Document Type

Thesis

Degree Name

Master of Kinesiology (MKin)

Department

Kinesiology and Physical Education

Faculty/School

Faculty of Science

First Advisor

Dr. Diane Gregory

Advisor Role

Supervisor

Abstract

Introduction: The intervertebral disc (IVD) is composed of the annulus fibrosus (AF), which surrounds and contains the nucleus pulposus (NP). It is hypothesized that when the IVD becomes injured, in the case of IVD herniation, a localized innate immune response is initiated. Although the presence of pro-inflammatory cytokines in injured IVDs has been well documented, the extent to which inflammation affects the mechanical properties of the IVD remains poorly understood. The purpose of this study was to determine the effect of IVD damage (via puncture) and inflammation (via lipopolysaccharide (LPS) exposure) on the mechanical and structural properties of the IVD.

Methods: Four functional spinal units (FSUs) (bone-IVD-bone) were dissected and removed from the tails of 20 Sprague Dawley rats (80 FSUs in total). In half of the FSUs, a puncture model (using a 19G needle) was used to mimic IVD herniation. Each FSU (two punctured and two non-punctured) was subsequently cultured in media to support cell viability. Further, 10ug/mL of LPS was added to the media of one punctured and one non-punctured FSU. FSUs were then cultured for either 24 hours or 6 days after which they were removed and mechanically tested in order to determine changes in IVD mechanics as a result of exposure to LPS and/or IVD puncture. All FSUs were subjected to cyclic mechanical loading in compression/tension using a displacement-controlled protocol. From the force displacement data obtained, the neutral zone (NZ) size (mm) and stiffness (MPa/mm) were calculated and compared. Four additional FSUs cultured for 6 days were subsequently used for histological analyses in order to visually identify changes to the structural properties. Media was collected and analyzed in order to quantify concentrations of pro-inflammatory cytokines (TNFa, IL-6) and chemokines (MCP-1, MIP2, RANTES).

Results: When collapsed across damage and media condition, NZ size increased and stiffness decreased from 24 hours to 6 days. Further, there was an increase in NZ size as a result of puncture, which is indicative of an increase in joint laxity. Interestingly, there was a decrease in NZ stiffness in the punctured FSUs observed at 24 hours however by 6 days, punctured FSUs were more stiff than the non-punctured FSUs indicating the presence of a potential reparative mechanism in the IVDs. All cytokines and chemokines measured in the media, regardless of condition, peaked at 24 hours and dropped off by 6 days. There was an interaction of media (LPS versus no LPS) and day (24 hours versus 6 days) where the FSUs placed in LPS had a higher concentration of TNFa and RANTES at 24 hours but no significant difference at 6 days. Comparatively, IL-6 concentrations were higher in the control group at 6 days with no changes at 24 hours. Histological analysis showed proteoglycan staining in the puncture tract, AF inward bulging and disorganization in the control media/puncture group. Moreover, in the LPS/no puncture FSU there was a significant lack of proteoglycan staining in the NP.

Discussion and Conclusion: This study demonstrated that punctured FSUs might initiate a reparative mechanism within the IVD that is not present in non-punctured FSUs. Further, the findings of this study indicate that the inflammatory profile created by LPS is different than the inflammation that might ensue as a result of IVD herniation. The increase in TNFa and RANTES in the LPS group and the increase in IL-6 in the control group is different than what has been found in previous research and may be due to differences in the regulatory mechanisms of TNFα and IL-6 or due to differences in the dose dependent response to LPS exposure

Convocation Year

2017

Convocation Season

Fall

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