| Acute spinal cord injury(ASCI) often results in traumatic paraplegia which is one of the most severe trauma. The impaired mechanism of ASCI include primarily and sequently spinal cord injury(SSCI). The pathological lesion degree of SSCI exceed the primarily spinal cord injury,and edema of spinal cord is one of the most important changes of physiology and biochemistry in SSCI. It is well known that aquaporin family plays an important role in water transmembrane transport in biological organism. Aquaporin-4(AQP4) belongs to the aquaporin family, which includes 13 subtypes at present. AQP4 is more abundant in the central nervous system (CNS) and mainly located at membrane of astrocyte cells in brain and spinal cord. AQP4 not only involves in water transport and regulation on water,electrolyte and osmotic pressure balance of brain and spinal cord in normal physiological state, but also in CNS edema originated from different causes such as trauma, ischemia, hemorrhage, inflammation and tumors. But there is a few research for the AQP4 expression of spinal cord after ASCI, thus this study is to investigate the molecularbiologic mechanism of spinal cord edema and magnetic resonance imaging (MRI) after ASCI by employing molecular biological techniques and Dynamic MRI scan. Objective To study the time course changes of aquaporin-4 (AQP4) and AQP4mRNA of spinal cord after ASCI in rabbits by improved self-made Allen method. To research the relationship between AQP4 expression, spinal cord edema and MRI after ASCI, in order to investigate the molecularbiologic mechanism of spinal cord edema and MRI.Methods 1. To Build acute incomplete spinal cord injury model by improved self-made Allen method. Fourty eight healthy rabbits were divided into six groups randomly: control group, 6h, 1d, 3d, 5d and 7d after ASCI groups, eight subjects in each group. Tarlvo scales were performed after ASCI to test the function of the rear limbs. 2. MRI and signal intensity evaluation were performed. Relative signal intensity(SI(r)) was measured on the clearest scan in sagittal plane. SI(r)=(Ss-Sn)/Sn, Ss and Sn indicate the signal intensity of the injury region and the background nosiy signal intensity respectively. Water quantity in injured area of spinal cord was obtained according to the formula: water quantity of spinal cord =(wet quantity-dry quantity)/wet quantity×100%. 3. Pathologic changes were observed by Optical microscope, immunohistochemical method was used to assess the protein expression of AQP4, and reversed transcriptive enzyme-polymerase chain reaction (RT-PCR) was used to assess the AQP4 mRNA. 4. Statistic software SPSS11.0 was used to analysis results. Data was expressed in: mean±standard deviation, the comparison between two sample mean adopts t-examination, P<0.05 shows the difference is significant. Correlation analysis was carried out between water quantity of spinal cord,SI(r), AQP4 protein and mRNA expression respectively, P<0.05 shows the difference is significant.Results 1. Compared with control group, The Tarlvo scales in five spinal cord injury groups had obvious difference (P<0.01). 2. SI(r) on T2WI of spinal cord increased obviously, reaches peaks 3d after injured, then decreases in 5d and 7d groups, SI(r) on T2WI in each injury group appear significant deviation compared with control group (P<0.05). The change of SI(r) on T1WI of each group compared with control has not significant deviation(P>0.05).3.The water quantity of spinal cord also increases obviously, reaches peaks 3d after injured,and decreases in 5d and 7d groups , but still higher than that in control group. The water quantity in each injury group appear significant deviation compared with control group(P<0.05). The SI(r) on T2WI and the water quantity (%) of spinal cord at different time after impairation appears remarkable positive correlation (r=0.953, p<0.01). 4. Experimental spinal cord injury with dynamic state MRI scan. After injury 6h to 1d, we can see the punctiform or lamellar high signal on T2WI, rabbits neurologic function appear incompletely damage; the signal intensity and extent on T2WI reach the highest and largest in 3d groups, but both neurologic function of lower extremities appear no changes; After injury 5d to 7d, signal intensity of the injured region begin to decrease, the neurologic function recovery partly at this time. 5. The early pathological change after ASCI appears focus bleeding, capillary engorgement, surrounding tissue edema, intercellular space widening and pericaryon swell.The bleeding and edema increases continuously and reaches peaks 3d after ASCI,but decreases in 5d and 7d groups, accompanied with glial cell proliferation. 6. The staining of AQP4 protein of control group was mainly located on the cell membrane of astrocyte cells,and it is of polar contribution of AQP4 in spinal pia mater and around of spinal cord central canal and blood vessel. 7. After injury, AQP4 protein and mRNA of spinal cord increased obviously , and reached peaks 3d after injured, then decreased in 5d and 7d groups, the expression of the AQP4 protein and mRNA of spinal cord in each group appear significant deviation compared with control group (P<0.05). AQP4 protein expression has a remarkable positive relationship with AQP4mRNA after ASCI(r=0.943,p<0.05). 8. AQP4 protein and mRNA expression were both remarkable positive correlated with the spinal cord water quantity after ASCI(r=0.993,p<0.01;r=0.950,p<0.05). AQP4 protein and mRNA expression were both remarkable positive correlated with SI(r) on T2WI after ASCI(r=0.972,p<0.01;r=0.887,p<0.05).Conclusions 1. We have set up acute incomplete spinal cord injury model by improved self-made Allen method successfully. 2. The rule of spinal cord edema from pathology after injury is: spinal cord edema increased obviously, and reached peaks 3d after injured,then decreased in 5d and 7d groups, the injured section of spinal cords stills has edema. The change of water quantity coincided the pathological change,which suggests that spinal cord edema is the important change of biochemistry of the spinal cord tissue, and meanwhile is the basis on pathology of SSCI. 3. The staining distribution of AQP4 protein of control group has a close relationship with water transport of spinal cord edema, which suggest that AQP4 participate the regulation of water metabolism mainly. AQP4 protein and mRNA expression were both remarkable positive correlated with the spinal cord water quantity after ASCI. These results suggest that up-regulation of AQP4 has a close relationship with the early stage of the injury spinal cord edema,and it is one of the the mechanism of molecular biology of spinal cord oedema after ASCI, which provides a theory basis on developping new prevention and cure method of spinal cord edema. 4. The SI(r) on T2WI and the water quantity of spinal cord (%) at different time after impairation appears remarkable positive correlation,which proved T2WI high signal has a close relationship with spinal cord oedema after ASCI. AQP4 protein and mRNA expression were both remarkable positive correlated with SI(r) on T2WI after ASCI, which show us the up-regulation of AQP4 is also one of the the mechanism of molecular that T2WI high signal, The T2-weighted-imaging could be used to estimate spianl cord edema, and show the position,extent and signal intensity of spinal cord edema after ASCI clearly. Dynamic MR scan can detect spinal cord edema developing process after ASCI.In one word,up-regulation of AQP4 has a close relationship with the early stage of the injury spinal cord edema, and it is one of the the mechanism of molecular biology of spinal cord oedema after ASCI. These findings suggest that AQP4 inhibitors or down regulators could lessen the degree of spinal cord edema, cut down the mortality and traumatic paraplegia rate. At the same time, up-regulation of AQP4 is also one of the the mechanism of molecular that T2WI high signal,MRI could show the position,extent and signal intensity of spinal cord edema after ASCI clearly, and Dynamic MRI can be used for research of spinal cord edema developing process after ASCI. |
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