SPINE Volume 36, Number 21, pp 1728–1736 ©2011, Lippincott Williams & Wilkins BASIC SCIENCE ISSLS Prize Winner: Adaptations to the Multi dus Muscle in Response to Experimentally Induced Intervertebral Disc Degeneration Stephen H. M. Brown , PhD , * § Diane E. Gregory , PhD , * J. Austin Carr , MS , * Samuel R. Ward , PhD , * Koichi Masuda , MD , * and Richard L. Lieber , PhD * Conclusion. Multi dus becomes stiffer, both in individual Study Design. Basic science study of the rabbit multi dus muscle bers and ber bundles, in response to experimentally induced response to intervertebral disc degeneration. intervertebral disc degeneration. This cannot be explained by change Objective. To assess changes in passive mechanical properties, in ber-type due to reduced muscle use, nor by the increased size of associated protein structure, and histology of multi dus in response the protein titin (which would reduce stiffness). We hypothesize that to disc degeneration produced by experimental needle puncture. ber bundles become stiffer by proliferation and/or reorganization of Summary of Background Data. Relationships have been collagen content within the muscle but the basis for ber stiffening reported between muscle dysfunction and low back injury; however, is not known. little is known about the cause and effect of such relationships. Key words: adaptation , connective tissue , extracellular matrix , Methods. Twelve rabbits were studied; 4 in each of 3 groups: intervertebral disc , muscle , spine , stiffness. Spine 2011 ; 36 : control, 4-weeks postintervertebral disc injury (4-week disc 1728 1736 degeneration), and 12-weeks postintervertebral disc injury (12-week disc degeneration). Single multi dus bers and bundles of bers were isolated and tested for slack sarcomere length and elastic umerous studies have linked chronic low back pain 1 – 3 modulus. Titin isoform mass, myosin heavy chain distribution, and (LBP) with abnormal muscle use and abnormal 4 – 8 muscle histology were also examined. Nmuscle structure. However, cause-effect relation- Results. Compared to control, individual muscle bers were ships among LBP, injury, and muscle dysfunction remain elu- 34% stiffer and ber bundles 107% stiffer in the 12-week disc sive. Defi ning these relationships is essential for understand- degeneration group. No changes were detected at 4-week disc ing as well as treating LBP, and is the goal of this study. 9 , 10 degeneration. No statistically signi cant change was found for To date, two studies by Hodges et al have elucidated MHC distribution in the 12-week disc degeneration group when some cause-effect relationships between back injury and mus- compared to control, whereas titin isoforms were larger (P < 0.05) cle function. In this porcine model, experimentally induced in the 12-week disc degeneration group. Histology revealed select intervertebral disc injuries were used to demonstrate multifi - 9 regions of multi dus, at 12-week disc degeneration, with increased dus muscle atrophy and fatty infi ltration 3 days postinjury, space between bundles of bers, which in some instances was partly and increased multifi dus motor excitability 15 minutes postin- 10 occupied by adipose tissue. jury, respectively. A third, prospective study, by Cholewicki et al 11 demonstrated in a population of varsity athletes that slower onset of abdominal muscle electrical activity, in From the Departments of * Orthopaedic Surgery ; Bioengineering ; and Radiology, University of California San Diego, San Diego, CA ; and response to spinal perturbations, increased the likelihood of § Department of Human Health and Nutritional Sciences, University of sustaining a low back injury. Thus, it appears that muscle and Guelph, Guelph, Ontario, Canada . the motor system have the ability to both adapt in response, Acknowledgment date: October 18, 2010. Revision date: January 17, 2011. and predispose to, spine injury. Although each of these stud- Acceptance date: January 18, 2011. ies provides important information regarding the relation- The manuscript submitted does not contain information about medical device(s)/drug(s). ship between spine injury and muscle control and structure, a Federal and institutional funds were received in support of this work. No number of questions remain. bene ts in any form have been or will be received from a commercial party Experimentally induced intervertebral disc injury has been related directly or indirectly to the subject of this manuscript. used in animal models to approximate degenerative changes Supported by NIH grants HD050837 and P01AR48152. SHM Brown and that naturally occur in the human lumbar spine with age. 12 – 14 DE Gregory were supported by NSERC postdoctoral Fellowships. These structural and biochemical degenerative changes are Address for correspondence and reprint requests to Richard L. Lieber, PhD, 15 Department of Orthopaedic Surgery (9151), UC San Diego and VA Medical related, in humans, to pain and dysfunction. Despite the rela- Center, 9500 Gilman Drive, Mail Code 0863, La Jolla, CA 92093; E-mail: tively routine use of such models to study changes in the cellu- [email protected] lar and mechanical components of the disc and spine, only the 9 DOI: 10.1097/BRS.0b013e318212b44b aforementioned Hodges et al study used this model to examine 1728 www.spinejournal.com October 2011 Copyright © 2011 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. BBRS204436.inddRS204436.indd 11728728 223/09/113/09/11 11:17:17 PPMM BASIC SCIENCE Adaptations to the Multifi dus Muscle • Brown et al Figure 1. MR im- ages displaying the location of mul- ti dus harvest at the approximate L3 level from two dif- ferent rabbits. ( A ) Rabbit 1: transverse plane and ( B ) rabbit 2: saggital plane. Arrows indicate lo- cation of multi dus removal. structural changes in the spine musculature. Specifi cally, Hodges Rabbits were positioned in a lateral prone position, and a pos- et al 9 employed ultrasound imaging of the multifi dus muscle terolateral incision was made to enter the retroperitoneum. preinjury 3 and 6 days postinjury, and reported reduced muscle After palpating and exposing the anterior surface of the L2– size by 3 days postinjury, along with histological evidence of L3 and L4–L5 intervertebral discs, an 18-gauge needle was increased fatty infi ltration within the muscle. However, these are guided linearly through the right-anterior annulus fi brosus rough estimates of muscle morphology and, at such short time into the nucleus pulposus of each disc. 14 After removing the points postinjury, it is still not known how these changes will needle the wound was thoroughly rinsed with sterile saline progress or regress over longer periods of time. Furthermore, and closed with layered sutures. Animals were monitored previous research has hypothesized that muscles can adapt to during recovery and allowed to carry out their normal rou- surrounding injury by undergoing what appears to be atrophy, tines under daily observation. yet maintain their physiological cross-sectional area (thus force producing capability) through structural remodeling. 16 Thus, it Harvesting Multi dus Muscle is still uncertain whether multifi dus undergoes remodeling, in Rabbits were sacrifi ced and immediately afterwards two response to disc injury, which could alter or compromise its multifi dus biopsies were taken at each of two locations (four functional capabilities. Passive mechanical properties help defi ne biopsies total). The fi rst two were taken at the approximate such functional capabilities in muscle, and have been shown to level of the L3 vertebral body (just caudal to the most cranial differentiate between healthy and dysfunctional skeletal muscle disc injury site) ( Figure 1 ); the second two were taken at the in some populations. 17 , 18 Thus, the purpose of this study was to approximate L7 level (two vertebral levels caudal to the most assess changes in passive mechanical properties, associated pro- caudal disc injury site). One biopsy from each site was imme- tein structure, and histology of multifi dus in response to experi- diately placed in a storage solution 19 to prevent hyperpolariza- mentally induced intervertebral disc degeneration in the rabbit. tion and destruction, and stored at − 20° C; this tissue was used for passive mechanical testing. The second biopsy was pinned MATERIALS AND METHODS to cork and frozen with liquid nitrogen and stored at − 80° C; Twelve female New Zealand White rabbits (9 months old at this tissue was used for histological and protein analyses. sacrifi ce) were separated into three groups of four. The fi rst group (Control) received no intervertebral disc injury prior Passive Mechanical Testing to sacrifi ce. The second and third groups incurred experimen- All muscle was tested within 2 weeks of harvest. Testing was tally induced disc injury and recovered for 4 weeks (4-week performed in a relaxing solution 19 to ensure muscle proper- disc degeneration) and 12 weeks (12-week disc degeneration), ties were assessed in a completely passive state. Single fi bers respectively. Procedures were approved by the local IACUC. and small fi ber bundles (composed of approximately 4–8 fi bers ensheathed in their connective tissue matrix) were iso- Experimentally Induced InterverteBral Disc lated and tied with 10–0 silk thread to pins secured on one Degeneration side to a microlevel force transducer (Model-405A, Aurora Rabbits were anaesthetized via subcutaneous injection of Scientifi c, Aurora, Ontario, Canada),