| Purpose To these patients with end-stage heart disease or congenital heart disease that can not be cured by surgery,heart transplantation is an effective way. The patients number waiting for heart transplantation are increased year by year. However, the sources of hearts are limited. Currently, heart preservation is limited to 6 hours with the method of cold ischemic storage.The development of heart transplantation is limited. By improving the composition of the preservation solution, such as add the calcium ions and antioxidant, can reduce ischemical reperfusion injury to some extent,but the results is not satisfied.Finding a new way to preserve isolated hearts is instantly,this can alleviate the shortage of donor hearts.A completely new method,High-pressured mixed gas preservation is studied in this article.In this paper,we are supposed to observe whether the effect of high-pressured mixed gas preservation is better than HTK preservation,and to look into the possible mechanism of the high-pressured mixed gas preservation method.Method 1 Using 84 C57BL/6 male mice to establish model of mice cervical heterotopic heart transplantation.48 donor mice aged 4-6 weeks, weight 21±2g were randomly divided into four groups (n=12) and subjected to naive operation (Group A), sham operation (Group B), standard control (Group C) and experimental control (Group D).36 recipient mice aged 6-8 weeks,weight29±2g were randomly divided into three groups (n=12) and subjected to sham operation, standard control and experimental control.All the mice were provided by the Center of Experiment Animal of Sun Yat-sen University.Operation on donor mice:All the donor mice ate and drank as usual.5% chloral hydrate was injected to the mice by intraperitoneal injection.The surgical site was shaved and sterilized with 1% Povidone Iodine. Open chest wall and diaphragm to expose heart and inferior vena cava. lmL (100 u/mL) of 4℃ heparin saline through was injected through inferior vena cava. Hyperkalemia cardioplegic solution were injected through inferior vena cava slowly until the heart stopped beating.Remove heart and lung and put them into a culture dish with 4℃ heparin saline.Operation on donor hearts:Group A:hearts were isolated and the blood was removed; Group B:hearts were isolated and preserved in HTK solution at 4℃ for 24 h and transplanted; Group C:hearts were isolated and preserved in high pressured gas (PO2:3200hPa+PCO:800hPa=4000hPa) at 4℃ for 24 h and transplanted,establish model of mice cervical heterotopic heart transplantation:All the recipient mice were anaesthetized, shaved and sterilized as was mentioned above. By using modified Cuff technique we did the cervical heterotopic heart transplantation(under 16x double binocular operating microscope). When the procedure was done, recording the time when the whole donor heart start to re-beating. After transplantation, Penicillin (50 U/10g) was injected to all the recipient mice. The recipient mice were raised warm and alone after surgery.Testing after transplantation:Group A:when the donor hearts were isolated to make myocardial tissue slices from the apex of the heart; Group B and C: Myocardial tissue slices from the apex of the heart were made after transplantation for 24 h. Using hematoxylin and eosin (HE) staining to observe the pathological change of the donor hearts.Using Terminal-deoxynucleotidyl transferase mediated nick end labeling (Tunnel) test to dentify apoptotic myocardial cells,and count apoptotic cell.The expression of B cell lymphoma/leukemia-2 (Bcl-2) and microtubule-associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ wasted by Western blotting (WB).Results 1 hours after transplantation,9,1 and 8 hearts were rebeating in group B,C and D,the rebeating rate is 75%、8.3%、66.7% respectly.There is significant difference between Group B,C and D (r2=12.667, P=0.002<0.01).The rebeating rate of group B and group D is not significant (P=0.653>0.0167),while the rebeating rate of group D is significant higher than group C(P=0.003<0.0167) and the rebeating rate of group B is significant higher than group C(P= 0.001<0.0167).The donor heart graft function scores of group B,group C and group D is [4.5 (4.0～4.5)、0.75 (0.5-1.0)、2.5 (2.0～2.875)],the difference is significant(P<0.01).The donor heart graft function scores of group B is higher than than group C and group D (P<0.01).The difference of group D is higher than group C (P<0.01)The test of western blot showed that the expression of Bcl-2 is (0.187±0.018)、 (2.065±0.319)、(0.261±0.075)、(2.058±0.292) in group A,B,C and D.Among groups the difference is significant (F=280,P<0.05).The expression of Bcl-2 in group B is highest,while there is no significant difference in group B and group D (P >0.05),the expression of Bcl-2 in group D is higher than group C.The test of western blot showed that the expression of LC3-Ⅱ is (0.125±0.03)、(1.243±0.20)、 (0.157±0.06)、(0.867±0.18) in group A,B,C and D.Among groups the difference is significant (F=187, P<0.05).The expression of LC3-Ⅱ in group B is highest,while there is no significant difference in group A and group C(P>0.05)we analyzed the pathologic changes:myocardial fibers were regularly arranged with clear striations. No local swelling, neutrophil infiltration,or any other pathologic changes were found in group A; no obvious edema or any other pathologic changes were found in group B;cell edema, inflammatory cell infiltration can be found both in Group C and Group D, but in Group C the edema is more serious and the number of inflammatory cells is larger.Tunnel test shows that:there is no apoptotic cells in myocardial tissue in group A;there is seldom apoptotic cells in myocardial tissue in group B; large amount of apoptotic cells in myocardial tissue in Group C; a small amount of apoptotic cells in myocardial tissue weere found in Group D decreased deeply than group C.The apoptosis index was1.08±0.56%、 2.13±1.71%、26.72±5.23%、5.04±1.77% in each group. The difference between each groups is significant (F=209,P<0.01).The apoptosis index in Group C and Group D were significantly higher than that in Group A and B(P<0.01).While,in Group C,the apoptosis index increased significantly (P<0.01) when compared with Group D.There is no significant difference in group A and B (P>0.05)Method 2 Using 60 C57BL/6 male mice to establish model of mice cervical heterotopic heart transplantation.36 donor mice aged 4-6 weeks, weight 21±2g were randomLy divided into four groups (n=12) and subjected to naive operation (Group A), standard control (Group B) and experimental control (Group C).24 recipient mice aged 6-8 weeks,weight29±2 g were randomly divided into three groups (n=12) and subjected to sham operation, standard control and experimental control.All the mice were provided by the Center of Experiment Animal of Sun Yat-sen University.Operation on donor mice:All the donor mice ate and drank as usual.5% chloral hydrate was injected to the mice by intraperitoneal injection.The surgical site was shaved and sterilized with 1% Povidone Iodine. Open chest wall and diaphragm to expose heart and inferior vena cava.l mL (100 u/mL) of 4℃ heparin saline was injected through inferior vena cava. Hyperkalemia cardioplegic solution were injected through inferior vena cava slowly until the heart stopped beating.Remove heart and lung and put them into a culture dish with 4℃ heparin saline.Operation on donor hearts:Group A:hearts were isolated the blood was removed; Group B:hearts were isolated and preserved in HTK solution at 4℃ for 16 h and transplanted; Group C:hearts were isolated and preserved in high pressured gas (PO2:1600 hPa+PCO:400hPa=4000hPa) at 4℃ for 16 h and transplanted.Establishing model of mice heterotopic heart transplantation:All the recipient mice were anaesthetized, shaved and sterilized as was mentioned above. Using microforcep to expose abdominal aorta and inferior vena cave. When the procedure was done, recording the time when the whole donor heart start to re-beating. After transplantation, Penicillin (50U/10g) was injected to all the recipient mice. The recipient mice were raised warm and alone after surgery.Testing after transplantation:Group A:when the donor hearts were isolated to make myocardial tissue slices from the apex of the heart; Group B and C: Myocardial tissue slices from the apex of the heart were made after transplantation for 24h. Using hematoxylin and eosin (HE) staining to observe the pathological change of the donor hearts.Using Terminal-deoxynucleotidyl transferase mediated nick end labeling (Tunel) test to dentify apoptotic myocardial cells,and count apoptotic cell. The expression of tumor necrosis factor a (TNF-a), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-10(IL-10) was observed by reverse transcriotion-polymerase chain reaction (RT-PCR).Results 2 hours after transplantation,3and 10 hearts were rebeating in group Band D,the rebeating rate is 25%、83.3% respectly.The difference is significant(.P=0.012<0.05).The donor heart graft function scores of group B and group C is [1.30 (0.963-1.54)、3.50 (3.125～3.938)],the difference is significant (Z=-3.447,P<0.01).The donor heart graft function scores of group C is higher than than group B(P<0.01).The results of RT-PCR test:Group B:Relative units of TNF-a gene expression in group B and C is (5.095±0.421)and(3.602±0.219),the TNF-a expression level was greatly decreased in Group C compared with Group B (t=10.902, P<0.01); Group B:Relative units of IL-1β gene expression in group B and C is(4.924±0.825)and (2.891±0.278), the IL-1β expression level was greatly decreased in Group C compared with Group B (t=8.084, P<0.01);Group B:Relative units of IL-6 gene expression in group B and C is (5.345±0.667)and (3.033±0.320),the IL-6 expression level was greatly decreased in Group C compared with Group B (t=10.821, P<0.01); Group B:Relative units of ICAM-1 gene expression in group B and C is (4.892±0.817)and (3.073±0.524), the ICAM-1 expression level was greatly decreased in Group C compared with Group B (t=6.493, P<0.01);Group B:Relative units of IL-10 gene expression in group B and C is (3.617±0.419) and (7.190±1.048), the IL-10 expression level was greatly decreased in Group B compared with Group C (t=10.971, P<0.01).we analyzed the pathologic changes:myocardial fibers were regularly arranged with clear striations. No local edema, neutrophil infiltration, or other pathologic changes were found in group A; edema, inflammatory cell infiltration can be found both in Group B and Group C, but in Group C the edema is slighter and the number of inflammatory cells is smaller.Tunnel test shows that:there is no apoptotic cells in myocardial tissue in group A; Group B:large number of apoptotic cells in myocardial tissue; Group C:a small amount of apoptotic cells in myocardial tissue, decreased significantly than Group B. The apoptosis index in Group B was14.18±4.75% and in group C was 6.61±1.06%,which were both significantly higher than that in Group A(1.36±0.30%).The difference between each group is significant(F=62.963, P<0.01).In Group B,the apoptosis index increased significantly (P<0.01) when compared with Group C.Conclusions In conclusion,High-pressured mixed gas is a new and effective way to preserve donor hearts, better than the current organ preservation method which is mostly an immersion method by using HTK solution.The anti-inflammatory and anti-apoptosis biological effect of CO can protect isolated hearts.High-pressured mixed gas preservation can reduce cold and reperfusion injury of donor hearts, possibly by enhancing autophagy to product energy and reducing myocardial cell apoptosis, but further study should be done to figure out the true mechanism of this new preservation method. |
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