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- MATERIALS AND METHODS
The developmental course of musculoskeletal deformities in neonatal brachial plexus palsy (NBPP) has not been studied extensively. The goals of this study were to: (1) evaluate a new animal model of NBPP, (2) characterize the development of musculoskeletal abnormalities in paralyzed shoulders, and (3) investigate the expression of myogenic and adipogenic genes in paralyzed rotator cuff muscles. Neonatal mice were divided into neurotomy and sham groups. The neurotomy group underwent surgical transection of the superior trunk of the brachial plexus within 24 h of birth. The sham group underwent the same surgical exposure, but the brachial plexus was left intact. Musculoskeletal deformities were evaluated with radiological and histological assays at 2, 4, 8, 12, and 30 weeks after birth. The supraspinatus muscles of a separate group of mice were used to examine expression of myogenic and adipogenic genes at 8 weeks. The neurotomized forelimbs developed deformities similar to those seen in human NBPP. The deformities progressed with age. The denervated supraspinatus muscles showed intramuscular fat accumulation and upregulation of both myogenic and adipogenic genes compared to the normal. The current study presents a useful animal model for future research examining musculoskeletal changes secondary to neonatal nerve injury. Published by Wiley Periodicals, Inc. J Orthop Res 28:1391–1398, 2010
Most children affected by neonatal brachial plexus palsy (NBPP) recover spontaneously during the first few months, but approximately 20% of affected children are left with permanent deficits.1, 2 The permanent deficits commonly described in the literature include internal rotation and adduction contracture of the shoulder, loss of active elbow flexion, osseous deformities (e.g., hypoplasia and dysplasia of the humerus and scapula, posterior subluxation or dislocation of the glenohumeral joint), and muscle atrophy.3–7 In our previous studies, the developmental course of the musculoskeletal deformities secondary to neonatal shoulder paralysis was investigated with the use of botulinum-toxin-A-induced paralysis in neonatal mice.8, 9 Those studies showed that shoulders injected with botulinum toxin develop deformities similar to human NBPP and rotator cuff muscle atrophy and accumulate fat. However, it was not clear whether the deformities and intramuscular fat accumulation seen in the botulinum-toxin-injected shoulders were truly due to the muscle paralysis or due to the effects of the toxin. In order to answer this question, it is necessary to develop an animal model that represents neurogenic paralysis of the shoulder muscles in the immediate postnatal period. The goals of the current study were to: (1) evaluate a new animal model of NBPP utilizing surgical transection of the brachial plexus, (2) characterize the development of musculoskeletal abnormalities in paralyzed shoulders, and (3) investigate the expression of myogenic and adipogenic genes in paralyzed rotator cuff muscles. We hypothesized that: (1) transection of the brachial plexus in postnatal mice would cause musculoskeletal deformities similar to those seen in human NBPP, (2) the deformities would progress with age, and (3) myogenic genes would be downregulated and adipogenic genes would be upregulated in the paralyzed rotator cuff muscles.
- Top of page
- MATERIALS AND METHODS
The data from the current study demonstrate that transection of the superior trunk of the brachial plexus in neonatal mice reproduces many important pathological conditions of human NBPP.3, 4, 7, 12 Paralysis of the forelimb was induced within 24 h of birth by microsurgical nerve transection, reproducing the immediate postnatal paralysis seen in human infants with NBPP. The neurotomized forelimbs developed a loss of active external rotation, flexion, and abduction of the shoulder, active flexion of the elbow, and active extension of the wrist and digits, which led to adduction and internal rotation contracture of the shoulder and extension contracture of the elbow. There were abnormalities of the humeral head such as hypoplasia, delayed mineralization, and flattening of the posterosuperior aspect. Humeral version was altered (i.e., increased introversion) in the neurotomized forelimbs. There were also abnormalities of the scapula such as hypoplasia and increased retroversion of the glenoid. There was profound atrophy of the supraspinatus, infraspinatus, subscapularis, teres minor, deltoid, and biceps brachii muscles in the neurotomized shoulders. The transection of the brachial plexus in this study resulted in a lower motor neuron lesion causing flaccid paralysis of the affected muscles. Bone and joint deformities are typically seen in lower motor neuron lesions (e.g., due to poliomyelitis, myelomeningocele, or brachial plexus injury) that occur early in the postnatal developmental of musculoskeletal tissues.
The glenohumeral joint developed anterior subluxation or dislocation with erosion of the anteroinferior glenoid. The direction of humeral head subluxation is different from that of human NBPP, in which the humeral head is subluxated posteriorly.4, 6, 7 The location of glenoid erosion is also different from human NBPP, which has erosion of the posteroinferior glenoid.4, 6, 7 This difference may be attributed to the different anatomies of the humerus and scapula between mice and humans as previously described.8 The articular surface of the human humeral head faces medially when the forearm is in the neutral position, whereas the mouse humeral head faces posteriorly. The mouse scapula is oriented more sagittally than humans, perhaps resulting in different shoulder kinematics.
Our data demonstrate that shoulder deformity secondary to NBPP progresses with age, which is consistent with the findings of clinical studies.6, 7 The increase in severity of deformity was particularly evident in bony and muscle outcomes. The soft tissue contracture also showed the same trend, but this did not reach statistical significance, which may be due to the small sample size of the study or may indicate a plateau in soft tissue contracture at a certain point. Interestingly, glenoid version showed an opposite trend; the difference between the shoulders decreased with age, and the glenoid was found to be anteverted rather than retroverted at 30 weeks, leading to anterior subluxation of the humeral head. This finding is different from clinical studies, which showed the increase of retroversion of the glenoid with age in children with NBPP.4, 6, 7 This difference may again be attributed to differences in kinematics between mouse and human shoulders.
Denervation of neonatal rotator cuff muscles caused intramuscular fat accumulation and muscle atrophy. The supraspinatus muscle of the neurotomized shoulders showed substantial atrophy at 2 weeks and started to show fat accumulation at 4 weeks. Although it was not quantified, the relative amount of fat appeared to increase with age compared to the muscle. At 30 weeks, the muscle was found to be almost entirely replaced by fat. This finding is consistent with the study of Poyhia et al.5 that showed atrophy and fatty deposition of the rotator cuff muscles in children with NBPP. This microscopic observation was supported by our gene expression studies. Adipogenic genes were upregulated in the neurotomized shoulders compared to normal. Interestingly, myogenic genes were also all upregulated in the neurotomized shoulders. Our previous study9 showed similar intramuscular fat accumulation in addition to muscle atrophy in botulinum-toxin-injected supraspinatus muscles. This finding is similar to the study of Frey et al.13 that showed upregulation of both adipogenic and myogenic genes in tenotomized infraspinatus muscles of adult sheep. It is not clear why adipogenic and myogenic genes were both upregulated in denervated neonatal muscles. This may represent an abortive regenerative process of the denervated muscle and resultant loss of the negative feedback inhibition by end products of the process, which leads to the constant upregulation of myogenic genes.
Only a few studies describe the use of animal models for NBPP. Our previous study8 used botulinum toxin A to paralyze the shoulders of neonatal mice and reported similar deformities as seen in the current study. The duration of paralysis was varied by controlling the dose of botulinum toxin A, which demonstrated the recovery potential of neonatal shoulders after the return of muscle function. The study of Li et al.14 utilized C5, 6 root neurotomy in rats and reported similar shoulder deformity to the current study. However, they performed the neurotomy at 5 days after birth allowing a substantial delay before the onset of paralysis. They measured glenoid version in histological sections, which likely was not as accurate as the µCT methods used in the current study.
Our study had several limitations. First, differences in shoulder kinematics of mice compared to humans resulted in humeral version change and humeral head subluxation in directions that were different than those seen in humans. Second, the mice in the neurotomy group could not bear weight with their neurotomized forelimb because of the paralysis while those in the sham group used their operated forelimb for weight bearing. It is possible that non-weight bearing may have contributed to the deformity development in the neurotomized shoulders in addition to muscle paralysis. Third, we performed a nerve transection in this study resulting in an irreversible plexus injury. Human NBPP, in contrast, is typically a reversible traction-type injury. A traction injury model would reproduce the pathology of human NBPP more accurately and will to be examined in future studies. The strengths of the present study include the creation of shoulder paralysis immediately after birth, quantitative, and rigorous outcome measurements with validated methods, and the use of real-time PCR to provide adjuvant data for the histological observation of intramuscular fat accumulation.
In conclusion, we report an animal model utilizing surgical transection of the superior trunk of the brachial plexus of mice in the immediate postnatal period to reproduce many important musculoskeletal deformities of human NBPP. As in the human condition, the deformities progressed with age. Intramuscular fat accumulation was found in addition to muscle atrophy in the denervated rotator cuff muscles. The adipogenic and myogenic genes were both upregulated in these muscles. This study presents a useful animal model that may provide a better understanding of musculoskeletal changes secondary to an early postnatal nerve injury and lead to better management of the condition.