| Cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) is one of the serious complications with higher incidence and mortality rate. Until now its pathogenesis is not clear, which makes the treatment difficult and unsatisfactory. Hence the pathogenesis should be researched by animal experiment, so that we can find an effective method of CVS treatment. By now there is no satisfactory model to help us understand the mechanism of the CVS. This experiment chooses chick embryo chorioallantoic membrane vessel (CAM) as CVS model, because this type of vessel resembles cerebral vessels in morphology. The CAM vessels will be observed dynamically at any time. The advantage of this model includes good continuity, short cycle, high success rate and repeated operation, so that it can overcome a lot of shortcomings of other animal models. This experiment successfully established CVS model of chick embryo's CAM vessel after a numbers of continuous exploration.Some studies have shown that Calcitonin Gene-Related Peptide (CGRP) is the strongest substance to dilate the vessels and even some studies reported that CGRP can be used to prevent and relieve the CVS after SAH. Since exogenous CGRP does not across the blood brain barrier (BBB), the concentration is not well maintained and thus the treatment is not affected well. This experiment successfully packaged out adeno-associate virus with NT4-CGRP fusion gene. The virus produced endogenous CGRP stably after the chick embryo CAM endothelial cells were infected by it to treat CVS.(1) The establishment of CVS model for chick embryo CAMObjective:To find the most appropriate concentration of the Oxyhemoglobin (OxyHb) which induces chick embryo CAM vasospasm and to establish the CVS model of chick embryo CAM. Method:Different concentration OxyHb (0umol/L; 1umol/L; 5umol/L; 10umol/L; 20umol/L; 100umol/L; 200umol/L) were added to the chick embryo CAM vessels according to the method of randomized controlled grouping, and nimodipine was applied to it. These CAM vessels were recorded by digital camera at different periods, including 6 hours (h), 12h,24h,2 days (d),3d,5d, and 7d. By means of Image-Pro Plus microscope and Image Analysis Software, the percentage of vasospasm of chick embryo CAM vessel area was measured. All data were indicated by average number plus or minus standard deviation, and data in the group and between the groups were analyzed by complete randomized design of variant analysis with statistical software SPSS 10.0. When P value was less than 0.05, the statistical significance was apparent. The pathological results of chick embryo CAM were obtained with the microscope to observe vascular morphology and determine whether the endothelium shredded or dropped, and so on.Result:1) Chick embryo CAM vessels died in relatively high concentrations (100umol/L; 200umol/L), but in relatively low concentrations (1umol/L; 5 umol/L) there was no significant difference compared with normal CAM vessel (P>0.05). Finally, we found out that the vasospasms of CAM were relatively obvious and survived under the concentration of 10umol/L of OxyHb.2) By taking pathological specimens at seven different periods, chick embryo CAM showed that the endothelial cell were flat and elastic membrane layer were smooth in the control group. After adding OxyHb solution for the 3,5, and 7 days, most of the endothelial cells were severely degenerated with some dropped and irregular appearances and obvious increment in the intercellular spaces.3) After administrating nimodipine the pathological results got better and there was a statistical difference between groups of CAM area percentage in comparison with control group (P<0.05).Conclusion:The best concentration of OxyHb that caused chick embryo CAM vasospasm was 10 umol/L, and the pathological results were similar with those with other models of CVS. The CVS could be relieved by the intervening with nimodipine. It indicated that chick embryo CAM vasospasm model was successful and it provided a new method for the research CVS pathogenesis.(2) To establish the recombinant adeno-associated virus that expressed CGRP Objective:To construct the recombinant adeno-associated virus that expressed CGRPMethod:Gene fusion technique was used to construct the recombinant adeno-associated virus that produced CGRP.1) To obtain CGRP cDNA by Polymerase chain reaction (PCR).2) To establish the NT4-CGRP with CGRP and expression vector PBV220-NT4.3) To package the recombinant adeno-associated virus with fusion gene NT4-CGRP by means of 293 cell transfection.4) To determine the virus titer by RT PCR.5) To infect 3T3 cells by recombinant adeno-associated virus and to measure CGRP expression by immunohistochemical method.Result:The results of Agarose gel electrophoresis confirmed that fusion gene had been correctly inserted into the virus carrier PSS-CMV. The titer of virus was 2×108 by RT PCR. Immunohistochemistry determined virus expression.Conclusion:Recombinant adeno-associated virus that expressed the CGRP was successful established.(3) To treat the chick embryo CAM vasospasm with recombinant adeno-associated virus that expressed the CGRPObjective:To study the affection that recombinant adeno-associated virus that expressed the CGRP treats the chick embryo CAM vasospasm.Method:160 seventh chick embryos were applied,20 randomly divided into saline control group,70 in OxyHb group:randomly divided into seven sub groups with10 per group and other 70 in OxyHb-CGRP virus group:randomly divided into seven sub groups with 10 per group. Photographs were taken by digital camera at different periods, including 6 hours (h),12h,24h,2 days (d),3d,5d, and 7d. By means of Image-Pro Plus microscope and Image Analysis Software, the percentage of chick embryo CAM vessel area was measured. All data were indicated by average number plus or minus standard deviation, and data in the group and between the groups were analyzed by complete randomized design of variant analysis with statistical software SPSS 10.0. When P value was less than 0.05, the statistical significance was apparent. The pathological results of chick embryo CAM were obtained with the microscope to observe vascular morphology and determine whether the endothelium shredded or dropped, and so on. Result:In the early period (6-24hours), there were no significant difference between OxyHb+CGRP group and OxyHb group in chick embryo CAM area, percentage and pathological results (P>0.05). In the late period (3d-7d), the statistical difference in vascular area percentage was obvious (P<0.05). Meanwhile the pathological results showed that elastic membrane layer was not obviously ruffled or thickened and no new cell dropped.Conclusion:Recombinant adeno-associated virus that expresses CGRP is effective for treating CAM vasospasm. It provides a new way for preventing and treating CVS. |
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