| Background:Surgical therapy is an important and effective treatment for thoracoabdominal aortic aneurysm (TAAA). Paraplegia is considered the major devastating complication of operations with the reported incidence between 4%-40%. The factors involed are various but they all come to one, i.e., spinal cord ischemic injury induced by aortic cross-clamping and the following local and systematic ischemic pathophysiologcal changes. Numerous measures have been clinically taken to prevent spinal cord from injury and some protective effects been observed. But there is no significant decrease in paraplegic incidence. This is mainly because that they are not the very way to resolve the radical problems brought about by ischemic damage to the spinal cord. Also most of them are practically complicated, some are even associated with certain side effects and complications as well. There is still a need to find a simpler and more effective method protecting the spinal cord during the perioperation period.ObjectiveTo research for an effective way to protect the spinal cord from ischemic damage during TAAA repairing operation, the spinal cord ischemic models are created in Wistar rats.Variations of the serum levels of endostatin, vascular endothelial growth factor (VEGF), brain derived neurotrophic factor (BDNF), spinal cord TrkB receptor and VEGF/KDR receptor mRNA expression were tested at different time after spinal cord ischemia-reperfusion. Meanwhile the histologic examinations were made. Recombinant cytokines were administered via subarachnoid cavity and its protective effect against spinal cord ischemic injury was observed.MethodsWistar rats weighing 200-250 gram were subjected spinal cord ischemia model with the Naslund's abdominal aorta occlusion method.1. Control Group(C). Eight rats were only performed abdominal aorta dissection without occlusion.2. Model group (Z). To creat the spinal cord ischemia model, forty-eight rats were undergone aortic cross clamping inferior to the left nephritic artery branch. The cross clamping time were 30 minutes for group ZI (24 rats) and 90 minutes for group ZII (24 rats). Blood samples were delivered six to eight hours (6-8h) after spinal ischemia, on the second day (2d) and the seventh day (7d) postoperatively. Meanwhile the lumbar spinal cord specimens were taken for morphological examinations.3. Treatment group (group V, B, VB, NS). Ninety rats were randomly assigned into five groups, 16 in each. Before the 90 minutes aortic cross clamping or after declamping, rats were administered via subarachnoid cavity injection with l)80ng/80 u 1 of recombinant rat vascular endothelial growth factor (rrVEGF165) for group V, 2) 40ng/80 u 1 of recombinant human brain derived neurotrophic factor (rhBDNF) for group B, 3) 80 u 1 of mixture contained 80ng rrVEGF165 and 40ng rhBDNF for group VB, 4) 80 U 1 of normal saline as placebo for group NS respectively. Besides a non-ischemia control treatment group (VBO) ofeight rats received spinal administration of 80 V 1 mixture of 80ng rrVEGF165 and 40ng BDNF were not performed aortic clamping intervention. Blood samples and spinal cord biopsy specimens were taken 6-8h and 7d after the spinal ischemia reperfusion.4. Laboratory testing: 1) Spinal cord pathological changes were observed under light microscope. 2) VEGF/KDR receptor and TrkB receptor mRNA expressions in the spinal cord were quantitative analyzed by means of reverse-transferent polymerase chain reaction (RT-PCR). 3) Serum level of BDNF, endostatin, and VEGF were tested using enzyme linked immunosorbent assay (ELISA).Results1 Spinal cord biopsy examinations.1) In group ZI there was only slight tissue edema. Nissle body dissolution was observed in only a small amount of neuronal cells 6-8h postreperfusion. On the second day, there was slight inflammatory response and recovered to normal on the seventh day.2) In group ZII, there was more serious ischemic cellular edema at 6-8h with enlarged cells in which nissle body lightly stained or disappeared. Pre-necrotic deeper nucleus staining was observed in some motor neurons. At 2d more motor neurons showed necrotic changes with severer inflammatory infiltration. At 7d there were significant individual disparities in rats. The worse ones had significant decrease in the amount of motor neurons, neuronal necrosis with sponge-like appearance in gray substance. While the less damaged ones had nuclear dissolved, nuclear disappeared, or obscured nuclear membrane in that only an absurd nuclear shadow discernable in some neuronal cells. In some of the motor neurons, the cell boundaries could not be seen clearly, showing a view of quasi-dissolvent. However there were still some normal neurons remained.3) The result was similar in group NS.4) The ischemic necrotic damage was less severe in groups V, B, and VB than that in group ZII. It was especially mild in group VB in which the majority of the motor neurons survived well on the seventh day after spinal cord ischemia-reperfusion.2. Serum BDNF level and the expression of TrkB receptor in spinal cord1). In group C the mean serum BDNF level was 2393.22i427.31pg.ml"1, TrkB gene expression quantification is 0.6417 ± 0.2365.2). For group ZI there was almost no variation in BDNF level at 6-8h or 2d after reperfusion, but a sharply increasing had been observed at 7d, to a level of 3414.61 ± 492.98 pg.ml"1, the difference was highly significant, p < 0.01 compared with that in group C or that of the two previous time points (6-8h, 2d). TrkB mRNA expressions were slightly up regulated, but of no significant difference compared to group C.3) After 90 minutes' ischemia, the serum BDNF concentration in group ZII declined markedly to 1895.93±271.16 pg.ml"1 at 6-8h, considerably lower than the normal level and that in group ZI, p<0.05. And then further declined to 1415.35±205.98 pg.ml"'at 7d, which was only 59.1% of the normal level. When compared with group C and that of group ZI, the differences were both of highly significance, pO.Ol. Nevertheless, such declining trend was not constant, on the second day post reperfusion, serum BDNF climbed back up to normal level ever once. TrkB mRNA expression had regulated up at 6-8h with a quantification of 0.8099±0.1426, then went down to normal on the 2nd day andt further down to 0.5304±0.1780 on the 7thd, which was much lower than the value of 6-8h point, pO.Ol when compared statistically, but had no significant difference to C and that of group ZI (p>0.05).4) Treatment group Serum BDNF levels:Group V elevated at 6-8h (3279.6±491.4 pg.ml"1, pO.Ol compared with group C) andback to normal at 7d (2548.1+382.4 pg.ml"1).Group B elevated at 6-8h and 7d (2875.03 + 476.15 pg.ml'1, p<0.05, and 3616.64 + 419.96 pg.ml"1, p<0.01 compared with group C).Group VB kept normal with the concentrations of 2490.8 + 472.8 pg.ml"1, 2315.43 + 533.37 pg.ml"1 at 6-8h and 7d points, respectively.Group VBO elevated at 6-8h (2783.51+234.12 pg.ml'1, p<0.05 compared with group C) and back to normal at 7d (2337.94 +357.5lpg.ml'1).The results in group NS were similar to that in group ZII it came to serum BDNF levels of 2005.84+ 392.66 pg.ml"'at 6-8h point and 1551.60 + 292.08 pg. ml"1 on the 7th day. To compare to group ZII and NS, p<0.01 for all values in group V, B, VB, VBO, the differences were highly significant.Expression of spinal cord TrkB mRNA.There were up-regulations at 6-8h in group V, B, VB, VBO. The quantitative value were of0.9586±0.2371, 0.8479 + 0.1503, 1.1685 + 0.2204, 1.1215 + 0.2222, respecctively, which had no significant difference to group ZII (p>0.05 for all). But they all, except for group B, significantly higher than that of group C, p <0.01.When on 7th day after reperfusion, TrkB mRNA remained over-expressed in group V and group B with the quantifications of 0.9594±0.0929 and 0.9613 ± 0.1550. Both were significantly higher than that of Group C and group ZII (p<0.01). While in group VB and VBO, the gene expressions down regulated to normal level with values of 0,6790±0.2046 and 0.6367 + 0.1971, respectively. Different to group ZII, group NS had not regulate down at 7d, but up to a quantification of 0.8443 + 0.1767, p<0.05.3. VEGF/KDR receptor mRNA expression in spinal cord1). Group C, the mean quantification value of the spinal cord VEGF/KDR mRNAexpression was 0.2571 ±0.0708.2) Group ZI, VEGF/KDR gene expression did not varied after 30 minutes' spinal ischemia. Although there was a slight fluctuation—up regulation at 2d and then down at 7d (0.2982 + 0.0694, 0.2109 + 0.0824, respectively), the differences were not statistically significant when compared to group C and 6-8h in the same group(p >0.05).3) In group ZII, the expression of VEGF/KDR mRNA tended to regulate up at 6-8h to 0.4005 + 0.1498, significantly higher than that in group C (pO.Ol) and group ZI (pO.Ol); but turned down to 0.1664 + 0.0375 at 2d, which had no difference to group C (P>0.05). At 7d the expression again regulated even up to 0.6316± 0.1490,( pO.Ol when compared with group C or group ZI). Inner the group, pO.Ol among the three time points, the differences were highly significant.4) Placebo injection (NS)to the subarachnoid cavity had showed almost the same influence on spinal cord VEGF/KDR expression as the model group ZII. It regulated up to 0.3350 + 0.0341, 0.6225 + 0.1534 respectively at 6-8h and 7d post-reperfusion.5) For those real treatment groups, at 6-8h after reperfusion, group V and VB0 had normal expression (0.2257 + 0.0207, 0.2716 + 0.0553, respectively), while group B and VB had distinctively regulated up (0.4983 ±0.0938, 0.4362±0.0984, pO.Ol, compared to C). At 7d after reperfusion, VEGF/KDR expression in group V, B, VB, and VB0 were nearly of the same range of up regulation with the quantifications of 0.3827±0.1404, 0.4161 ±0.0683, 0.4131 ±0.1802, 0.4039±0.1196, respectively, but still less much than that in either ZII or NS, p<0.05, the differences were statistically significant.4. Serum endostatin level and e/V ratio 4.1. Endostatin. 1) The serum endostatin level of group C was 56.52 + 10.28 ng.ml"1.2) In group ZI, it slightly lowered at 6-8 h, raised at 2d, and then returned to normal till 7d.3) In group ZII, endostatin maintained unchanged at the early stage after reperfusion, but increased markedly at 2d to 84.80±16.96 ng.ml"1, higher than that in group C, p<0.05; and then further increased to 92.48 ±25.SOng.ml"1 at 7d, p<0.01 when compared with group C, the difference was of highly significance.4) As for group NS, which received placebo injection, serum endostatin elevated to 106.36 + 20.08 ng.ml"1 at 7d, similar to group ZII.5) For group V, which was treated with rrVEGF1 5, serum endostatin levels were 62.48 ±21.56ng.mr' and 62.20+ 18.48ng.ml"1 respectively at 6-8 h and 7d after declamping, either with no significant difference to that in group C, p>0.05; But lower than that in group ZII at 7d, p<0.01.6) In groups VB and VB0, the serum endostatin level slightly rose. But there were not different significances. 4.2. e/V ratio.The e/V ratios for group ZI, ZII, V, VB, VB0 and NS were ( 2.9960±l .7182) X 103, (4.9028±2.0675) X 103, (2.1585±0.8498) X 103, ( 1.2892±0.9214) X 103, (2.0969±1.0993) X103, (4.8977±2.0256) X103, respectively. Among them, ZII & NS were the highest two, p<0.01 when compared with the other three groups, — group V, VB and VB0, the differences were of high significances. The lowest one was VB, but it had no significant difference compared to both V and VB0.Conclusions1. Abdominal aortic occlusion for thirty minutes renders slighter influence on the morphology and functions of motor neurons of the spinal cord and can get recovered in seven...
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