title: High-molecular-weight hyaluronan inhibits macrophage proliferation and cytokine release in the early wound of a preclinical postlaminectomy rat model.
abstract: BACKGROUND
  Failed back syndrome, a condition that affects 3-14% of postoperative spine patients, is characterized by the recurrence of radicular pain after spinal decompression. The source of this pain in some patients is thought by many investigators to be the result of epidural scarring and nerve root tethering, but this is controversial. We have previously demonstrated that in a disc-injury model the untreated postlaminectomy rats develop a significant proliferative fibrous response at 8 weeks with spinal nerve scarring to the disc and adjacent pedicle, and increased sensitivity to tactile allodynia testing in the related sensory dermatome. Topical high-molecular-weight hyaluronan (HMW HA) moderates both the proliferative fibrosis and the behavioral pain response.
  
  
  OBJECTIVE
  Our purpose is to study the time-related changes in the proinflammatory cytokine and monocyte/macrophage profiles in the epidural space in the early postlaminectomy untreated and HMW HA gel treated groups.
  
  
  METHODS
  A modified rat laminectomy with disc injury model was employed to assess epidural fibrosis between and around the spinal nerves using a quantitative immunohistochemistry assessment approach along with correlative enzyme-linked immunosorbent assay analysis.
  
  
  METHODS
  Lumbar laminectomies at L5 and L6 with a L5-L6 disc injury were performed on 120 adult male Sprague-Dawley rats. The rats were then randomized into one of two groups: untreated and treated. The treatment group received a one-time topical application of 0.1 cc of HMW HA gel directly to the laminectomy site just before wound closure. The rats were then randomly subdivided into survival periods of 24 hours, 72 hours, and 7 days. Immunohistochemistry was performed on fresh frozen sections and stained for interleukin-1 beta (IL-1beta) and monocytes/macrophages (ED-1) using monoclonal antibodies and 3, 3' diaminobenzidine (DAB) chromogen. The amount of stain in each specimen was then quantified using the National Institutes of Health computer imaging analysis system.
  
  
  RESULTS
  The semiquantified data from the histological specimens demonstrated significant decreases in the IL-1beta and IL-6 infiltration observed at 24 hours in the epidural space and around the right nerve root (p=.0296 and 0.0195, respectively) in the HA gel treated group. Additionally, significant decreases in the monocyte/macrophage infiltration were observed at 72 hours in the epidural space around the left nerve root (p=.0039) and right nerve root (p=.0072) in the HA gel treated group. At 7 days, IL-1beta, IL-6, and macrophage infiltration of the wound had declined in both the HA gel and the untreated groups. The enzyme-linked immunosorbent assay data support the same pattern as seen in the histological results.
  
  
  CONCLUSIONS
  These results demonstrate that treatment of postlaminectomy wounds with HMW HA gel decreases the number of monocytes and macrophages and the concentration of certain cytokines in the early inflammatory phase of healing. There are several plausible explanations for this effect. First, the HMW HA may block the interaction of short-chain low-molecular-weight HA with proinflammatory cell surface receptors. The interaction of these short-chain oligo-HA fragments, upon cell-surface receptor binding, induces changes in inflammatory cells that lead to increased cell motility and migration into the wound area. Second, the addition of exogenous HMW HA may cause a dilution effect in the wound, thereby decreasing the concentration of inflammatory cells in the extracellular matrix of the region of injury. Finally, the migration of inflammatory cells may be decreased in the viscous environment of the HMW HA. The first explanation is believed by the authors of this paper to be the more likely mechanism. HMW HA probably mutes the proinflammatory effects of the low-molecular weight fragments, leading to decreased inflammation, and thus decreased fibrosis and scar formation noted in the chronic model.
authors.affiliation: Department of Orthopaedics, Veterans Administration San Diego Health Care System, 3350 La Jolla Village Drive, San Diego, CA 92161, USA.
authors.forename: Aimee L
authors.initials: AL
authors.lastname: Schimizzi
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Massie
authors.forename: Mark
authors.initials: M
authors.lastname: Murphy
authors.forename: Andrew
authors.initials: A
authors.lastname: Perry
authors.forename: Choll W
authors.initials: CW
authors.lastname: Kim
authors.forename: Steven R
authors.initials: SR
authors.lastname: Garfin
authors.forename: Wayne H
authors.initials: WH
authors.lastname: Akeson
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D016207:Cytokines / Q000378:metabolism*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005355:Fibrosis / Q000209:etiology / Q000473:pathology / Q000517:prevention & control; D006820:Hyaluronic Acid / Q000494:pharmacology*; D007124:Immunoenzyme Techniques; D007403:Intervertebral Disc / Q000293:injuries / Q000473:pathology; D007796:Laminectomy / Q000009:adverse effects*; D008159:Lumbar Vertebrae / Q000293:injuries / Q000601:surgery; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008970:Molecular Weight; D011183:Postoperative Complications; D051381:Rats; D017207:Rats, Sprague-Dawley; D014945:Wound Healing / Q000187:drug effects* / Q000502:physiology
chemical_list: D000276:Adjuvants, Immunologic; D016207:Cytokines; D006820:Hyaluronic Acid