Qin Bian

Cervical Intervertebral Disc Degeneration Induced by Unbalanced Dynamic and Static Forces: A Novel in Vivo Rat Model

Study Design. Establishment of a novel in vivo animal model of cervical spondylosis. Objective. To investigate apoptotic, degenerative, and inflammatory changes occurring in the cervical intervertebral discs of rats. Summary of Background Data. 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. Methods. 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. Results. 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. Conclusions. 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.

Oleanolic acid exerts an osteoprotective effect in ovariectomy-induced osteoporotic rats and stimulates the osteoblastic differentiation of bone mesenchymal stem cells in vitro.

ObjectiveOleanolic acid (OA) and its glycosides have been reported to prevent bone loss by inhibiting the formation of osteoclasts. However, because bone formation and resorption are balanced processes in bone metabolism, no studies have described the effect of OA on osteogenesis. The aim of the present study was to evaluate the osteoprotective effect of OA in rats with ovariectomy (OVX)-induced osteoporosis and to search for the molecular targets of OA in bone mesenchymal stem cells (bMSCs). MethodsTwo-month-old female mice that underwent OVX were treated with OA (20 mg/kg a day). After 2 weeks and after 3 months, bone mass was evaluated by micro-CT, morphometry, and immunohistochemical detection. In addition, the expression of 256 genes was measured via microarray and confirmed by real-time reverse transcription–polymerase chain reaction. The effects of OA on the activities of bMSCs were also observed in vitro using alkaline phosphatase and cell proliferation assays. ResultsMicro-CT displayed only a tendency for bone loss at 2 weeks but a decrease in bone mass at 3 months after OVX. OA treatment increased osteoblast number, increasing osteocalcin and runt-related protein 2 protein levels in vivo and facilitating the osteoblastic differentiation of bMSCs in vitro at doses of 10−6 and 10−5 M. Gene expression profile analysis revealed that OVX caused a marked dysregulation of gene expression, especially at 2 weeks, some of which was rescued by OA. Few of these genes overlapped, but their functions were involved in the Notch signaling pathway between two phases of the osteoporotic process. ConclusionsOA exerts an osteoprotective effect in OVX-induced osteoporotic rats and stimulates the osteoblastic differentiation of bMSCs in vitro. The molecular mechanism of this effect might be related to the Notch signaling pathway and requires further investigation.

Leg amputation accelerates senescence of rat lumbar intervertebral discs.

Study Design. Several senescence biomarkers were observed to investigate cell senescence in degenerative intervertebral lumbar discs of foreleg-amputated rats. Objective. To determine if cell senescence is accelerated in degenerative intervertebral lumbar disc cells in an upright-rat model. Summary of Background Data. Cellular senescence was accelerated in human and sand rat degenerative intervertebral disc (IVD) cells. Repeated use of upright posture by rats contributed to degenerative disc changes. No convincing evidence of cell senescence was observed in the lumbar disc of the foreleg amputated rat. Methods. The forelimbs of 20 rats were amputated at 1 month of age such that they maintained an upright stance; rats were housed in custom-made cages. Nonamputated rats, also 1 month of age, were kept in regular cages and served as a control group. The lumbar IVDs were harvested from rats in 2 groups, at 5 or 9 months following amputation. Senescence-associated-&bgr;-galactosidase-positive staining was used to detect cell senescence. p16INK4a and p27KIP were assessed by immunohistochemistry analysis. Total RNA isolated from these samples was used to measure the gene expression of p16INK4a, RB, cyclin D1, CDK4, PTEN, p27KIP, p19ARF, p21, TERT, and RAGE by real-time polymerase chain reaction assay. Results. The highest levels of SA-&bgr;-GAL activity were detected in 9-month amputated rats. Quantitative immunohistochemical analysis showed that there were highest rates of p16INK4a and p27KIP protein expression in the cartilage endplate and anulus fibrosus of 9-month amputated rats. The mRNA levels of p16INK4a, RB, PTEN, p27KIP, p19ARF, and RAGE were upregulated. The increased cyclin D1 mRNA level was statistically significant only at the ninth month following amputation; CDK4 and TERT mRNA levels were downregulated to a similar extent at both points compared with nonamputated controls. mRNA expression of p21 was significantly downregulated. Conclusion. Accelerated cell senescence was associated with forelimb amputation that causes abnormal loading in rat lumbar IVDs.

The osteoprotective effect of psoralen in ovariectomy-induced osteoporotic rats via stimulating the osteoblastic differentiation from bone mesenchymal stem cells.

Objective Psoralea corylifolia extract has been reported to promote bone formation in osteoporotic animals. Psoralen (PSO), a flavonoid glycoside, as the active component of P corylifolia L, is effective in increasing new bone-forming osteoblasts in parietal bone defects. However, the effect and molecular mechanisms of PSO on bone mesenchymal stem cells (bMSCs) in the osteoporotic state are widely unknown. This study was designed to evaluate the osteoprotective effect of PSO in ovariectomy (OVX)-induced rats and to seek possible molecular mechanisms of PSO in bMSCs. Methods We observed the osteogenic effect of PSO (3-month treatment) on osteoporotic rat models induced by OVX via testing bone densitometry, histomorphometries, and immunohistochemistry in vivo. Alkaline phosphatase staining and colony-forming unit-fibroblast and colony-forming unit-adipocyte assays were performed to evaluate the differentiation potential of bMSCs ex vivo. In addition, the molecular targets of PSO in bMSCs were detected by stem cell microarray analysis of 256 genes and confirmed by real-time reverse transcription–polymerase chain reaction. Results Micro-CT morphometry analysis showed that PSO significantly improved bone mass indicators including increased trabecular thickness and decreased trabecular space. Meanwhile, PSO elevated the well-known osteogenic marker osteocalcin level in OVX-induced osteoporotic rats. Next, in ex vivo studies, we revealed that PSO facilitated alkaline phosphatase staining and increased the colony-forming unit-fibroblasts. Based on gene expression profile analysis, we screened a set of genes dysregulated in OVX but reversed by PSO treatment. These genes were highly enriched in the Notch signaling pathway, which was documented to play a role in bMSC differentiation. Conclusions Our findings show that PSO promotes bone mass in OVX-induced osteoporotic rats. This effect of PSO is highly related to the stimulation of differentiation of bMSCs to osteoblasts.

The expression of osteoprotegerin is required for maintaining the intervertebral disc endplate of aged mice

OBJECTIVE Human chondrocytes and annulus fibrosus cells of intervertebral disc (IVD) express osteoprotegerin (OPG), but the effect of OPG on the pathogenesis of IVD degeneration remains unknown. Here we assessed the phenotype change of IVD in OPG(-/-) mice. METHODS The IVDs from 12-, 20-, and 28-week-old OPG(-/-) mice and WT controls were subjected to histologic analyses including TRAP staining for osteoclasts, immunostaining for OPG and type I collagen protein expression, and TUNEL staining for apoptosis. The IVD tissues were also subjected to real time RT-PCR for mRNA expression of genes for osteoblast-osterix, ALP, and osteocalcin; for osteoclasts-trap, rank, mmp9 and cathepsin K, and for chondrocytes-aggrecan, mmp13 and Col10. RESULTS OPG protein expresses at the cells of endplate cartilage and annulus fibrosis in IVDs of WT mice. Compared to WT mice, OPG(-/-) mice developed aging related cartilage loss and bony tissue appearance at the endplate. Stating from 20 weeks of age, IVDs from OPG(-/-) mice expressed significantly increased mmp13 and Col10 levels, which is associated with increased osteoblast number and elevated expression of osteoblast marker genes. Furthermore, TRAP+ osteoclasts were presented in the endplate cartilage of OPG(-/-) mice. These osteoclasts localized adjacently to and erosion into the cartilage. Increased expression of RANK, mmp9 and cathepsin k was detected in OPG(-/-) IVDs. CONCLUSIONS OPG at IVD plays an important role for maintaining the integrity of endplate cartilage during aging by preventing endplate cartilage from osteoclast-mediated resorption.

Prolonged upright posture induces calcified hypertrophy in the cartilage end plate in rat lumbar spine.

Study Design. Both forelimbs of rats were amputated and these rats were kept in the custom-made cages to keep prolonged and repeated upright posture. Changes of bone were observed in the lumbar vertebrae at three different time points after the surgery. Objective. To investigate the effect of prolonged and repeated upright posture on the cartilage end plate of rat lumbar vertebrae. Summary of Background Data. Previous studies show calcified hypertrophy is related to mechanical stress, but there are no clear evidences to indicate whether or not long-term and repeated assumption of the upright posture could result in calcified hypertrophy in cartilage end plate of rat lumbar spine. Methods. The forelimbs of 30 rats were amputated when they were 1 month old. These rats were kept in the custom-made cages and were forced to stand upright on their hind-limbs and tails to obtain water and food. Normal rats of the same ages kept in regular cages were used as control. The rats were killed at 5, 7, and 9 months after the surgery and lumbar vertebrae samples were harvested for micro-CT, histologic, and immunohistochemical studies. Total RNA isolated from these samples were used for real-time RT-PCR of type X collagen (Col10&agr;1), vascular endothelial growth factor (VEGF), and transforming growth factor &bgr;1 (TGF-&bgr;1). Results. Micro-CT showed increased inner part of cartilage end plate. Histologic revealed peripheral hypertrophy of disc after the surgery. Immunostaining and real-time RT-PCR showed increased protein and mRNA expression of type X collagen, VEGF, and TGF-&bgr;1. Conclusion. Prolonged upright posture induces cartilage end plate calcification and hypertrophy in rat lumbar spine.

Prolonged and repeated upright posture promotes bone formation in rat lumbar vertebrae.

Study Design. Both forelimbs of rats were amputated and these rats were kept in the custom-made cages to keep prolonged and repeated upright posture. Changes of bone were observed in the lumbar vertebrae at three different time points after the surgery. Objective. To investigate the effect of prolonged and repeated upright posture on bone formation of rat lumbar vertebrae. Summary of Background Data. Previous studies show long-term and repeated load-induced bone formation, but there are no clear evidences to indicate whether or not long-term and repeated assumption of the upright posture could result in bone formation at rat lumbar vertebrae. Methods. The forelimbs of 30 rats were amputated when they were 1 month old. These rats were kept in the custom-made cages and were forced to stand upright on their hindlimbs and tails to obtain water and food. Normal rats of the same ages kept in regular cages were used as control. The rats were sacrificed at 5, 7, and 9 months after the surgery and lumbar vertebrae samples were harvested for micro-computed tomographic (CT), histological, and immunohistochemical studies. Total RNA isolated from these samples were used for real-time polymerase chain reaction of type I collagen (Col1&agr;2), type X collagen (Col10&agr;1), transforming growth factor-&bgr;1 (TGF-&bgr;1) and its related nuclear transcript factor runt-related transcription factor 2 (Runx2), as well as the biomarker of angiogenesis and vascular invasion, which is also a prerequisite for endochondral bone formation: vascular endothelial growth factor (VEGF). Results. Micro-CT and histological studies showed increased trabecular bone density and increased osteoblast quantities of lumbar vertebrae after surgery. Immunostaining revealed increased protein expression of type I collagen, type X collagen, TGF-&bgr;, and VEGF. Real-time polymerase chain reaction showed upregulated expression of Col1&agr;2, Col10&agr;1, VEGF, TGF-&bgr;1, and Runx2 mRNA. Conclusion. Upright posture induces bone acquisition in the rats’ lumbar spine, primarily through the mode of the endochondral ossification, which is associated with increased loading, activated VEGF, and TGF-&bgr;1 signaling.

Establishment of a rat model of cervical syndrome with kidney deficiency

OBJECTIVE To establish a rat model of cervical syndrome (CS) with kidney deficiency. METHODS A group of 30 three-month-old female Sprague-Dawley rats were randomly divided into normal control group, CS group and CS with kidney deficiency group (combined group), with 10 rats in each group. Rats in the normal control group received no treatment, rats in the CS group underwent only resection of cervical muscles and ligaments as unbalanced dynamic and static animal model, and rats in combined group underwent resection of both cervical muscles and ovaries as kidney deficiency model. Serum and cervical intervertebral discs were collected. Kidney deficiency was determined by observing the morphologic changes of uterus and appendages, detecting the weight of uterus and appendages and the content of serum estradiol (E(2)). The degeneration of intervertebral discs was determined by detecting the histopathology, the expressions of type II collagen and type X collagen proteins, and the expressions of aggrecan-1 (Agc1), type II procollagen gene (Col2a1), matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNAs. RESULTS Compared with those in the normal control group and CS group, the rats in the combined group were noted with the uterus atrophied, the caliber of oviduct thinned, the weight of uterus and appendages diminished obviously, the content of serum E(2) decreased, cervical intervertebral disc degenerated more seriously, type II collagen protein expression decreased, type X collagen protein expression increased, Agc1 and Col2a1 mRNA expressions in intervertebral disc decreased, and the MMP-13 mRNA expression increased. CONCLUSION The rat model of CS with kidney deficiency is established. Kidney deficiency can aggravate cervical intervertebral disc degeneration.

Mechanism of Yiqi Huayu Bushen Recipe in treating cervical syndrome with kidney deficiency in rats

OBJECTIVE To research the action mechanism of Yiqi Huayu Bushen Recipe (YHBR), a compound of traditional Chinese herbal medicine, in treating cervical syndrome (CS) with kidney deficiency in rats. METHODS A total of 30 three-month-old female Sprague-Dawley rats were randomly divided into normal control group, CS with kidney deficiency model group (untreated group) and YHBR group, with 10 rats in each group. Rats in the normal control group received no treatment, while rats of the other two groups underwent resection of both cervical muscles and ovaries to establish the model of CS with kidney deficiency. Three months after surgery, rats in the YHBR group were intragastrically administered YHBR for one month. Another one month later, all rats were sacrificed. The content of serum estradiol (E2) was detected by radio-immunoassay; contents of plasma cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were tested, and the ratio of cAMP/cGMP was also calculated. Hemorheology was detected by Weissenbergs method; expression of alpha-granular membrane protein (CD62p) was detected by flow cytometry; HE staining was used to detect the histopathology of cervical intervertebral disc degeneration; type II collagen protein was detected by immunohistochemistry and aggrecan-1 (Agc1), type II procollagen gene, Col2a1, tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-13 (MMP-13) mRNAs were detected by fluorescent quantization polymerase chain reaction. RESULTS Compared with the untreated group, rats in the YHBR group showed an obvious increase in serum E2 content (P<0.05), and an increase in plasma cAMP and cGMP content without significant difference; hemorheological parameters and percentage of CD62p expression were significantly decreased in the YHBR group (P<0.05, P<0.01). YHBR could improve the degeneration of cervical intervertebral discs, decrease the Miyamoto scores (P<0.05), and increase the type II collagen. The expressions of Agc1, Col2a1 and TIMP-1 mRNAs were significantly increased and MMP-13 mRNA significantly decreased in YHBR group (P<0.05, P<0.01). CONCLUSION YHBR may improve CS with kidney deficiency and delay the degeneration of cervical intervertebral disc by regulating the immune-metabolism system, coagulation system and endocrine system.

Ligustrazine Inhibits Cartilage Endplate Hypertrophy via Suppression of TGF-β1.

CEP hypertrophy is one of the characteristics of intervertebral disc degeneration (IDD). LIG exerts a protective effect on IDD in animal model. The effect of LIG on CEP hypertrophy is further investigated in the present study. Cells were isolated from hypertrophic samples obtained from patients during vertebral fusion surgery. Cellular proliferation and the expression of type I collagen (Col I) and TGF-β1 were tested. In the bipedal rats, the edges of the CEP and the sizes of noncartilaginous outgrowth, as well as the expression of osteogenic markers, Col1a, ALP, Runx2, and TGF-β1, were detected. Within two passages, the condensed hypertrophic CEP cells exhibited osteogenic capacity by bony-like nodules and ALP positive staining, along with increased expression of Col I and TGF-β1. LIG inhibited proliferation of CEP cells and downregulated the expression of Col I and TGF-β1 in vitro. Furthermore, LIG attenuated CEP hypertrophy on the lumbar spine of bipedal rats by reducing Col1a, ALP, Runx2, and TGF-β1 mRNA expression and TGF-β1 distribution in vivo. We concluded LIG exerted a preventive effect on CEP hypertrophy via suppression of TGF-β1 levels. This information could be used to develop alternative therapeutic methods to treat spinal CEP hypertrophy.