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A divergent efficient assembly of disubstituted 1,2,4-triazoles was established by cyclization of readily accessible N ′-nitro-2-hydrocarbylidene-hydrazinecarboximidamides with moderate to excellent yields under mild reaction conditions. This divergent synthetic strategy was achieved simply by varying the reaction conditions. Under acidic conditions, amino-1,2,4-triazoles were obtained by an intramolecular redox reaction involving the NO2 group. Control experiments and DFT studies revealed that this transformation proceeds via an intramolecular 1,3-hydride transfer pathway leading to HNO2 elimination. Under neutral conditions with water as the solvent, nitroimino-1,2,4-triazoles were obtained by oxidative intramolecular annulation under air. [ABSTRACT FROM AUTHOR]

Objective To reveal the function of CB during disc inflammation due to cumulative damage by detecting CB4R and CB-2R. Methods Wistar rats were randomly divided into experiment group and control group. The experiment group was prepared according to Jill A Ulrich method. Control group was prepared by skin cutting and suturing. Six rats were taken from each group on 3th day, 7th day, and 10th day after the operation. The intervertebral discs were collected. Morphology was observed. Expression of CB4R and CB-2R was detected using real-time PCR. CB-2R protein content was examined using Western blotting. Results Compared with the control group, the annulus rupture and nucleus bleeding were observed in experiment group in 10 days. The expression levels of CB receptor increased significantly in a time-dependent manner. CB-2R protein expression increased more significantly at the 3th day, 7th day, and 10th day in experiment group than that in control group (P < 0. 01). Conclusion CB is involved in the regulation of the rat bone and disc inflammatory reaction due to the cumulative damage. [ABSTRACT FROM AUTHOR]

Objective: To investigate the expression pattern of hypoxia-inducible factor 1alpha (HIF-1alpha) in experimental secondary spinal cord injury (SSCI) in rats and its potential effects on SSCI., Methods: A total of 66 SD rats (female or male) with weight (250 +/- 20) g were randomly divided into 3 groups: normal control group (group A, n = 6), pseudo injury group (group B, n = 6), and spinal cord injury (SCI) group (group C, n = 54). In group A, no treatment was given as normal control. In group B, only laminectomy was applied. In group C, laminectomy was applied and static compression model of SCI was built at T10 level. The expression of HIF-1alpha was measured with HE and immunohistochemical staining in groups A, B (1 hour after pseudo injury), and C (1, 3, 6, 12 hours and 1, 2, 3, 7, 14 days after SCI). Results All rats survived to the end of the experiment. HE staining showed that the spinal tissue of groups A and B were dense and the nucleus were round and big with light staining and clear nucleolus. The injured neuron at 1-12 hours after SCI of group C presented pyknosis and deep eosin staining. The swelling axon with bubbles and the disintegrated and disorganized medullary sheath in white matter appeared at 1-3 days after SCI. The hyperplasia of glial cells were obvious and gray matter cells were broken and apoptosis with cavities in injured spinal segment was observed at 7 and 14 days after SCI. Immunohistochemical staining showed that HIF-la was poorly expressed in group A and increased a little in group B. The positive expression in group C increased at 3 hours after SCI, which was found in spinal cord anterior horn neurons and a small amount of ganglion cells. It reached peak at 1 day, maintained at a high level during 1-3 days and then declined. At 14 days, it appeared only in a small amount of ganglion cells of white matter. There was no significant difference in the number of HIF-1alpha positive cells between groups A and B (t = 1.325, P = 0.137). The number of HIF-1alpha positive cells at each time point in group C was more than those in groups A and B (P < 0.05), and there were significant differences between all time points in group C (P < 0.05)., Conclusion: The expression of HIF-1alpha increases after SCI, it is related to the ischemia hypoxia after SSCI, and the expression pattern was correlated with the injury time.

Objective: To explore the survival time of autogeneic BMSCs labeled by superparamagnetic iron oxide (SPIO) in rabbit intervertebral discs and the rule of migration so as to prove bases of gene therapy preventing intervertebral disc degeneration., Methods: Twelve rabbits were used in this experiment, aged 8-10 weeks, weighing 1.5-2.0 kg and neglecting their gender. BMSCs were separated from rabbits bone marrow by density gradient centrifugation and cultivated, and the 3rd generation of BMSCs were harvested and labeled with SPIO, which was mixed with poly-l-lysine. The labeling efficiency was evaluated by Prussian blue staining and transmission electron microscope. Trypanblau stain and MTT were performed to calculate the cell's activity. Rabbits were randomly divided into experimental group (n = 8) and control group (n = 4), the labeled BMSCs and non-labeled BMSCs (5 x 10(5)/mL) were injected into their own intervertebral discs (L1,2, L2,3, L3,4 and L4,5), respectively. At 2, 4, 6 and 8 weeks, the discs were treated with Perl's fluid to observe cell survival and distribution., Results: The labeling efficiency of BMSCs with SPIO was 95.65% +/- 1.06%, the cell activity was 98.28% +/- 0.85%. There was no statistically significant difference in cell proliferation within 7 days between non-labeled and labeled cells (P > 0.05). After 8 weeks of operation, the injected cells was alive., Conclusion: Labeled BMSCs with SPIO is feasible in vitro and in vivo, and the cells can survive more than 8 weeks in rabbit discs.

Objective: To investigate the feasibility and effect of inducing adipose-derived stem cells (ADSCs) treated with growth differentiation factor 5 (GDF-5) to undergo chondrogenic differentiation in vitro., Methods: Six healthy Japanese rabbits aged 3 months (2-3 kg) of clean grade were chosen, irrespective of sex. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by vimentin immunohistochemistry and CD44, CD49d, CD106 immunofluorescence staining. ADSCs at passage 3 were used and the cell density was adjusted to 1 x 10(6)/mL, then the ADSCs were treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium, respectively. The morphology changes of the induced ADSCs were observed by inverted contrast phase microscope and their growth state were detected by MTT. The mRNA quantities of Col II and proteoglycan expressed by the induced ADSCs were detected with RT-PCR. The Col II proteoglycan synthesized by the induced ADSCs were detected with alcian blue staining, toluidine blue staining, immunohistochemistry staining, and Western blot method., Results: ADSCs mostly presented small sphere, fusiform and polygon shape with positive expression of CD44 and CD49d and negative expression of CD106 and vimentin. The ADSCs treated with 100 ng/mL GDF-5 presented sphere or sphere-like change and vigorous proliferation. The mRNA quantities of Col II and proteoglycan synthesized by the induced ADSCs treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium increased in a dose-dependent manner at 7 days. There were significant differences among all the groups (P < 0.05), except that no significant difference was evident between the 0 ng/mL group and the 10 ng/mL group (P > 0.05). When ADSCs were treated with 100 ng/mL GDF-5 for 14 days, the Col II and the mRNA and protein quantities of proteoglycan reached the peak, and the results of alcian blue, toluidine blue and Col II immunohistochemistry staining were positive., Conclusion: ADSCs treated with certain concentration of GDF-5 have higher expression of Col II and proteoglycan and possess partial biological function of chondrocyte.

Objective: To determine the contents of matrix metalloproteinase 3 (MMP-3) and interleukin 1 (IL-1) in the tissues of the lumbar disc herniation and to investigate their roles in the pathogenesis., Methods: The tissues of the herniated lumbar disc were obtained from 30 patients undergoing surgery for persistent radiculopathy from June 2003 to December 2004 and at the same time these samples were divided into the following three experimental groups: the bulge group (n =11), the protrusion group (n = 9), and the prolapsus group (n = 10), 14 males, 16 females, aged 33-64 years. As the control group, 9 lumbar disc specimens were harvested from 9 patients (4 males, 5 females, aged 21-58 years) suffering from bursting fracture of the lumbar spine. The specimens were analyzed by the ELISA method for the contents of MMP-3 and IL-1., Results: The contents of MMP-3(14.25+/-1. 32, 19.89+/-2.97, 20.69+/-2.18 ng/ml in the bulge group, protrusion group and prolapsus group, separately) and IL-1 (8.52+/-0.22, 11.88+/-0.52, 11.90+/-0.73 pg/ml in the bulge group, protrusion group and prolapsus group, separately) in the experimental groups were significantly higher than those in the control group. The contents of MMP-3 and IL-1 in the protrusion group were not significantly higher than those in the prolapsus group, but they were significantly higher than those in the bulge group(P<0.01). The contents of MMP-3 had a significant relationship with the contents of IL-1 in the three experimental groups and the control group(P<0. 01)., Conclusion: The result demonstrates that the tissues of the lumbar disc herniation can produce both MMP-3 and IL-1, which may have an unknown but important relationship with each other.

Objective: To observe ultrastructural changes of the intervertebral disk in the corresponding area after internal fixation of spinal column., Methods: Twenty-four Japanese big ear rabbits were divided into internal fixation of spinal column group (n=12) and control group (n=12). The internal fixation model was made as follows: The spinous processes and erector spinal muscle were exposed and the T10-L3 spinous processes and the relevant two-side articular processes under the periosteum were isolated. With the help of L-shaped Kirschner wires, the steel wire was threaded through the articular of T11, T12, LI and L2, and were connected with L-shaped Kirschner wires. After 6 months of operation, the following intervertebral disk tissues were observed with transmission electron microscope: nucleus pulposus, internal anulus fibrosus and external anulus fibrosus of L1 intervertebral disk. The T12 and L2 intervertebral disk surface structure was observed horizontally and longitudinally with scanning electron microscope, respectively., Results: After internal fixation of spinal column, the structural changes of cells in nucleus pulposus and internal annulus fibrosus occurred earlier than that in the external annulus fibrosus. Proteoglycan and special structure were found in nucleus pulposus and matrix of annulus fibrosus. However, the forms of special structure in nucleus pulposus and internal layer of annulus fibrosus were different. In the degeneration matrix of intervertebral disc, the proteoglycan particles and special structure were obviously decreased., Conclusion: Abnormal stress environment can result in the degeneration of intervertebral disk. There is a regular distribution of the special structure in nucleus pulposus and matrix of annulus fibrosus, which is related to biology behaviour of proteoglycan particles in the degeneration of intervertebral disk.