Software, which objectively identifies foreground versus background pixels based on the distribution of intensities in the image, resulting in images D, E, and F. Distinct boundaries were reproducibly identifiable relative to background using this approach. A line drawn through the NMJ (G) provided the corresponding histogram (H) which displayed the intensities of AChR and AChE staining across the NMJ. The pixels above the preset threshold levels were categorized and counted to determine appositional scores.
Table 1.
Monoclonal Antibodies to Myosin Heavy Chains.
Figure 2.
Typical staining patterns for idiopathic scoliosis (IS) and CP patients with the corresponding threshold images.
Spinalis samples were triple stained for AChR (red), AChE (blue), and either laminin β2 or SV2 (green). Representative staining patterns with two of the three stains are shown. Compared to idiopathic scoliosis patients, CP patients generally had more AChE present outside AChR (A), more AChE present outside laminin β2 (B), less laminin β2 present outside AChE (B), more AChR present outside laminin β2 (C), and more AChE present outside SV2 (D). Both CP and IS samples had a similar distribution of SV2 relative to AChR (E).
Table 2.
Median values and ranges for NMJ component comparisons; patients pooled by diagnosis.
Table 3.
Median values and ranges for NMJ non-colocalization scores; each patients received a score equivalent to the median values within their NMJs.
Table 4.
Comparison of individual CP patients with the idiopathic scoliosis group.
Figure 3.
Comparison of neuromotor maturation in idiopathic scoliosis (IS) and CP patients.
A) Spinalis sample from a control patient showing distinctly stained type I (dark) and type II (light) fibers using an ATPase assay at low pH (4.65). B) Spinalis sample from a patient with CP showing type I and type II fibers with an overall mature appearance similar to that seen in panel A, except for an increased predominance of type I fibers. C) Analysis of fiber typing: at least 100 fibers were enumerated as either type I or type II fibers for each patient sample (n = 10 per group) and compared for fiber type predominance. The average frequency of each type is shown. Patients with CP had a significantly higher frequency of type I fibers than patients with IS (p = 0.013 by Mann-Whitney) and a significantly lower frequency of type II fibers (p = 0.044 by Mann-Whitney). D) CP (lanes 6–8) and IS (lanes 1–4) muscles were analyzed for the presence of fast, slow, and embryonic MYHs by Western blot. Fetal muscle (lane 5) was used as a positive control for immaturity and the presence of embryonic MYH. Antibodies used are as follows: A4.74 (fast) for MYH1, MYH2, and MYH4; F1.652 (embryonic) for MYH3; A4.951 (slow type I) for MYH7. Neither CP nor control muscle contained the embryonic MYH isoform. E) RNA was extracted from spinalis muscle and subjected to real time RT-PCR. Data are presented as mean (± SD) fold differences compared to fetal tissue (n = 12 CP, 12 IS). AChRα1 (CHRNA1), AChRδ (CHRND), AChRε (CHRNE), AChRγ (CHRNG), AChRα7 (CHRNA7), myogenin (MYOG), AChE (ACHE), and β2 syntrophin (SNTB2), did not show significant differences between the CP and control groups. β1 syntrophin (SNTB1) expression was significantly higher in CP samples than control samples (p<0.001 by Mann-Whitney).
Table 5.
Telomere lengths expressed as T/S ratio relative to normal leg muscle in patients with CP, idiopathic scoliosis, or DMD.
Figure 4.
NMJs from CP Patients by Electron Microscopy.
A) Top panel shows an electron micrograph of a CP NMJ. A zoomed out image is shown in the upper right corner with a red line marking a Schwann cell nucleus, cyan marking the Schwann cell membrane, magenta marking the nerve terminal, yellow marking the postsynaptic folds, and green marking the postsynaptic nucleus. Scale bars = 1 µm. B) A zoomed image of primary folds in the CP NMJ is also shown. The blue lines indicate the general depth of postsynaptic fold penetrance into the muscle. For data analysis, each fold was measured separately using ImageJ software to draw trace lines for individual folds following the methods outlined by Banks et al. [27]. C) A CP NMJ with a single mitochondrion in the nerve terminal (arrow). The total area occupied by mitochondria was calculated with ImageJ software by tracing each mitochondrion segment and measuring the summed mitochondrial area relative to the total area of the terminal.
Figure 5.
CP NMJs have a greater distance between postsynaptic folds, longer postsynaptic folds, and reduced mitochondrial load.
A,B) Two examples of control NMJs with zoomed out images in the corners. The cyan lines label Schwann cell membrane, magenta lines label the nerve terminals, and yellow lines label the postsynaptic membranes. Note the presence of mitochondria in the nerve terminals. C) Graph depicting the average distance between folds in CP (0.655 µm±0.0368 SEM, n = 25 NMJs) as compared to controls (0.392 µm±0.0299 SEM, n = 25 NMJs). There was a statistically significant (p<0.0006) increase in the distance between primary folds in CP NMJs as compared to control NMJs. D) Graph indicating the mean length of primary folds in CP and control sample in microns. Mean primary fold length in CP (1.536 µm±0.128 SEM, n = 25 NMJs) was significantly (p = 0.0062) higher in CP as compared to controls (1.012 µm±0.0636 SEM, n = 25 NMJs). E) Graph depicting a significant (p = 0.0001) reduction in mitochondrial area per square micron of the nerve terminal is evident in CP (7.8%+1.8 SEM, n = 25 NMJs) as compared to control samples (23.8%±1.4 SEM, n = 25 NMJs). Error bars in all the graphs are +/−SEM.
Figure 6.
CP NMJs have a reduced mitochondrial load.
A) Confocal images showing double staining for acetylcholine receptor by α-bungarotoxin (red) and mitochondria (green) using a mouse monoclonal antibody for complex VI subunit I of cytochrome c oxidase (Mitosciences, Oregon). Images represent the mean intensities of fluorescence signal across 10 z-stacked images by confocal microscopy. CP NMJs show a decreased intensity for synaptic mitochondria. B) Average mitochondrial fluorescence intensities of NMJs of CP (93.25±8.017 SEM, n = 25 NMJs) and control (156.70±12.035 SEM, n = 25 NMJs) samples. Bars represent the mean ± SEM of immunofluorescence intensity in arbitrary fluorescence units based on the signal from the mitochondrial stain at the NMJs of CP and control samples.