The Role of Subscapularis Muscle Denervation in the Pathogenesis of Shoulder Internal Rotation Contracture after Neonatal Brachial Plexus Palsy: A Study in a Rat Model Vasco V. Mascarenhas, 1 Marcelo Casaccia, 2 Alejandra Fernandez-Martin, 2 Mario Marotta, 2 Cesar G. Fontecha, 2 Sleiman Haddad, 2 Jorge Kno ¨ rr, 3 Francisco Soldado 2 1 Hospital da Luz Lisboa, UIME, Centro de Imagiologia, Portugal, 2 Vall Hebron Research Institute (VHIR). Universitat Auto ` noma de Barcelona, Barcelona, Spain, 3 Pediatric Hand Surgery and Microsurgery, Department of Orthopaedic Surgery, Hospital Sant Joan de De ´u, Barcelona, Spain Received 15 March 2014; accepted 8 July 2014 Published online 13 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22709 ABSTRACT: We assessed the role of subscapularis muscle denervation in the development of shoulder internal rotation contracture in neonatal brachial plexus injury. Seventeen newborn rats underwent selective denervation of the subscapular muscle. The rats were evaluated at weekly intervals to measure passive shoulder external rotation. After 4 weeks, the animals were euthanized. The subscapularis thickness was measured using 7.2T MRI axial images. The subscapularis muscle was then studied grossly, and its mass was registered. The fiber area and the area of fibrosis were measured using collagen-I inmunostained muscle sections. Significant progressive decrease in passive shoulder external rotation was noted with a mean loss of 58˚ at four weeks. A significant decrease in thickness and mass of the subscapularis muscles in the involved shoulders was also found with a mean loss of 69%. Subscapularis muscle fiber size decreased significantly, while the area of fibrosis remained unchanged. Our study shows that subscapularis denervation, per se, could explain shoulder contracture after neonatal brachial plexus injury, though its relevance compared to other pathogenic factors needs further investigation. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1675–1679, 2014. Keywords: neonatal brachial plexus palsy; glenohumeral dysplasia; shoulder internal rotation contracture; subscapularis muscle; muscle growth Shoulder impairment is the most common long-term complication and the major cause of morbidity in children with upper trunk neonatal brachial plexus palsy (NBPP). 1 The subscapularis muscle has been implicated as a primary deforming force leading to the development of shoulder internal rotation contracture (SIRC). This constant internal rotation posture leads to early glenohumeral joint deformity (i.e., glenohumeral dys- plasia). 2,3,4 While the natural history of the changes in bone and articular alignment has been extensively studied, the pathogenesis of muscular changes preced- ing them remains unclear and is currently receiving more attention. 5–7,8,9,10 The mechanism leading to subscapularis muscle growth impairment with subse- quent volume loss and contracture has been explained by two differing hypotheses: denervation 8,11,9,12 and muscle imbalance. 5,6,7,13,14,10 The imbalance theory infers that the subscapularis changes are secondary to the lack of passive stretching in the absence of active shoulder external rotation, which leads to progressive shortening and stiffness of subscapularis muscle due to an abnormal muscle growth or muscle hypoplasia. 5 The denervation hypothesis states that denervation of the subscapularis muscle causes progressive muscle fibrosis, shortening, and contracture. 9 Recent experi- ments in a mouse model of upper trunk neurectomy supported this second hypothesis, showing that muscle reinnervation prevents contracture. 8,12 However, these studies showed that muscle shortening and contracture occurred without fibrosis. We assessed the contribution of selective subscapular denervation through surgical neurectomy in the neonatal period in a rat model. METHODS This study was conducted following NIH Guidelines for the use of laboratory animals and with the approval of the local ethics committee for experimental animal use. Seventeen rat newborns from one pregnant Sprague–Dawley Oncins France Strain A (OFA) rat were used. A sham group, undergoing a surgical approach without neural injury, was not considered necessary as previous studies proved this group had no alterations in passive shoulder external rotation (PSER) or subscapularis growth. 13 PSER measurement, magnetic resonance imaging (MRI), and subscapularis muscle weighting were done 4 weeks after surgery as described below and following the methodology of previous studies. 13 Sectional muscle fiber size and muscle area of fibrosis of subscapularis muscles were determined using images from collagen-I inmunostaining and also followed the methods of a previous study. 13 Rat Newborn Surgery Five-day-old rat pups underwent right brachial plexus surgery under general anesthesia with isofluorane. A surgi- cal microscope was needed for dissection. A transverse incision below the clavicle was made, splitting the pectoralis major and minor muscles to expose the brachial plexus. The superior subscapular nerve was identified, as it branched from the suprascapular nerve. The inferior subscapular nerve was also identified as it branched from the posterior cord. 13 A neurectomy with complete excision, from the origin This study was approved by the Ethic commission of VHIR center, act number 24, extraordinary session 20th Novem- ber 2008. Grant sponsor: Instituto de Salud Carlos III; Grant number: FIS PI10/01357. Correspondence to: Francisco Soldado (T: þ34688999890; F: þ34 93 253 21 49; E-mail: drsoldado@cirugiamanoinfantil.com) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. JOURNAL OF ORTHOPAEDIC RESEARCH DECEMBER 2014 1675