| Background and Objective:Spinal cord injury (SCI) is a kind of serious central nervous system (CNS) diseases, often lead to serious or permanent disability. It can not only bring great pain to the patient, but also brings heavy burden to the family and society. In recent years, rehabilitation and treatment of SCI has been a hotspot and difficulty of research in the world and the main reason is the complicated pathological and physiological mechanisms of SCI.Pathophysiological process of SCI mainly includes two phases:primary injury and secondary injury. The primary injury is caused by mechanical damage and incontinuity of the nerve tissue and vessels. The secondary injury involves a complex cascade of molecular events, including spinal cord hemorrhage, edema, imbalance in ionic homeostasis, vascular abnormalities, ischemia, reperfusion, lipidperoxidation, mitochondrial dysfunction, glutamate excitotoxicity and inflammatory responses, eventually leading to necrosis and the apoptosis of neurons and oligodendrocytes, the formation of the glialscar, impeding of axonal regeneration, reducing the self-repair ability of organization. Among them, the inflammation plays an important role in the secondary SCI. The direct damage of vessels or inflammation will cause the destruction of blood spinal cord barrier (BSCB) after SCI. Then, the damage of BSCB caused a series of permeability changes, resulting in spinal cord hemorrhage, edema and inflammation, produce and secrete large amounts of toxic factors, initiate the oxidative stress reaction, leading to tissue edema and influence the prognosis of patient badly. Tight junction is one of the most key structure for BSCB function, the changes of MMP-9 expression and activity may be lead to abnormal changes of Tight junction protein molecular components and barrier damage.So far, the treatments of SCI mainly include early-stage surgery(decompression of the compressed segmental spinal canal), drug therapy(Hormone stosstherapy, dehydration and Neurotrophic drugs, et.al) and late-stage rehabilitation. Although there are certain effectiveness of these treatments, it will leaving over inordinately dysneuria and disability. Therefore, it’s urgent to find a new method to improve nerve regeneration and function recovery.Stem cell transplantation has been identified as a potential therapeutic modality for treating SCI in recent years. Several cell types, including embryonic stem (ES) cells, olfactory ensheathing cells, Schwann cells, mesenchymal stem cells (MSCs), neural stem cells (NSCs) and induced pluripotent stem (iPS) cells have been used to the treatment of SCI. But so far, the seed cells for clinical research is still less, the main problems including:limited cells, the low survival rate of the grafted cells, the immune rejection, ethical issues, tumorigenicity and so on. Therefore, it is particularly important to find the suitable seed cells to improve the survival rate of transplanted cells and in-depth understanding of the mechanism for the treatment of spinal cord injury. A large number of literatures show that adipose tissue-derived marrow stromal cells (ADSCs) have a potential to the multiple layer tissue differentiation and can differentiate into fat cells, bone cells, cartilage cells, muscle cells, nerve cells under appropriate conditions. Moreover, compared with other sources of stem cells, ADSCs represented a promising cell type for the treatment after SCI because it can be easily obtained in ubiquitously distributed adipose tissue and expandable in vitro.Studies have shown that ADSCs transplantation can secrete neurotrophic factor, reduce inflammation, reduce apoptosis and glial scar formation and promote endogenous neurogenesis in a certain extent, so as to reduce secondary injury and promote the recovery of motor function. However, the effect of ADSCs transplantation on blood spinal cord barrier in rats after SCI and the related mechanism has not been reported. The purpose of this experiment is to explore the effects and relevant mechanisms of ADSCs on blood-spinal cord barrier disruption induced by SCI.METHODS:1. ADSCs collect, cultivate, flow cytometry identification.2. Adult healthy male Sprague Dawley (SD) rats were used to establish the model of T10 spinal cord injury using the improved clamping method. They were randomly divided into:sham operation group, spinal cord injury (SCI) group and stem cell transplantation group. After injury, a total of 1×105 cells were instantly transplanted into the rostral and caudal from lesion epicenter of the cell transplantation group using Hamilton micro syringe, rats in SCI group received injection of the same quantity of PBS at the same time point in the same place, rats in sham operation group accepted only laminectomy, without SCI.3. Behavioral assessment3 days before operation and 1,3,5,7,10,14 days after SCI, BBB scale was used to evaluate hind limbs locomotor function recovery of rats.4. The detection of spinal cord tissue water contentDetermination of spinal cord tissue water content by dry wet weight method. Animals were decapitated with a sharp blade in 1,3,5 and 7 days after injury. Take head and tail about 1 cm of injury centre of the spinal cord tissue to determine the water content of spinal cord. Each sample was weighed immediately after removal (wet weight) and after drying at 80℃ for 72 h (dry weight) using an electronic analytical balance. The percentage of water content in the spinal cord was calculated using the following equation:Water content in the spinal cord= [(wet weight -dry weight)/wet weight] ×100%.5. Assess of blood spinal cord barrier (BSCS) permeabilityBSCB integrity was evaluated by Evans-blue staining. After anesthesia,2% EB-dye solution was injected (4ml/kg, i.v.) via femoral vein before 1 hours of killing, normal saline transcardially perfused until the drainage was colorless. Removed about 1 cm of injury centre of the spinal cord tissue and weighted carefully. Then incubated the samples in trichloroacetic acid solution. After homogenization and centrifugation (14,000 r×20 minutes), diluted the supernatants with ethanol (1:3), and determined its fluorescence intensity (excitation at 620 nm and emission at 680 nm) using an automatic microplate reader. The EB leakage was represented as ug/g spinal cord weight.6. Western blotAfter anesthetized and decapitated, tissues (about 1cm) around the lesion sites in spinal cord were obtained and tissues were homogenized in liquid nitrogen and diluted in RIPA Lysis Buffer. The protein concentration of the supernatant was measured using a Bicinchoninic Acid Protein Assay kit after centrifugation at 14,000 r for 20 min at 4℃. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies:ZO-1、Claudin-5 and MMP-9. Secondary antibodies were used to detect the primary antibodies. GAPDH was employed as an internal control to determine and normalize the relative intensity of each band.7. Preparation of paraffin sections and HE stainingAfter anaesthetized, rats were perfused transcardially with saline followed by 4% paraformaldehyde solution. About 1 cm of the spinal cord tissue were then removed and fixed by immersion in the same solution for 24 hours. After dehydrated and vitrified, they were embedded in paraffin, and 3 um sections were prepared. Sections were de-waxed in xylene and rehydrated in graded ethanol and deionized water and then stained by hematoxylin for 2 min and eosin for 30 s. Images were captured with a microscope.8. TUNEL staining was used to detect the cell apoptosis.Sections were de-waxed and rehydrated and then performed by heat treatment in a microwave oven in citrate buffer solution (PH=6.0) for antigen retrieval. After incubated 30 min by serum at room temperature, sections were washed with PBS and added 10 μl of TUNEL reaction mixture to the section, and then incubated for 60 minutes at 37℃ in a humidified atmosphere. Images were acquired with a fluorescence microscope.9. ImmunofluorescenceSections were de-waxed and rehydrated and then performed by heat treatment in a microwave oven in citrate buffer solution (PH=6.0) for antigen retrieval. After incubated 30 min by 5% donkey serum at room temperature, sections were incubated overnight at 4℃ with primary antibody (rabbit anti MPO, rabbit anti Iba-1). After washed with PBS, sections were then dark incubated with the second antibody (goat anti rabbit -Alexa Fluor 488) at 37℃ for 1 hour. Images were acquired with a fluorescence microscope.RESULTS:1. Cell growth, morphology, identificationCultures of ADSCs are round, unequal in size, suspended, which mixed with a large number of red blood cells. The cells began to paste the wall after static culture for 24 h, gradually increased after 48 h, cells were round in irregular polygon. Adherent cells continued to increase with the prolongation of time, the fusiform cells have small protrusions. The primary cell culture for 7 days can achieve a 80% to 90% fusion, which characterized by a spiral or radially arranged and can be passaged at the first time. After passage, cell proliferation is fast and cell purification is high.Flow cytometry showed high expression of CD29 and CD90, were 99.97% and 99.04%, while the low expression of the CD45 and CD49d, were 2.37% and 2.67%, respectively, which is in line with the general expression of mesenchymal stem cell surface markers.2. Behavioral assessmentAll rats BBB score were 21 points preoperation and all rats showed paralysis(except sham-operation group) postoperative, BBB score were 0. Hind limbs motor function score in SCI group and cell transplantation group rats increased gradually with the extension of time. The cell transplantation group showed the better recovery compared with SCI group at the same time point (P<0.05). The recovery of hind limbs motor function is obvious on the fifth postoperative day, there were significant difference in any two groups among three groups (P<0.05).3. The detection of spinal cord tissue water contentTo evaluate alterations in spinal cord edema, water content in the spinal cord was measured. The water content of spinal cord in sham operation group basically remain unchanged, on the third postoperative day, edema reached a peak and then gradually decreased, water content on the SCI group was higher than cell transplantation group at the same time point, the water content of the spinal cord tissues in the two groups was higher than that of the sham operation group at seventh days after injury, there were significant difference in any two groups among three groups (P<0.05).4. Assess of blood spinal cord barrier (BSCS) permeabilityEvans blue leakage in spinal cord tissues was used to assess alterations in BSCS permeability. Compared with sham operation group, BSCS permeability was significantly increased after SCI, which suggesting the increasing of the permeability of BSCS after SCI, while ADSCs transplantation could alleviate the leakage of Evans blue, there were significant difference in any two groups among three groups (P<0.05).5. Western blotWestern blot showed that the expression of MMP-9 protein was rare in the third days after operation in sham operation group, the expression of MMP-9 protein was significantly increased after SCI, while stem cell transplantation can significantly inhibit the expression of MMP-9 protein, there were significant difference in any two groups among three groups (P<0.05).The expression level of ZO-1 and Claudin-5 protein were the highest in sham operation group, than cell transplantation group, the lowest were SCI group, there were significant difference in any two groups among three groups (P<0.05). 6.TUNELWe detection apoptosis by TUNEL kit and found that apoptosis cells were least in sham operation group, SCI group were the most, while cell transplantation group was significantly lower than that of SCI group, there were significant difference in any two groups among three groups (P<0.05).7. ImmunofluorescenceThe results of MPO Immunofluorescence:there was basically no positive cells in sham operation group, the expression of positive cells was the most in SCI group, while cell transplantation group was significantly lower than that of SCI group, there were significant difference in any two groups among three groups (P<0.05).The results of Iba-1 Immunofluorescence:the microglial cells were in static state mostly in sham operation group, the cell body was small, and there were bumps in all directions. After SCI, microglia were activated, activated microglia showed neurite retraction and the cell body were relative increase, but the activated microglial cells in the cell transplantation group were less than SCI group, there were significant difference in any two groups among three groups (P<0.05).8. HE stainingHE staining showed that the boundary of the gray matter and white matter of spinal cord tissue were clear in sham operation group, the morphological and structure of neurons were normal, nerve fibers arranged closely and continuous, there were no bleeding, inflammatory cell infiltration and cavity formation. The SCI group and cell transplantation group were found different degrees of infiltration of inflammatory cells and nerve fiber dissolution, syringomyelia and hemorrhage, but the pathology change of cell transplantation group were smaller than SCI group.Conclusion:ADSCs transplantation promote the recovery of motor function of hind limbs in rats after SCI, the mechanism may be through inhibiting the expression of MPO and activation of microglia, downregulation of MMP-9 expression, reducing loss of tight junction protein ZO-1 and Claudin-5, reducing the spinal cord edema and apoptosis, which plays an important role in protection BSCS. |
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