| Objective:Myelopathy induced by progressive compression of spinal cord is a frequently encountered clinical problem. The pathogeny may be spinal column trauma, epidural tumor of spinal cord, myelopathy induced by degeneration of cervical vertebrae, ossification of yellow ligaments or spinal column tuberculosis, which may compress spinal cord with a progressive process. Compression of spinal cord may cause both of progressive ischemic and mechanic injuries of spinal cord, ultimately resulting in paralysis and malfunction of sphincters. Various methods have been tried to induce spinal cord compression in previous studies, but none of them is satisfying enough. Therefore, it is high time for us to establish a new model, so as to provide a basis to study the pathological process and mechanism of the injury.Methods(1) the vertebrae and vetebral canal of rat were dissected. The external feature of vertebrae,spinal column and the anatomic relationship of spinal cord with vertebra body werw observed. (2) Based upon the morphological characteristics of vertebrae and spine of rat, a spinal cord compression device was designed.(3) The rat spinal cord compression model was established by the self-made compression device.(4) The model was evaluated by methods of X-ray imaging, Behavioral testing,TTC staining, HE and Nissel staining.Results(1) The conus medularis was at the level of L3 vertebra and between L3 and L4. The lumbosacral enlargement was located behind the vertebral bodies of L2 and L3.(2) A vertebra near to the lower portion of the thoracic region was taken for example. It was made up of 2 principal parts, a vertebral body and vertebral arch. The vertebral arch had a pair of pedicles and a pair of laminae articular, 2 transverse and a spinous processes. Two anterior and four posterior articular processes springed from the junction of the pedicle and lamina. The former were triangular cuneiform in shape; the latter, including 2 posteriosuperior and 2 posterioinferior processes, formed 2 triangular notches in either side projecting backward to fit the neighbor anterior articular processes.(3) The spinous processes from T10 to L5 were directed from posterioinferior to anteriosuperior. Based upon the morphological characteristics of the vertebrae and spine of rat, a spinal cord compression device was designed by us. The device includes 3 parts: a fixer, a compression steel board and a screw.(4) With the increase of compression degree and duration of compression time, the all rats gradually presented with progressive paraplegia, laggard in walking, hind-limbs paraplegia and malfunction of sphincters in compression group.(5) The TTC showed that spinal cord ischemia was observed at each investigation time after compression,the degree of spinal cord ischemia was correlated with the compression degree and the duration of compression time . HE staining showed that one week after compression the edema of neurons and gliocytes, vacuolation in some neurons and'spongiform'degeneration in the white matter of the compressed spinal cord were observed.Nissl staining showed the reducing size of some neuron, loss of some Nissl bodies and pyknosis in some neurons,which characterized by nuclear shrinkage and increased bosophilla, when compression growing up.Conclusions(1) The structural characteristics of rat thoracic and lumbar vertebra provide the appropriate mechanical fulcra for the fixation of the self-made spinal cord compression device.(2) The advantages of self-made spinal cord compression device are its scientific configuration, firm fixation, and so on. (3) The progressive compressed spinal cord injury model established by self-made device can satisfactorily replicate the development of clinical progressive spinal cord compression. It is simple and replicable.(4) The compression degree could be conveniently adjusted according to different research purposes.(5) The progressive compressed spinal cord injury model is a pretty good model for studies on mechanism of ischemia impairments induced by spinal cord compression.(6) The results of behavior testing, X-ray imaging, TTC staining and histologic examination imply that the characteristic of the rat spinal cord in operation group were consistent with those of progressive compression spinal cord ischemic injury. Objective: The study was to observe the changes of apoptosis and Caspase-12 expression and the changes of ultrastructures of cell in the progressive compression spinal cord of rats to clarify the molecular mechanism of apoptosis after compression of rat spinal cord. Methods 120 adult Wistar rats (250-300g), male or female, were randomly divided into 2 groups, including control group (n=60) and operation group (n=60). The progressive compression spinal cord model of rat was established with a self-made device in the operation group. The operation process in control group was the same as the operation group except the compression of spinal cord. The segments of compressed spinal cord were properly taken at an interval of 1 d, 3 d, 7 d, 14 d, 21 d, 28 d after compression (n=10 for each group), respectively. The compressed segments of spinal cord were examined by the methods of Tunnel staining as well as transmission electromicroscopy, IHC test, Western blotting and X-ray imaging, so as to explore the rules of apoptosis, caspase-12 expression and morphological changes ofconpressed spinal cord. The data were analyzed by the computer image analysis software and SPSS10.0 statistic software. .results(1) Under electronic transmission microscope,the cytomembrane vesicle, cytoplast shrinkage, chromatin condensation, pyknosis, a large scale of DNA fragmentation,various degrees of edema of neurons and gliocytes, vacuolation in some neurons and spongy degeneration in the white matter were observed. Moreover, different degrees edema of endoplasmic reticulum in neurons was observed at each investigation time.(2) The Tunnel staining showed that apoptosis was observed in the early stage of spinal cord compression.With the duration of compression time,the apoptotic neurons and gliacytes were obvously increased.The rate of apoptosis on the 1st, 3rd, 7th, 14th, 21st and 28th days after compression was (12.5±2.3)%, (13.0±3.6)%, (17.2±4.3)%, (17.2±4.3)%, (36.1±6.5)%, and (2.3±7.9)%, respectively. There was significant difference between the compression group and the control group (P<0.05).(3) Correspondingly,the number of Caspase-12 positive cells in different investigation times of 1st, 3rd, 7th, 14th, 21st and 28th days was(18.47±2.16), (22.03±2.53), (25.12±3.12), (30.70±4.26), (31.37±4,82), (26.62±3.84), respectively. There was a significant difference between the compression group and the control group(P<0.05). The results showed that the rate of apoptosis was correlative with the increase of expression of Caspase-12 compression.(4) The Western blot showed that the expression of Caspase-12 was increased with the duration of compression time and the degree of compression.ConclusionThe results suggest that apoptosis of neurons is the essential pathological factor of the spinal cord injury during the process of the progressive compression of spinal cord. Apoptosis may induce the secondary degeneration of neurons in the compression spinal cord, which results in paralisis and/or malfunction of sphincters. Caspase-12 might be involved in apoptosis induced by ischemia of progressive compressed spinal cord. Objective:To observe the rules of Ca2+ concentration changes as well as Ire1, Grp78 and Caspase-12 expression changes in progressive compression spinal cord injury of rats, so as to explore the signal transduction pathway of apoptosis induced by endoplasmic reticulum stress in progressive compression spinal cord ischemic injury and to provide bases for clinical intervention treatment.Methods:Adult Wistar rats of both sexes weighing 250-300g were randomly divided into 2 groups, including control group and operation group. The progressive compression spinal cord model was established with a self-made device in the operation group. The operation process in control group was the same as the operation group except the compression of spinal cord. The segments of compressed spinal cord were properly taken at an interval of 1 d, 3 d, 7 d, 14 d, 21 d, 28 d after compression, respectively. The changes of Ca2+ concentration as well as Ire1, Grp78 and Caspase-12 expression in the neurons of progressive compression spinal cord of rats were observedResults: After progressive compression of spinal cord, the Ca2+ concentration in neurons was increased. The Ca2+ concentrations at each investigation time were 371.78±69.35, 383.26±10.47, 402.6±62.23, 417.15±67.56, 313.34±12.39, 297.45±28.71 nmol/L, respectively. There was a significant difference between operation group and control group (P<0.05). The expression of Grp78 mRNA and Grp78 was increased in 1 d, 3d, 7d, 14d, 21d after compression, and had its peak in 21d .The expression of Grp78 mRNA and Grp78 in 28d was obviously declining after operation, but still higher than that in control group.there was a significant difference between the operation group and the control group (P<0.05) in all of investigation time. The expression of Ire1 mRNA and Ire1 was obviously increased in 1 d after opration. The expression of Ire1 mRNA and Ire1 was rapidly elevated in 3 d, 7 d, 14 d after opration, and then the expression of Ire1 mRNA and Ire1 was decreased in 21 d and 28 d after opration. There was a significant difference between the operation group and the control group (P<0.05). The expressions of Caspase-12 mRNA was gradually increased in 1d, 3d and 7d after opration and its peak in 14 d. The expression of Caspase-12 mRNA and Caspase-12 was obviously by increased in 21 d and 28 d after opration. There was a significant difference between the operation and the control group (P<0.05).Conclusion:(1)The increase of Ca2+ concentration in the neurons might be a factor trigering the endoplamic reticulum stress induced by progressive compression spinal cord.(2) The increase of Ire1 expression suggest that the unfold protein accumulated in the ER, indicating the neurons were in the stage of ERS.(3) The increasing of higher expression of Grp78, which may protect the cells from apoptosis. reflected the degree of endoplasmic reticulum stress.(4) The Caspase-12 experssion was increased at each investigation time . The changes of Caspase-12 expression were correlative with those of apoptosis.(5) Double Immunofluorescence testing showed that IRE1 and Grp78 or Caspase-12 and Grp78 were coexistence in the neurons at each investigation time.In summary, the above mentioned results imply that Ca2+,Ire1,Grp78 and Caspase-12 might participate in the signal transduction of apoptosis induced by endoplasmic reticulum stress in progressive compression spinal cord ischemic injury.
|
PDF Full Text Request