Cervical Intervertebral Disc Degeneration Induced by Unbalanced Dynamic and Static Forces: A Novel In Vivo Rat Model
University of Rochester, Rochester, New York, United States Spine
(Impact Factor: 2.3).
07/2006; 31(14):1532-8. DOI: 10.1097/01.brs.0000222019.84095.23
Establishment of a novel in vivo animal model of cervical spondylosis.
To investigate apoptotic, degenerative, and inflammatory changes occurring in the cervical intervertebral discs of rats.
Cervical degeneration occurs as the result of imbalance of both static and dynamic spinal stabilizers. The disc degeneration that occurs is characterized by increased local inflammation and increased apoptosis of intervertebral disc cells.
By excising the paraspinal musculature and posterior cervical spinal ligaments of rats, both static and dynamic cervical stabilizers were disrupted. The resultant biomechanical imbalance resulted in biochemical and histologic changes, which were characterized by light microscopy, electron microscopy, immunostaining, enzyme-linked immunosorbent assay, polymerase chain reaction, and in situ hybridization.
Histologic analysis showed characteristic degenerative changes of the intervertebral discs and vertebral endplates following surgery. Ultrastructural examination revealed apoptotic changes, which were verified by immunostaining. Instability also resulted in significant up-regulation of inflammatory factors, as shown by enzyme-linked immunosorbent assay, polymerase chain reaction, and in situ hybridization.
By creating static and dynamic posterior instability of the cervical spine, this novel model of cervical spondylosis results in rapid intervertebral disc degeneration characterized by increased apoptosis and local inflammation, such as that seen clinically.
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- "Abnormal stresses caused by spinal instability have been previously shown to precipitate apoptosis of disc cells and endplate chondrocytes[ 28 ]. Increased apoptosis engenders decreased production of extracellular matrix proteins, which in turn reduce the osmotic forces within the nucleus pulposus, prompting loss of water content and structural integrity, and degeneration of intervertebral discs. Normal disk height is considerably reduced, and subluxation of the joint surface occurs-a phase marked by an increase of abnormal spinal segment movement and potential involvement of spinal cord and nerve roots. Vertebral column stability is maintained by the integrity of all the anterior elements of support plus one posterior element, or conversely, all the posterior and one anterior element. "
Available from: ncbi.nlm.nih.gov
- "Degenerative changes in the IVD can lead to nerve root compression, resulting in radiculopathy (Hacker and Miller, 2003; Chen et al., 2005). To investigate whether electroacupuncture protected AF cells from apoptosis caused by cervical IVD degeneration , we established a rat model of cervical intervertebral disc degradation induced by the application of unbalanced dynamic and static forces, as described in the literature (Wang et al., 2006). TUNEL staining showed that EA treatment could reduce the signs of apoptosis after sur- gery. "
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ABSTRACT: The purpose of this study was to investigate whether treatment with electroacupuncture (EA) inhibited mitochondria-dependent apoptosis in annulus fibrosis (AF) cells in a rat model of cervical intervertebral disc degradation induced by unbalanced dynamic and static forces. Forty Sprague-Dawley rats were used in this study, of which 30 underwent surgery to induce cervical intervertebral disc degradation, 10 rats received EA at acupoints Dazhui (DU 14) and Shousanli (LI 10). TUNEL staining was measured to assess apoptosis in AF cells, immunohistochemistry was used to examine Bcl-2 and Bax expression, colorimetric assays were used to determine caspase 9 and caspase 3 activities and RT-PCR and western blotting were used to assess the mRNA and protein expression of Crk and ERK2. Treatment with EA reduced the number of AF-positive cells in TUNEL staining, increased Bcl-2-positive cells and decreased Bax-positive cells in immunohistochemical staining, significantly inhibited the activation of caspases-9 and -3, and enhanced the mRNA and protein expression of Crk and ERK2. Our data show that EA inhibits AF cell apoptosis via the mitochondria-dependent pathway and up-regulates Crk and ERK2 expression. These results suggest that treatment with may be a good alternative therapy for preventing cervical spondylosis.
Available from: Lei-Sheng Jiang
- "The rats were randomly divided into six groups, with eight rats in each group. To develop lumbar IVDD, rats in three of six groups received a surgical procedure, which is a modified version established by Wang et al. (2006a) and leads to unbalanced dynamic and static forces of the lumbar spine. Briefly, through a dorsal medial approach under general anesthesia, the sacrospinal muscles, spinous processes , supraspinous ligaments, interspinous ligaments, posterolateral 1/2 of bilateral zygapophysial joints of the lumbar spine were removed. "
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ABSTRACT: Intervertebral disc cell apoptosis occurs through either death receptor or mitochondrial pathway, but whether disc cell apoptosis is also mediated by the endoplasmic reticulum (ER) pathway remains unclear. The objective of this study was to investigate whether ER and mitochondria are co-involved in disc cell apoptosis and intervertebral disc degeneration (IVDD) in rats. Forty-eight rats were used for in vivo experiments. IVDD was characterized by X-ray and histomorphology examination, disc cell apoptosis was detected by TUNEL staining, and the co-involvement of ER and mitochondria in apoptosis was determined by immunohistochemical staining for GRP78, GADD153, caspase-12, and cytochrome C. Additional eight rats were used for annular cell isolation and culture. After sodium nitroprusside treatment, annular cell apoptosis was observed morphologically and quantified by flow cytometry; the expression of biomarkers of ER stress and mitochondrial dysfunction were analyzed by reverse transcriptase PCR (RT-PCR), fluorescence double labeling, and Western blot; and mitochondrial membrane potential was detected by 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbo cyanine iodide (JC-1) staining. Finally, NS3694 and Z-ATAD-FMK were employed to inhibit the formation of apoptosome complex and the activation of caspase-12, respectively, and apoptotic incidence and caspase-9 activity were assayed. We found that IVDD, induced by unbalanced dynamic and static forces in the rats, was accompanied by increased disc cell apoptosis and enhanced expression of GRP78, GADD153, caspase-12, and cytochrome C. Annular cell apoptosis induced by sodium nitroprusside was confirmed by morphologic observation and flow cytometry. With increased apoptosis, the expression of GRP78, GADD153, and caspase-12 upregulated, mitochondrial membrane potential decreased, and accumulation of cytochrome C in the cytosol manifested. Furthermore, NS3694 and Z-ATAD-FMK dramatically suppress annular cell apoptosis and caspase-9 activity. In conclusion, disc cell apoptosis mediated simultaneously by ER and mitochondria plays a potent role in IVDD.
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