Longitudinal Comparison of Enzyme- and Laser-Treated Intervertebral Disc by MRI, X-Ray, and Histological Analyses Reveals Discrepancies in the Progression of Disc Degeneration: A Rabbit Study

Abstract

Regenerative medicine is considered an attractive prospect for the treatment of intervertebral disc (IVD) degeneration. To assess the efficacy of the regenerative approach, animal models of IVD degeneration are needed. Among these animal models, chemonucleolysis based on the enzymatic degradation of the Nucleus Pulposus (NP) is often used, but this technique remains far from the natural physiopathological process of IVD degeneration. Recently, we developed an innovative animal model of IVD degeneration based on the use of a laser beam. In the present study, this laser model was compared with the chemonucleolysis model in a longitudinal study in rabbits. The effects of the treatments were studied by MRI (T2-weighted signal intensity (T2wsi)), radiography (IVD height index), and histology (NP area and Boos' scoring). The results showed that both treatments induced a degeneration of the IVD with a decrease in IVD height and T2wsi as well as NP area and an increase in Boos' scoring. The enzyme treatment leads to a rapid and acute process of IVD degeneration. Conversely, laser radiation induced more progressive and less pronounced degeneration. It can be concluded that laser treatment provides an instrumental in vivo model of slowly evolving IVD degenerative disease that can be of preclinical relevance for assessing new prophylactic biological treatments of disc degeneration.

Figure 1

MRI images (T2-weighted midsagittal) and analysis of rabbit lumbar spines. Intervertebral discs of 1-year-old rabbits were treated according to either the enzyme technique (enzyme) or the laser procedure (laser) as described in Materials and Methods. Untreated IVDs were used as an internal control. (a) After the indicated times, a magnetic resonance imaging (MRI) scan of the spine was performed. Representative MRI scans are shown. Bar: 1 cm. (b) T2-weighted signal intensity was measured after the indicated times as described in Materials and Methods. ^∗ P < 0.05 as compared with day 0. Values are expressed as mean ± SEM.

Figure 2

X-ray images and analysis of rabbit lumbar spines. Intervertebral discs of 1-year-old rabbits were treated according to either the enzyme technique (enzyme) or the laser procedure (laser) as described in Materials and Methods. Untreated IVDs (control) were used as an internal control. (a) After the indicated times, X-ray images of spines were taken. Representative X-ray images are shown. Bar: 1 cm. (b) Decrease in IVD height (%) was measured after the indicated times as described in Materials and Methods. ^∗ P < 0.05 as compared with day 0. Values are expressed as percentage of decrease ± SEM.

Figure 3

Histological analysis of rabbit intervertebral discs (IVDs) using hematoxylin phloxine safran (HPS) staining. IVDs of 1-year-old rabbits were treated according to either the enzyme technique (enzyme) or the laser procedure (laser) as described in Materials and Methods. IVDs were processed for histological analysis at days 0 (untreated IVD from two rabbits as internal control) and 7 and 90 days after treatment as described in Materials and Methods. Representative hematoxylin phloxine safran is shown at days 0, 7, and 90. For clarity reasons and regarding similarities in follow-up, only the control at day 0 is illustrated. Bar: 5 mm for macroscopic section image and 500 μm for magnification image.

Figure 4

Histological analysis of rabbit intervertebral discs (IVDs) by Alcian blue/PAS staining (BA-PAS). IVDs of 1-year-old rabbits were treated according to either the enzyme technique (enzyme) or the laser procedure (laser) as described in Materials and Methods. IVDs were processed for histological analysis at days 0 (untreated IVD from two rabbits as internal control) and 7 and 90 days after treatment as described in Materials and Methods. Representative Alcian blue/PAS staining is shown at days 0, 7, and 90. For clarity reasons and regarding similarities in follow-up, only the control at day 0 is illustrated. Bar: 5 mm for macroscopic section image and 500 μm for magnification image.

Figure 5

Evaluation of NP area and Boos' scoring for rabbit intervertebral discs (IVDs). IVDs of 1-year-old rabbits were treated according to either the enzyme technique (enzyme) or the laser procedure (laser) as described in Materials and Methods. Untreated IVDs from two rabbits were used as an internal control. NP area (a) and Boos' scoring (b) were determined for the different conditions at 0, 7, and 90 days as described in Materials and Methods. ^∗ P < 0.05 compared to control. Values are expressed as mean ± SEM.