Resistance to Neuromuscular Blockade by Rocuronium in Surgical Patients with Spastic Cerebral Palsy

Individuals with spastic cerebral palsy (CP) often exhibit altered sensitivities to neuromuscular blocking agents (NMBAs) used for surgical intubation. We assessed usage of the NMBA rocuronium in patients with spastic CP and evaluated potential modifiers of dosing including gross motor function classification system (GMFCS) level, birthweight, gestational age, and the use of anticonvulsant therapy. In a case-control study, surgical patients with spastic CP (n = 64) or with idiopathic or non-neuromuscular conditions (n = 73) were enrolled after informed consent/assent. Patient data, GMFCS level, anticonvulsant use, and rocuronium dosing for intubation and post-intubation neuromuscular blockade were obtained from medical records. Findings reveal participants with CP required more rocuronium per body weight for intubation than controls (1.00 ± 0.08 versus 0.64 ± 0.03 mg/kg; p < 0.0001). Dosing increased with GMFCS level (Spearman’s rho = 0.323; p = 0.005), and participants with moderate to severe disability (GMFCS III-V) had elevated rocuronium with (1.21 ± 0.13 mg/kg) or without (0.86 ± 0.09 mg/kg) concurrent anticonvulsant therapy. Children born full-term or with birthweight >2.5 kg in the CP cohort required more rocuronium than preterm and low birthweight counterparts. Individuals with CP exhibited highly varied and significant resistance to neuromuscular blockade with rocuronium that was related to GMFCS and gestational age and weight at birth.


Introduction
Cerebral Palsy (CP-see table of abbreviations below) is a group of disorders occurring in approximately 2.9 per 1000 children [1,2]. CP is characterized by a static, non-degenerative encephalopathy arising in the developing brain with associated musculoskeletal and peripheral effects that often worsen over time [1,[3][4][5]. Personalized care for children with CP is challenging due in large part to the complex, multifactorial nature of the condition and a lack of understanding of the fundamental pathways contributing to disease. A number of risk factors for CP have been identified, including prematurity, hypoxia-ischemia, placental insufficiency, chorio-amnionitis or other prenatal infection, perinatal inflammation, genetic causes, and combinations of these factors [6][7][8][9]. In particular, the presence of genetic variants have been investigated in some types of CP [10,11] with de novo copy number variants [12][13][14][15][16][17], single nucleotide variants [18], and de novo mutations [19] implicated in as many as 33% of the CP cases evaluated in those studies. Additional studies suggest that epigenetic alterations are associated with CP [20][21][22][23]. Improvements in personalized care based on these advances will require more complete delineation of the characteristics and susceptibilities among individuals with different types of CP.

Participants and Data
Patients scheduled for spine surgery at the Alfred I. duPont Hospital for Children in Wilmington, DE, between 2013 and 2017 and having a diagnosis of spastic di-or quadriplegic CP (cases) or a non-neurodegenerative condition associated with either juvenile or adolescent idiopathic scoliosis or pars interarticularis defect (controls) were enrolled after consent and assent under the following IRB approved studies: The Pathophysiology of Physical Disability in Cerebral Palsy (IRB#687629), The Neuro-Orthopedic Tissue Repository (IRB#381494), and The Nemours Biobank (IRB#349465). 182 participants receiving ROC during surgery were enrolled; 45 participants were subsequently excluded due to missing medical record data or the presence of a secondary diagnosis of neuromuscular scoliosis or a chromosomal or genetic abnormality ( Figure 1). Patients with a diagnosis of CP were separated into two sub-groups based on whether they were receiving anticonvulsant therapy at the time of enrollment/surgery or not. Pre-operative levels of liver function markers (alanine transaminase (ALT) and bilirubin) were noted due to their association with resistance to neuromuscular blockade induced by chronic anticonvulsant therapy [39,41,42]. Diagnostic, demographic, and other descriptive data were collected from the EMR.
CP were separated into two sub-groups based on whether they were receivin vulsant therapy at the time of enrollment/surgery or not. Pre-operative levels of tion markers (alanine transaminase (ALT) and bilirubin) were noted due to the tion with resistance to neuromuscular blockade induced by chronic anticonvu apy [39,41,42]. Diagnostic, demographic, and other descriptive data were colle the EMR. Of the original 182 enrolled participants, a total of 137 were included in the analyses after 45 patients were excluded due to the appearance of ary secondary diagnoses, missing data in the electronic medical record (EMR), or a chan mary diagnosis. Data analysis included n = 73 controls and n = 64 participants with CP. ber of female (F) and male (M) participants included in each cohort are indicated. All en patients were utilized in the ROC dose analysis and for analyses comparing ROC dose a gestational data. Muscle sections from each diagnostic group were scanned for the prese least 10 nAChRs per section. Samples with fewer than 10 nAChRs per section were exclu NMJ analysis.

Tissue Preparation, Staining, and Imaging
Muscle samples were obtained during surgery using a surgical scalpel. Sam erector spinae were taken from the concave side of the scoliotic curve at the tho bar junction. Lower extremity samples were obtained from muscle tissue that cent to the tendon being modified or resected during the procedure. samples lected by the lead surgeon during the procedure and placed in a sterile specime less the surgeon decided that the progression of the case, the local anatomy, th the patient, or other considerations disallowed the collection. Tissue was tran the lab, trimmed and mounted using tragacanth to allow the preparation of ≤1 sections, cryopreserved by immersion in liquid nitrogen chilled isopentane, and −80 °C. Sections were prepared by the Nemours Histotechnology Core for imm ing; additional 8-10 µm sections were post fixed in 10% neutral buffered form stained with hematoxylin and eosin to assure tissue integrity, the presence of tioned muscle, and the absence of freezing artifact. Of the original 182 enrolled participants, a total of 137 patients were included in the analyses after 45 patients were excluded due to the appearance of exclusionary secondary diagnoses, missing data in the electronic medical record (EMR), or a change in primary diagnosis. Data analysis included n = 73 controls and n = 64 participants with CP. The number of female (F) and male (M) participants included in each cohort are indicated. All enrolled patients were utilized in the ROC dose analysis and for analyses comparing ROC dose against gestational data. Muscle sections from each diagnostic group were scanned for the presence of at least 10 nAChRs per section. Samples with fewer than 10 nAChRs per section were excluded from NMJ analysis.

Tissue Preparation, Staining, and Imaging
Muscle samples were obtained during surgery using a surgical scalpel. Samples from erector spinae were taken from the concave side of the scoliotic curve at the thoraco-lumbar junction. Lower extremity samples were obtained from muscle tissue that was adjacent to the tendon being modified or resected during the procedure. samples were collected by the lead surgeon during the procedure and placed in a sterile specimen cup unless the surgeon decided that the progression of the case, the local anatomy, the status of the patient, or other considerations disallowed the collection. Tissue was transferred to the lab, trimmed and mounted using tragacanth to allow the preparation of ≤1 cm 2 cross-sections, cryopreserved by immersion in liquid nitrogen chilled isopentane, and stored at −80 • C. Sections were prepared by the Nemours Histotechnology Core for immunostaining; additional 8-10 µm sections were post fixed in 10% neutral buffered formalin and stained with hematoxylin and eosin to assure tissue integrity, the presence of cross-sectioned muscle, and the absence of freezing artifact.
Samples were evaluated using 3 NMJ markers: nicotinic acetylcholine receptor (nAChR), acetylcholinesterase (AChE), and laminin β2 (Lβ2) with primary (monoclonal anti-Lβ2) and secondary (goat-anti-mouse IgG with Alexa 488) antibodies along with fluorescent probes for nAChR (α-bungarotoxin) and AChE (fasciculin-2) [43]. Slides were fixed for five minutes with 2% paraformaldehyde (Electron Microscopy Sciences) in Dulbecco's Phosphate-Buffered Saline (DPBS, pH 7.0-7.6, Thermo Fisher Scientific). All subsequent reagents were prepared in DPBS with DPBS rinses performed between steps. The tissue samples were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 15 min, and blocked with 3% bovine serum albumin (Sigma-Aldrich) for 30 min at room temperature. After tissue sections were fixed and permeabilized they underwent screening for the presence of at least 10 NMJs (Control = 36 and Cerebral palsy = 26) using fluorescently-tagged α-bungarotoxin. Sections containing 10 or more nAChRs (as labeled by α-bungarotoxin) were stained for one hour at room temperature with a monoclonal primary antibody to locate Lβ2 using a solution containing 4 mg/mL C4 supernatant prepared per instructions from the Developmental Studies Hybridoma Bank (DSHB) in Iowa City, Iowa (The hybridoma, C4 developed by J.R. Sanes, was obtained from the Developmental Studies Hybridoma Bank created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242). Sections were rinsed and incubated with a secondary antibody (Alexa Fluor 488-conjugated goat-anti-mouse IgG at 1:500 dilution, Life Technologies) and directly labeled peptides to illuminate acetylcholinesterase (AChE; Fasiculin-2, Abcam, at 1 µg/mL, directly labeled with Alexa Fluor 555 Microscale Protein Labeling Kit, Life Technologies) and nicotinic acetylcholine receptor (AChR; Alexa Fluor 647 conjugated a-Bungarotoxin at 0.16 µg/mL, Life Technologies). Slides were mounted with anti-fade solution (Life Technologies) and sealed under #1.5 glass coverslips (Fisher Scientific).
Images were acquired on a wide-field Olympus BX-60 fluorescence microscope fitted with a 12-bit Evolution QEi camera (Media Cybernetics). Optical sections were captured using a 40× PlanApo objective and a computer-controlled stage using Image Pro Plus Software 6.3 (Media Cybernetics). The co-mapping of marker pairs was determined as in previous studies [43,44]. Composite images of nAChR with AChE, nAChR with Lβ2, and AChE with Lβ2 were pseudo-colored red and green (respectively per pairing) such that colocalized areas were yellow. Pairwise NMJ organization scores were calculated as the proportion of voxels containing only one marker (red or green) to the total number of voxels with that stain (red + yellow or green + yellow) to estimate the degree of NMJ disorganization (a score of 1 indicated complete separation of the two components; a score of zero indicated complete co-localization).

Statistics
GraphPad Prism (Version 6.07) was used for statistical analysis. For continuous variables, parametric testing included two-way and one-way analysis of variance (ANOVA) with Holm-Sidak's multiple comparison test. Nonparametric testing included Mann Whitney U (MWU) and Kruskal Wallis (KW) testing. For correlations, Pearson's r was used to evaluate two continuous variables; otherwise, Spearman's rho was used. Fisher's Exact Test (FET) was employed to test contingencies. Statistical p-values below 0.05 were considered significant. The test applied is indicated for each comparison. Unless otherwise noted, data are provided as means with standard error of the mean (SEM) or medians with range. Cases with missing data were excluded from analyses.

Demographics
Of the 182 participants enrolled, 45 were excluded due to missing data or potentially confounding secondary diagnoses. Demographic data on the remaining 137 participants are summarized in Table 1. While there were significant differences in age and body mass index (BMI) between the case and control cohorts (p < 0.001 for both by MWU test), these characteristics did not correlate with the dose of ROC for either group (CP: age, p = 0.412 and BMI, p = 0.256; Control: age p = 0.220 and BMI p = 0.634, Spearman's rho). In addition, the distribution of females and males in the groups was not significantly different (p = 0.06, FET); the participants' sex within the cohorts was not significantly related to ROC dosing and did not have a significant interaction with diagnosis (p = 0.172 and p = 0.287, respectively, by 2-Way ANOVA).

Rocuronium Dose Determination
The doses of ROC utilized for intubation and for post-intubation maintenance of neuromuscular blockade were derived from electronic surgical records. Clinically, NMBA dosing is often titrated based on an individual's response to induced muscle contraction until optimum neuromuscular blockade is reached [45,46]. For participants in the present study, ROC dosages were determined in this way at the time of the procedure using a standard practice where an initial dose was calculated based on body weight and additional ROC added by titration if needed. In short, well-established methods of train-of-four ulnar nerve stimulation to induce contraction of the adductor pollicis were used [36,47]. This process allowed sufficient relaxation while avoiding the use of excessive ROC, which can be problematic in post-surgical recovery [48,49]. The primary use of ROC was to facilitate intubation, and ROC was generally discontinued thereafter to allow neurophysiologic monitoring of the spinal surgery. In cases where neurophysiologic monitoring was not being performed or was not possible, intraoperative doses of ROC were sometimes delivered to help maintain optimal surgical conditions and reduce involuntary patient movements, spontaneous respiration, or spasmodic breathing [31,50].
A total of 25 different anesthesiologists, with approximately equal CP and control cases, were responsible for administration of the intubating rocuronium dose. We found no significant effects on ROC dosing evaluating anesthesiologist alone or when comparing the effect of anesthesiologist on the ROC dose of patients categorized by GMFCS (data not shown: TWA, p = 0.24 and p = 0.27 respectively). Subsequent sensitivity analysis of total anesthesiologists was performed in which the correlation of mean ROC dose per GMFCS against mean ROC dose per GMFCS with groups of 5 anesthesiologists removed. This sensitivity analysis revealed no effect of anesthesiologist on rocuronium dose as each correlation yielded a significant Pearson correlation coefficient (Supplemental Figure S1).

Chronic Anticonvulsant Therapy
Chronic anticonvulsant therapy (CAT) is common among individuals with spastic CP due to a co-occurring seizure disorder or epilepsy [38,[52][53][54][55]. Resistance to neuromuscular blocking agents has been linked to the hepatoxicity of anticonvulsants and the upregulation of drug-metabolizing liver enzymes, especially CYP3A4, which is known to metabolize ROC [39,41,42], Although it was not possible to biopsy liver samples for the present study, elevated CYP3A4 generally correlates with increased circulating alanine transaminase (ALT) and total bilirubin [42,56]. Pre-operative serum ALT and bilirubin values were available from the EMR of a subset of study participants receiving CAT (n = 29 CP cases) and were evaluated as a surrogate measure against participants who were not on CAT (n = 14 CP cases). For patients on CAT, bilirubin and ALT correlated with each other (Spearman's rho = 0.599, p < 0.0003), but there was no significant correlation between intubating ROC dose and serum values of either ALT or bilirubin for any group. Interestingly, CP patients on CAT actually had significantly lower total serum bilirubin than those not on CAT (0.27 ± 0.03 vs. 0.54 ± 0.08 mg/dL, respectively, MWU p = 0.0006, Table 2). In addition, although different anticonvulsants have varying degrees of hepatotoxicity risk [57], no significant differences in ROC dose were observed when patient anticonvulsant use was categorized by hepatotoxicity classification (3 classification groups, KW, p = 0.718, Supplemental Table S1C). We also found that participants with moderate to severe CP (GMFCS III, IV, or V) had elevated intubating ROC doses whether they were receiving CAT (1.21 ± 0.13 mg/kg; n = 30) or not (0.86 ± 0.09 mg/kg; n = 19). Both groups differed significantly from each other and from controls (0.64 ± 0.03 mg/kg; n = 73) by ANOVA with post hoc Holm-Sidak test: CP-CAT vs. CP-Non-CAT, p = 0.03; CP-CAT vs. Con, p < 0.0001; CP-Non-CAT vs. Con, p = 0.04. Importantly, participants with CP who were not on CAT still required significantly more ROC than controls (Figure 2 and Supplemental Table S1A,B).
We also found that participants with moderate to severe CP (GMFCS III, IV, or V) had elevated intubating ROC doses whether they were receiving CAT (1.21 ± 0.13 mg/kg; n = 30) or not (0.86 ± 0.09 mg/kg; n = 19). Both groups differed significantly from each other and from controls (0.64 ± 0.03 mg/kg; n = 73) by ANOVA with post hoc Holm-Sidak test: CP-CAT vs. CP-Non-CAT, p = 0.03; CP-CAT vs. Con, p < 0.0001; CP-Non-CAT vs. Con, p = 0.04. Importantly, participants with CP who were not on CAT still required significantly more ROC than controls (Figure 2 and Supplemental Table S1A,B).  I, II, III, IV, and V, respectively). CP participants required significantly more ROC than controls (p < 0.0001, MWU) and well above the standard 0.6 mg/kg dose (represented by horizontal dashed bar). Within the CP cohort, patients who were on chronic anticonvulsant therapy (CAT) required significantly more ROC than those who were not using anticonvulsants (p = 0.007, 2WA). Participants with CP who were full-term or were normal birthweight required significantly more ROC than preterm or low birthweight (p = 0.0017 and p = 0.0011, respectively, MWU), and ROC dose was significantly correlated with increasing GMFCS score (p = 0.005; Spearman's rho). Filled circles represent outliers. Statistical significance is denoted by: p < 0.05 = *, p < 0.01 = **, and p < 0.001 = ***.

Risk Factors Associated with Cerebral Palsy
In many cases, the cause of CP is unknown, but for 46 of our participants, a suspected etiology was indicated in their EMR (Table 1). Among these 46, no significant associations , and children with CP across the spectrum of GMFCS scores (n = 3, 6, 6, 14, and 35 for GMFCS I, II, III, IV, and V, respectively). CP participants required significantly more ROC than controls (p < 0.0001, MWU) and well above the standard 0.6 mg/kg dose (represented by horizontal dashed bar). Within the CP cohort, patients who were on chronic anticonvulsant therapy (CAT) required significantly more ROC than those who were not using anticonvulsants (p = 0.007, 2WA). Participants with CP who were full-term or were normal birthweight required significantly more ROC than pre-term or low birthweight (p = 0.0017 and p = 0.0011, respectively, MWU), and ROC dose was significantly correlated with increasing GMFCS score (p = 0.005; Spearman's rho). Filled circles represent outliers. Statistical significance is denoted by: p < 0.05 = *, p < 0.01 = **, and p < 0.001 = ***.

Risk Factors Associated with Cerebral Palsy
In many cases, the cause of CP is unknown, but for 46 of our participants, a suspected etiology was indicated in their EMR (Table 1). Among these 46, no significant associations between GMFCS score and suspected etiology were observed (data not shown), and no effect of etiology was seen on ROC dose (Supplemental Table S2).

Rocuronium Dose and Neuromuscular Junction Microanatomy
Non-depolarizing NMBAs like ROC competitively inhibit nicotinic acetylcholine receptors (nAChRs) organized at NMJs on skeletal muscle [37], Histological screening of muscle samples by hematoxylin and eosin staining was performed to verify that samples were primarily muscle without significant freezing artifact, physical damage from processing, or tissue necrosis (Figure 3). Analysis of immunofluorescence results showed expected findings of dysmorphic NMJs in CP muscle samples such as an increased staining of laminin and disorganization of nAChRs [37,43,44]. To evaluate nAChR distribution and NMJ organization, nAChR was stained and analyzed against other NMJ components acetylcholinesterase (AChE), which is a tightly-regulated enzyme that halts neurotransmission by cleaving acetylcholine [59], and laminin β2 (Lβ2), which is a component of the muscle basal lamina associated with NMJ structure (Figure 4)    . Representative neuromuscular junctions from control and cerebral palsy. Skeletal muscle cryosections were immunofluorescently labeled to illuminate the co-localization of laminin β2 (green; panels (A,E)), AChE (blue; panels (B,F)), and nAChR (red; panels (C,G)). Co-staining displays as white or lavender tones (D,H) when the color channels are highly co-localized. On average, NMJs from participants with CP (e.g., right panel) exhibited significantly disrupted NMJ organization as compared to controls (left panel; n = 200 total NMJs, overall). Disorganization among CP NMJs was evident in Patients with CP whether they were on CAT or not, were found to have statistically larger areas of AChR, AChE, and Lβ2 per NMJ than controls (CP-Cat vs. Control, p < 0.000, p = 0.005, and p = 0.001; CP-Non Cat vs. Control, p = 0.006, p = 0.025, p = 0.003, respectively); however, the CP-subgroups were not significantly different from each other (CP-CAT vs. CP-Non CAT p = 1 for all measures, Figure 5D,F). Interestingly, patients with CP exhibited significant correlations between intubating ROC dose and measures of NMJ size as estimated by the extent of AChR, AChE, and/or Lβ2 staining. Both the CP-CAT cohort (Pearson r = 0.394/p = 0.043 for AChR; r = 0.226/p = 0.428 for AChE; r = 0.169/p = 0.030 for Lβ2) and the CP-Non CAT cohort (r = -0.840/0.009 for AChR; -0.851/ 0.008 for AChE; -0.908/0.002 for Lβ2) exhibited correlations between ROC and NMJ size ( Figure 5B,C and Supplemental Figure S3B). Among controls, on the other hand, there was no significant correlation in these measures (Pearson r = 0.077/p = 0.328 for AChR; r = 0.177/p = 0.150 for AChE; r = 0.129/p = 0.226 for Lβ2; Supplemental Figure S3A). Thus, ROC dosing seemed positively correlated with NMJ size in the cohort of CP patients on CAT but negatively correlated with NMJ size among CP cases that were not on CAT.  (panel (B).) correspond to tissues from which images represented in Figure 4 were obtained. Whole tiled images were captured at 10× magnification on an Evos microscope (scale bar in each photo represents 500 µM.) These images show the general morphology of the muscle samples used in the study. Figure 4. Representative neuromuscular junctions from control and cerebral palsy. Skeletal muscle cryosections were immunofluorescently labeled to illuminate the co-localization of laminin β2 (green; panels (A,E)), AChE (blue; panels (B,F)), and nAChR (red; panels (C,G)). Co-staining displays as white or lavender tones (D,H) when the color channels are highly co-localized. On average, NMJs from participants with CP (e.g., right panel) exhibited significantly disrupted NMJ organization as compared to controls (left panel; n = 200 total NMJs, overall). Disorganization among CP NMJs was evident in the ZY-ZX planes as well. Scale bar represents 10 µM.

Discussion
Our study expands on previous reports of general resistance to NMBAs in CP through detailed characterization of resistance to ROC in spastic CP [32,35,37]. Participants with CP exhibited elevated ROC requirements and increased variability in ROC dosing associated with GMFCS severity and select birth characteristics. Because higher GMFCS score may be indicative of worsening muscle pathology, we hypothesized that elevated ROC dose in participants with spastic CP, especially those not on CAT, would The degree of pairwise co-localization between nAChR, AChE, and Lβ2 was also analyzed for correlation with ROC dose. Not surprisingly, control subjects exhibited a correlation between intubating ROC dose and NMJ dysmorphism represented by the presence of AChE outside of the nAChR-defined NMJ (Pearson r = 0.338, p = 0.022, Figure 5A), and there appears to be variability in NMJ colocalization scores among individuals that may account for the relatively small range of doses seen in the control cohort. There were no correlations between intubating ROC dose and NMJ dysmorphism for either CP subgroup. (Supplemental Table S3). Quantification of NMJ dysmorphism showed significant variability (Supplemental Tables S3 and S4), but participants with CP generally showed disrupted NMJ staining patterns compared to controls (Figure 4 and Supplemental Video S1).

Discussion
Our study expands on previous reports of general resistance to NMBAs in CP through detailed characterization of resistance to ROC in spastic CP [32,35,37]. Participants with CP exhibited elevated ROC requirements and increased variability in ROC dosing associated with GMFCS severity and select birth characteristics. Because higher GMFCS score may be indicative of worsening muscle pathology, we hypothesized that elevated ROC dose in participants with spastic CP, especially those not on CAT, would be associated with NMJ disruption. Our results showed that measures of microanatomic NMJ dysmorphism were associated with ROC dosing; however, the associations varied between the two primary CPsubgroups-those receiving CAT and those not. The CP cohorts also exhibited significant differences in the area of NMJ component staining (AChR, AChE, and Lβ2) that was not see in controls. Together, these data indicate that individuals with CP (whether on CAT or not) may possess disrupted NMJs as has been suggested by previous studies [37,43,44]. More detailed assessments of NMJ component organization and NMJ function may be needed in the future to determine if NMJ disruption is causally related to an increase in the need for ROC in CP.
The use of anticonvulsant therapy further elevated ROC dosing when compared against controls, but patients with moderate to severe spastic CP required significantly more rocuronium for intubation (1.6×) whether they were on CAT (1.9×) or not (1.3×). Interestingly, we expected that participants with CP who were on CAT would show a correlation between elevated ROC dosing and serum ALT, which is a surrogate indicator of the liver dysfunction that has been linked increased ROC catabolism by CYP3A [39,41,42]. This expectation was bolstered by previous reports suggesting that individuals on CAT should exhibit a quicker recovery from neuromuscular blockade whether they received bolus dosing or continuous infusion [39,40]. We found that this expectation was only partly true for participants on CAT (Table 2) as intubating ROC doses did not correlate with ALT; whereas, in cases where ROC was used for extended periods during a case, the total ROC correlated with serum ALT. Regardless of CAT status, participants with CP in our study received significantly more ROC whether it was used for intubation alone or it was used throughout their procedure, as compared to controls. These findings expand previous reports (e.g., [33]) indicating that patients with CP have altered sensitivities to NMBAs and suggest that patients with CP, independent of CAT, require elevated intubating and maintenance doses to provide optimum surgical conditions. ROC doses in CP correlated with GMFCS scores and with the participant's gestational age and weight at birth (Figure 2). Elevated total ROC appeared to be independent of surgery duration (Table 1) and unrelated to the relative quantity of nAChR present at NMJs ( Figure 5). We also noted an incidental finding that serum levels of magnesium and triglycerides, (Table 2), which have been implicated in ROC dosing, did not correspond with elevated ROC use. In fact, the high-normal serum magnesium and low serum triglycerides seen in CP participants both suggest a lower requirement for ROC [61,62]. As expected, CAT had a significant effect on ROC dosing (Figure 2), but participants with CP who were not on anticonvulsants also had significant resistance to ROC. The non-CAT group showed significant positive correlations between ROC and GMFCS, birthweight, and gestational age at birth. Thus, resistance to ROC in CP appears to be a complex result of multiple inputs, including the use chronic anticonvulsant medication, but children with spastic CP appear also to have a fundamental resistance associated with their diagnosis. Importantly, ROC dosing in the CP cohort also had a high degree of variability, especially as disease severity (i.e., GMFCS score) increased. The combination of increased resistance to ROC and high variability may have implications when administering care to individuals with spastic CP, and clinicians should be prepared for inconsistent patient responses to standardized doses of ROC, and perhaps other drugs, that may vary to a great degree across individual patients.
Interestingly, although low BW and prematurity are risk factors for spastic CP [58], we found that children with CP who were full-term or who had normal birthweights tended to require higher ROC doses during surgery later in life than their preterm and LBW peers. The increased need for ROC in these cases may reflect differences in the etiologies of CP that prevail in late gestation and/or perinatal onset. Studies suggest that term-born individuals with CP are significantly more likely to have birth defects, perinatal stroke, poor fetal growth [63], placental disorders [64], and maternal or neonatal infection [65], while those with CP born pre-term are more likely to have intraventricular hemorrhage or periventricular white matter injury [66]. Although we did not see significant correlations between disease etiology and GA, BW, or total ROC dose in our cohorts, our analyses may be limited by the small number of participants for which an etiology was indicated in the EMR (n = 36) as well as an overrepresentation of severely affected participants (e.g., 76.6% of CP participants in our study were GMFCS IV or V compared to 27-31% of such patients that would be expected based on a published meta-analysis of prior CP studies) [67]. Nonetheless, the notion that CP is clinically different in children born preterm versus full-term is consistent with previous studies indicating that severe intellectual disability, epilepsy, and severe ambulatory impairments are more common in term-born than preterm children with CP [68,69].
The positive correlation between GMFCS score and ROC dose (Figure 2) in the CP cohort indicates to us that higher levels of disability in spastic CP are associated with increasing ROC requirements and suggests that functional status and drug resistance are related. Previous data indicate that NMJ dysmorphism is also related to GMFCS [44], and since ROC binds nAChR at NMJs [70], we hypothesized that NMJ disruption would be related to ROC dosing. Although we found some correlation between ROC dosing and total NMJ marker staining among the groups, correlations between intubating ROC dose and NMJ dysmorphism were only found in controls where AChE mismatches with nAChR (EOR) were significant ( Figure 5A). We found that among CP subjects, independent of anticonvulsant therapy required elevated ROC doses, there was an increased area of nAChR, and that the distribution of nAChR in patients with CP, depending on the subgroup, exhibited either a positive or a negative correlation with intubating ROC dose (positive for subjects with CP on CAT and negative for subjects with CP not on CAT; Supplemental Table S3). This complex landscape of NMJ morphologies may suggest that increased use of ROC in CP may be due to aspects NMJ morphology alteration but that we may need to consider other components than the AChR receptor target of ROC. These data also indicate that altered distribution of synaptic components are may be associated with elevated ROC, and although ECM expansion was not assessed directly in the current study, it appeared that all participants with CP had elevated Lβ2 in the area of the NMJ (values that were found to correlate with intubating ROC dose; Supplemental Table S4). These differences in the quantity and distribution of AChE and Lβ2 in CP imply a role for altered NMJ microanatomy, which we have seen in other studies [37,43,44,71], and which may be indicative of NMJ dysfunction [72]. Overall, our data are suggestive that NMJ conformation may be related to the amount of ROC needed during surgery, but the pairwise NMJ measures assessed here may not be the best indicators for functional dysmorphism among participants with CP.
The findings of our current study are compelling but necessarily limited by the data available in participant medical records, which made some comparisons challenging. Another potential limitation exists in our reliance on patients presenting for surgery who received ROC, which may represent a sub-population of all individuals with spastic CP, and our reliance on recorded ROC utilization, which is an accurate number that may have been influenced by subjective or complex considerations associated with individual cases. The interpretation of results in this paper is necessarily bounded by these limitations, and extrapolation to the broader CP community may require additional research.

Conclusions
Overall, our findings provide insight into ROC usage in patients with CP and highlight manifestations of spastic CP. The detailed mechanisms accounting for ROC resistance in children with CP requires further investigation, but a set of complex individual traits may be related to ROC resistance, and ROC resistance may typify a set of broader issues impacting personalized treatment plans for children with spastic CP and considerations around variable drug responses associated with medications targeting NMJs or peripheral neuromotor function. In particular, clinicians should be aware of the possibility that individuals with CP have varied resistance to ROC that may be disease severity and disease etiology dependent with possible relationships to NMJ microstructure.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/jpm11080765/s1, Figure S1: Sensitivity Analysis of Rocuronium Dosage by Anesthesiologist, Figure S2: Correlation of total rocuronium dose with birth statistics and serum analytes, Figure S3: Non-Significant findings for NMJ measures vs. intubating ROC dose, Table S1: Rocuronium utilization by category and anticonvulsant type, Table S2: Mean Intubating and Total ROC dose by Etiology, Table S3: Correlation of Rocuronium use and NMJ dysmorphism scores by group, Table S4: Correlation of Rocuronium use and NMJ dysmorphism scores by diagnosis, Video S1: Comparison of control and CP NMJ in 3D. Funding: The work presented in this manuscript was generously supported by funding from the Nemours foundation, the Swank Family Foundation, the NIH-funded Delaware CTR ACCEL Program (U54 GM104941), and the Pedal with Pete Foundation with the American Academy of Cerebral Palsy and Developmental Medicine.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Nemours (protocol numbers: 349465, which was initially approved 2 September 2012; 381494, which was initially approved 30 December 2012; 687629, which was initially approved 6 May 2015).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available from the corresponding author on reasonable request. The primary data are not publicly available to protect the privacy of research participants.