Establishment Of A Novel In Vivo Rabbit Model Of Disc Degeneration Through Axial Loading On The Spine And The Mechanism Exploration

Intervertebral disc degeneration(IDD) is thought to be the root cause of numerous musculoskeletal disorders of the spine, and its high treatment costs and impact on work performance leads to incalculable distress and tremendous economic loss. Although current surgical and non-surgical interventions may relieve discogenic pain, they do not restore disc function The causes of IDD are multifactorial, and there is no clear understanding of its pathophysiology and pathogenesis. Animal models of IDD play a crucial role in clarifying its pathological mechanisms and providing a means for evaluating new drug therapies and other treatment modalities.In the present study, we attempted to establish a simple in vivo rabbit model of early IDD through cumulative noninvasive axial loading on the spine. The axial load was achieved by placing the rabbit into a tube designed to maintain the animal’s an upright posture, and then applying an external load axially. This model simulated the IDD process in humans, and thus might constitute a suitable model for use in further studies.Part I. Noninvasive cumulative axial load-induced intervertebral disc degeneration- a novel in vivo rabbit modelBackground and objective: Intervertebral disc degeneration(IDD) is thought to be the root cause of numerous musculoskeletal disorders of the spine. However, there is no clear understanding of its pathophysiology. Animal models of IDD play a crucial role in clarifying its pathological mechanisms and providing a means for evaluating new treatment modalities. However, no ideal animal model of IDD currently exists. The purpose of this study is to establish a novel in vivo rabbit model of early IDD that can better simulate the IDD process in humans.Methods: Twenty-four male New Zealand white rabbits were randomly assigned to two study groups(experimental group and a control group). Each rabbit in the experimental group(n = 12) was placed into a plastic tube specially designed to maintain the animal in an upright posture. A specially designed device was used to monitor the friction force between the rabbit and the tube in the experimental group. Following a 1-week adaptation period, during which rabbits in the experimental group were confined in tubes for 4-6 hours a day, a collar composed of Styrofoam and plummets, weighing 600 g, was placed onto the neck of each experimental rabbit(Figure 1b) to begin the formal experiment. Rabbits in the experimental group were confined in their tubes for 6 hours every day(3 hours in the morning and 3 hours in the afternoon). When not confined in their tubes, the rabbits in the experimental group were regularly fed in their cages. Rabbits in the control group(n = 12) were regularly fed in their cages throughout the entire experiment. The body weights of all of rabbits in both groups were measured every four weeks to assess the general condition of each animal.Prior to the experiment, lateral radiographs were taken of each rabbit in the experimental group while the animal was sitting in the tube and maintained in an upright position to observe its bony positions in the tube. Next, lumbar lateral radiographs were taken of all rabbits in the experimental and control groups in the lateral decubitus position under general anesthesia. The differences in disc height as measured in the upright and lateral decubitus positions in the experimental group were analyzed by measuring the disc heights on the radiographs. At 4, 8, and 12 weeks after beginning the experiment, lumbar lateral radiographs were taken in the lateral decubitus position for both the experimental and control groups to monitor signs of disc degeneration. To evaluate differences between the different levels of lumbar discs, the data from 3 segments(discs L2-3, L4-5, and L6-7) were analyzed.Prior to beginning the experiment, and at 4, 8, 12 and 14 weeks after starting the experiment, all animals in both groups underwent an MRI scan. From 12 to 14 weeks, the rabbits in the experimental group did not receive any treatment, and were fed the same as the control group while allowing time for disc recovery. The hydration status of the nucleus pulposus(NP) was graded using a modified Schneiderman’s score. To evaluate differences among the different levels of lumbar discs, the data from 3 segments(discs L2-3, L4-5, and L6-7) were analyzed.At 14 weeks, all animals in both groups were sacrificed by injection with pentobarbital. The NPs of L5-6 were obtained for analysis with quantitative real-time PCR analysis. Real-time PCR was performed according to standard procedure. The log2(Δ-Δ-Ct) was performed by comparing the mean experimental Δ-Ct to the mean control animal Δ-Ct. The mean value of the three results for each sample was used in the final analysis.Following sacrifice, disc segment L6-7, which showed the most obvious degenerative changes on MRI examination, was removed using a mini electronic saw for later histologic analysis. The specimens were immediately fixed in 10% formaldehyde for one week, decalcified using Perenyi’s fluid for 6 days, and washed with running water for 12 hours to eliminate the acid. Tissue samples were then cut midsagittally, embedded in paraffin, sectioned to 5-μm thickness, stained with hematoxylin and eosin(H&E), and examined by light microscopy. Additional sections were stained with picrosirius red and examined by polarized light microscopy to observe changes in collagen fibers which may have occurred during disc degeneration. In human age-related disc degeneration, the diameters of the central fluidic regions of degenerated discs(the ?functional NP’) have been reported to decrease. To quantify such changes, the diameter of the functional NP and widths of the anterior annulus fibrosus(AAF) and posterior AF(PAF) in the midsagittal plane images of gross pathology were measured using Photoshop version 10.0(Adobe) software.Results: Two rabbits in the experimental group died during treatment and their data were not included in the final study results. The remaining 10 rabbits in the experimental group demonstrated good tolerance to their treatment. During the 14-week experiment, the mean body weight of animals in the experimental and control groups increased from initial values of 2.56 kg and 2.62 kg, respectively, to final values of 3.63 kg and 3.81 kg, respectively. No significant difference was observed between body weights in the experimental group and control group at any time point. The mean percentage of the friction force between the rabbit and the tube was 19% at the beginning(time 0), decreased to 11.1% at 20 min, and then fluctuated around that level during the following 40 minutes.The lateral radiograph of the rabbit in the experimental group which had been placed into the tube and maintained in an upright position showed that there was an obvious kyphotic curvature on the lower lumbar spine, and the disc height seemed much smaller than the height in the image of the same rabbit in the lateral decubitus position. All three segments showed obvious differences between the disc heights in the upright and lateral decubitus positions. In L2-3, the mean disc height in the upright position was 74.4% of that in the lateral decubitus position. This ratio dropped to 60.7% for L4-5 and 46.5% for L6-7. Significant difference on disc height reduction was also observed among different segments.Serial lateral radiographs of rabbits in the control and experimental groups obtained before treatment and at 4, 8, and 12 weeks after treatment showed that there was a steady decrease in DHI values with time for both the control and experimental groups. However, no significant difference was observed between the two groups at any lumbar level or any time points.Serial MRI scans of the lumbar spinal areas of animals in the experimental and control groups showed that animal in the experimental group present a progressive decrease in the signal intensities of their lumbar discs over the 14-week period, and especially in the lower lumbar discs. In contrast, NP signal intensities in the control group decreased more slowly during the same period. Grayscale values for the NP areas decreased gradually in both groups over time, especially in the first eight weeks. In disc L2-3, no significant difference was observed between the experimental and control groups at any time point. In disc L4-5, a significant difference between the two groups was observed at 12 weeks, and was maintained over the following 2-week recovery period. In disc L6-7, a significant difference between the two groups was observed at 8 weeks, and continued until the end of the experiment.The result of Real-time PCR showed that the collagen type I α m RNA expression was significantly increased(3.06-fold), whereas collagen type II α and aggrecan m RNA expression were significantly decreased(0.35-fold and 0.37-fold, respectively) in the experimental group as compared with the control group.Histologic examination showed significant difference between the control and experimental group. In the control group, H&E staining showed clear demarcation between the NP and AF, and scattered clusters of NP cells within an abundant gelatinous matrix. Picrosirius red staining revealed a nearly intact AF, with a normal pattern of fibrocartilage lamellas and a well-defined border between the AF and NP. In the experimental group, H&E staining showed a large increase in fibrocartilage lamellas in the inner border of the posterior AF, resulting in a significantly decreased gelatinous NP area. The increased inner lamellas had an appearance typical of fibrocartilage, and originated from cartilage endplates, while the outer border of the posterior AF had lost its fibrocartilage lamella structure and resembled hyaline cartilage. Picrosirius red staining showed newly formed birefringent collagen fibers infiltrating the NP from the margins of the cartilage endplates. The thickened posterior AF lacked the normal structure of fibrocartilage lamellas, and showed an uneven staining pattern. The newly formed fibrocartilage lamellas were integral components of the inner AF, whereas the size of the hydrostatic NP area was significantly decreased. Measurements showed that the mean widths of the functional AAF and PAF had increased by 26.5% and 37.6%, respectively,in the experimental group as compared with the control group, whereas the mean diameter of the functional NP had decreased by 14.5% in the experimental group as compared with the control group.Conclusion: The upright posture combined with noninvasive external loading method used in this study induced accelerated degenerative changes in rabbit lumbar discs from the result of MRI, real-time PCR and histology. These mild degenerative changes closely resembled those which occur in the early phase of disc degeneration in humans, suggesting the model’s usefulness in future studies.Part II. The impact of cumulative axial load on intervertebral disc degeneration-insights from a rabbit modelObjective: To observe the process of disc degeneration and explore the mechanism of IDD based on the cumulative axial load-induced rabbit model.Methods: 20 female New Zealand white rabbits(4month old, weighing 2.3~2.7 kg) were randomly divided into two groups. The experimental group(EG, n=12) were put into specially designed plastic tubes to make the rabbits maintain an upright posture and loaded with a collar weighing 600 g as described above. The EG rabbits were put into the tubes for 6 hours every day(3 hours in the morning and 3 hours in the afternoon). Apart from the “working time” in the tubes, the EG rabbits were regular feed in their cages. The control group(CG, n=8) were regular feed in their cages throughout the whole experiment. Additional 4 old animals of 24 months old were regular fed as the control group. The body weights of all groups of rabbits were measured every four weeks to assess the general condition of the animals.Before the experiment, MRI of all animals were taken under anesthesia by the intramuscular injection of xylazine(3 mg/kg) and ketamine(40 mg/kg).Then at 6 weeks, 12 weeks and 24 weeks after the experiment, all the animals or the remaining of all animals were taken MRI examination. Hydration status of the NP was graded with a modified Schneiderman’s score. “Silver PACS-ADViewer” software was used to determine the greyscale of the NP of the interested discs and the cerebrospinal fluid at the same image on a T2-weighted sagittal MRI scan. The greyscale of the NP was normalized against that of the cerebrospinal fluid, which was given an arbitrary value of one. To evaluate the difference among the different levels of lumbar discs, the data from 3 discs were analyzed.After 12 weeks of experimental treatments, 4 rabbits from EG and 4 rabbits from CG were killed by injection with an overdose of pentobarbital. The NPs of L5-6 were carefully removed by a close cut at the end plate and taking out only the gelatinous tissue under microscopic visualization. The NPs were immediately frozen in liquid nitrogen for quantitative real-time PCR analysis. At the end of the experiment(24 weeks after treatment), 4 rabbits from CG, 5 remaining rabbits from EG, and 4 30-month old rabbits were killed, and the samples were treated the same as above. Total RNA was extracted from the NP. The expressions of the matrix genes collagen type I α, collagen type II α and aggrecan, the anabolic gene BMP7, the catabolic gene MMP3 and the anti-catabolic gens TIMP-1 were analyzed by real-time PCR. The housekeeping gene glyceraldehyde phosphate dehydrogenase(GAPDH) was used as the internal control. The differences between the mean Ct values of the gene of interest and the housekeeping gene were denoted as(Δ-Ct), The log2(Δ-Δ-Ct) was used to determine the relative quantitation of the gene expression of either the experimental or old group as compared with the control group.After the animals were sacrificed at 12 and 24 weeks, disc segment L6-7, which showed the most obvious degenerative changes on MRI examination, including approximately 1 mm of the adjacent vertebral bodies was obtained by a mini electronic saw for histological analysis. The tissues were cut midsagittally, embedded in paraffin, sectioned to 5-μm thickness, stained with hematoxylin and eosin(H&E) and examined with light microscopy. Sections were also stained with picrosirius red and examined with polarized microscopy to observe changes in the collagen fibers during the process of disc degeneration.All the data were statistically analyzed by using SPSS 18.0 software and displayed as mean ± standard deviation. The One- Way ANOVA was performed, and the Homogeneity of variance test was firstly used to determine the homogeneity of variances. If the variances were homogeneity, Least-significant difference(LSD) was performed. Otherwise, Tambane’s T2 was used. P<0.05 was commonly considered to be statistically significant.Results: During the experimental treatment, 3 animals in the experimental group died and their data was not included in the final results. The remaining 9 rabbits in the experimental group tolerated the entire treatment well. During the 24-week experiment, the mean body weight of the experimental group increased from 2.54 kg to 4.06 kg, and no significant difference was observed at any time point between the experimental group and the control group which increased its mean body weight from 2.45 to 4.23 kg.MRI scans of the lumbar spine of the 3 groups showed significant differences. A progressive decrease in the signal intensity of the lumbar discs of the experimental group was apparent over the 24-week period, especially for the lower lumbar discs. In contrast, the NP signal intensity of the control group decreased more slowly during the same period. However, the old group showed obvious low signal intensity, and did not change obviously over time. The grayscale measurement results showed that the grayscale value of the NP area decreased gradually for both control and experimental groups over time. In L6-7, significant difference between the two groups could be observed at 12 weeks and this disparity increased significantly at 24 weeks. In L2-3, significant difference between the two groups could only be observed at 24 weeks, and the disparity of the signal was not as obvious as L6-7.The gene expression in the experimental and control groups is expressed as a relative value to the control group. The gene expression in the EG and OG is expressed as a relative value to the CG sample. At 12 weeks, the expression level of the collagen type I α1 m RNA increased significantly in the EG than in the CG(2.3 fold). At 24 weeks, this disparity further increased(5.4 fold). However, no significant difference between CG and OG was observed(Figure 3a). The expression level of collagen type II α1 m RNA decreased significantly in the EG than that in the CG in both 12- and 24-week periods. Similar to the findings of collagen type II α1 m RNA expression, the average aggrecan m RNA expression level was lower at 12 and 24 weeks in the EG comparing with that in the CG, with significant difference only at 24 weeks(0.58 fold). No significant difference was observed between OG and CG for the above two genes expression. The expression of anabolic gene BMP7 in the EG increased significantly both in 12 and 24 weeks comparing with the CG(5.0-fold and 4.3-fold respectively). Significant difference was also observed between EG and OG. The mean expression of catabolic gene MMP3 also seemed increase obviously in the EG and OG comparing with that in the CG; However, no statistical significance was observed(Figure 3e). The expression of anti-catabolic gene TIMP-1 in the EG decreased significantly both in 12 and 24 weeks comparing with that in the CG(0.09-fold and 0.17-fold respectively). Significant difference was also observed between EG and OG.At 12 weeks, in the control group, H&E staining showed clear demarcation between the NP and the AF. Notochordal cell clusters could be seen scattered within an abundant myxoid matrix. Picrosirius red staining displayed a nearly intact annulus fibrosus with a normal pattern of fibrocartilage lamellas and a well-defined border between the AF and the NP. In the experimental group, H&E staining showed an obvious increase in fibrocartilage lamellas in the inner border of the posterior AF, thereby significantly decreasing the gelatinous NP area. The fibrocartilage fibers invaded into the NP from both the superior and the inferior cartilage endplate, leading to a complete division of the NP between the central and posterior peripheral regions. Increased small chondrocyte-like cells could be observed in the central area of the NP, while the notochordal cells mainly distributed near the endplates. Picrosirius red staining showed the newly formed birefringent collagen fibers invading the NP from the margins of the cartilage endplates and encircling the inner border of the inner AF. The thickened posterior AF lost the normal structure of fibrocartilage lamellas, and stained unevenly. At 24 weeks, increase of fibrocartilage lamellas in the inner border of the posterior AF could be observed in the control group, however, the infiltrated fibrocartilage lamellas was thin, and the structure of the NP and AF was not basically changed. In the experimental group, however, the NP tissue in the central region became smaller with direct invasion of fibrocartilage fiber bands from the CE. The fibrocartilage fiber bands increased the thickness of the AF structures of the IVD, and divided the NP tissue into small pieces. At 12 weeks, in the CG, the notochordal cell clusters could be seen scattered within an abundant myxoid matrix, no obvious small cartilage like cell could be observed. In the EG, increased small chondrocyte-like cells could be observed in the central area of the NP, while the notochordal cells mainly distributed near the cartilage endplates. At 24 weeks, there were small cartilage like cells in the central area of the NP in the CG, while the NP was mainly composed of NC clusters. In the EG, however, the amount of cells in the NP decreased significantly. Small cartilage like cells occupied the central part of the NP, while the NCs could only be seen in the periphery areas.Conclusion: our study demonstrated that chronic cumulative axial load on the spine could significantly accelerate lumbar intervertebral disc degeneration. The degenerative changes include progressive dehydration of the NP of the lower lumbar disc, the structural and cellular changes of the NP and AF, and the changes on the expressions of the matrix genes. During the process of cumulative axial load-induced disc degeneration, the expression of both anabolic gene BMP7 and catabolic gene MMP3 increased, while the anti-catabolic gens TIMP-1 decreased significantly. Theoretically, the degenerative changes described above might be a remodeling process of the intervertebral disc to adapt to the altered environment. On the basis of physiological mechanisms, our rabbit model partially resembles the human cumulative occupational lumbar load-induced disc degeneration, and thus could lead to new investigations of mechanisms of disc degeneration.