The Variations Of Vascular Morphology And Tumor Necrosis Factor In Carotid Allografts Of Rabbit

PrefaceThe vascular damage is one of the surgical diseases commonly encountered in clinical work. Along with the rapid development technique of composite graft, the high quality and effect of composite graft could basically meet with the clinical want. But it commits some despair problem when applying in the repair of small vessels. Up to now, most of the repair of small vessels asks for autografts. Allografts are widely used in the reconstruction and repair of vessels. But the limited sources of autografts confine the effective rescue of some emergent traumatic patients. One practical ways is to get an alternative of autografts, the vigorous allografts, so as to effectively rescue more patients in time. However, the ischemic -reperfusion process and the recipients immune response of allografts infinitely limit a successful outcome of vascular transplantation. Tumor necrosis factor (TNF), a sort of autocrine proinflammatory factors, plays an important role in the preeess of isehemie -reperfusion process and recipients immune response of allografts, which could lead to early failure of the allogafts and transplanted organs.So as to find the practical and feasible methods for precondition and preservation of allografts, we established an animal model of carotid transplantation in Japan rabbits, and investigated the variation TNF on the level of pathological morphology, immunochemical enzyme, and mRNA expression, to evaluate the role of TNF in the vascular transplantation. Materials and MethodsGrouping and Precondition Methods: Thirty healthy homozygotic Japanese rabbits (male), body weight from 1.65 kg to 2.5 kg ( mean±SD, 1.89±0.19 kg), were taken as recipients and random assigned into four groups (Group A, n=3, null control; Group B, n=9, least interferences; Group C, n=9, precubated with antibiotics and banked under room temperature; Group D, n=9, preconditioned the same way as Group C but cryopreserved in liquid nitrogen). The rabbits in Group B, C, and D were divided into three subgroups (3 per subgroup) according the surviving days of postoperafion days (POD). Grouping criteria: subgroupⅠ: less than POD 7; subgroupⅡ: POD 12-15; subgroupⅢ: more than POD 15. Three rabbits in the Group A underwent in situ re-implanting of autografts and taken as the null control. Rabbits in Group B accepted in vivo allografts without any precondition but rinsed by normal saline (NS, 0.9％) with heparin (25 IU/ml). Those in Group C were in vivo implanted with allografts pre-incubated by antibiotics (1.04％Medium RPMI 1640 with Penicillin/Streptomycin, 50 IU/ml equally), airproof deposited under room temperature and used in less than 72 hours after harvesting. Rabbits in Group D were in vivo implanted by allografts cryopreserved after sterilization with antibiotics (the same way as done in Group C). Five healthy homozygotic New Zealand rabbits (male), body weight from 2.8 kg to 3.2 kg (mean SD, 3.02 0.21 kg), were random selected as donors of common carotid allografts (Group G).Major Instruments and Reagents: The equipments of this trial were microsurgery magnifier (Keeler 2.5), Type SSW-3 microsurgery instruments, deep-hypothermic freezer (86C FORMA-725 ), low- temperature centrifuge (HERMIF. Z383K), PTC-100 PCR amplifier, KODAK ID gel image analyzer, and Type S-500P ultraviolet/visible-light sequential spectrophotometer. All RT-PCR reagents, including total RNA extraction kit, cDNA synthesis kit, PCR amplification kit, and marker, were commercially purchased from TaKaRa Biotechnology Co., Ltd. PCR were undertaken with two paired primer sequences designed by Primer Premiere free -download software from Internet. The sequences of primers of rabbit TNF were respectively designed according to the known gene sequences of rabbit TNF free downloaded from GenBank of National Library of Medicine of USA. The other detective reagents and detection kits were Medium RPMI 1640 (Gibed BRL Co. ), Dimethyl Sulphoxide (DMSO, Merck Co. ), Penicillin (PC, 400 MIU per ampoule), Streptomycin (SM, 100 MIU per ampoule), immunohistochemistry detection kits for TNF, including anti-rabbit lamb serum (multicloning antibodies), hegoat serum (non-specific) and all-purpose SP detection kit)Animal Model: All the operations of compounding of reagents, harvesting or implanting of carotids were randomizedly undergone in sterilized room as bacteria-free as possible under the guidelines for human. Autografts in Group A were implanted immediately after rinsed the visible residential blood cells with NS with heparin (25 IU/ml). The ways of transplantation of aUografts in Group B, C, and D were very similar to what had been done in Group A except the preconditing ways of allografts were of awful difference. All the animals were observed, evaluated, and recorded per day such contents as the activity, and the dietetic and living quality. The grafts were harvested after 5 -21 days'followup. The harvested blood samples (about 5 ml) were centrifuged for 15 minute, 3 000 revolutions per minute (rpm) under room temperature, the plasma were extracted, infused into plastic tubes pre- syringed with EDTA-2Na)/aprotinin (30μl equally), and cryopreserved in a deep-hypothermic freezer for testing plasma NO concentration. Recorded the findings (graft's appearance and diameter) around the graft vessel and checked up if there was an obstruction, thrombosis, or stenosis in the graft fragment. Harvesting all the autografts/allografts, cut off a fragment about 3 mm, fixed it with 10％paraformaldehyde for 18-24 hors, embedded with wax after automatically serial dehydration, and slided it every 5 m for histomorphological stains and immunohistochemistry stains; the rest (around 1 cm) was deposited into a labeled plastic tube with a screwed cap proportionally.Observational data: (1) Histomorphological transformations (Hematoxylon/Esin staining): Single-blindly observated and recorded if there would be thrombosis and its quality, if the layers of vascular walls could be differentiated from each other, how about the infiltration intensity of leukocytes (granulocytes or Iymphocytes), if there would acidophilic or basophilic granules in the cellular cytoplasm, whether there would be varieties of cell nucleus of arterial tissues (e. g. chromatolysis, karyopycnosis, dissolution of nucleus, plasmolysis), and if there would be hyperplasia of intima or media. (2) Activities of TNF (Immunohistochemistry): Manipulated under the direction of the immunohistochemistry detection kit for TNFof rabbits. Single-blindly examined, evaluated and recorded the site, size, and density of granules (nigger-brown staining) of positive-stained ceils (cytomembrane or cytoplasm) with light microscope. Scoring of immunohistochemistry stain was expressed as follow as: 0 (negative, -), no visible staining; 1 (weakly positive, +), few cells with faint staining; 2 (positive, + + ), moderate staining; 3 (intensively positive, + + ), intense-diffuse staining. (3) Expression of TNF mRNA in the allografts (reverse transcription-polymerase chain reaction):Extraction total RNA from allografts and synthesis of cloning DNA (cDNA) were operated under super clean circumstances. The density of amplification bands of TNF was standardized by the densitometric dataof beta-actin before statistical analysis.5. Statistical Analysis: All the data were expressed as mean standard deviation and analyzed by the statistic software SPSS 10.0 For Windows with Student t test (single -tailed paired or unpaired double specimen with equal variance, ), or a Chi-Square test. Values of P＜0.05 were considered as significance.ResultsThe morphological variation of allografts: All the allografts manifested some extent inflammatory reactions or vascular structural damage. The allografts in Group B (without any precondition) were damaged the most obviously worse, and even included all sorts of structural damage represented in the trial groups, and especially appeared necrosis of vascular cells, infiltration of inflammatory cell, such as macrophages and lymphocytes. A great deal of organized thrombus within the lumen eroded into the wall of allografts o The vascular structure of al lografts kept the best in Group D, but also committed thrombosis, organization and dissolution of thrombus. The allgrafts in all the subgroups manifested utmost similar.The unobstructed ratio: The criterion of in - time unobstruction is both the proximal and distant of allografts kept open right after anostomosis while operation. The criterion of unobstruction at the end of the study is arterial blood flows out of the incision of allografts while harvesting the allografts. The unobstruction ratio is the ratio of the numbers of unobstructed allografts divided by all the num ber of allografts in the trial group or subgroup. The in - time unobstruction ratio were 100％(30/30). The unobstructed ratio is, respectively, 100％(3/3) in Group A, 44.4％(4/9) in Group B, 55.6％(5/9) in Group C, and 77. 8％(7/9) in Group D.The immunohistochemical staining of TNF: The density of immunohistochemical staining of TNF is one -blind observed and expressed as respectively, the null, without any staining particles; score 1, some cells in some observation fields full of staining particles; score 2, most of the cells in some observation fields full of staining particles; and utmost all the observation fields found cells full of staining particles. The scores of immunohistochemical staining of TNF in Group A and D were significantly than that of Group B and C. And the scoring of Group B was the highest in all the trial groups (P＜0.05). And TNF in allografts of Group D was significantly less than those without any precondition (P＜0.05).The results of the scores of immunohistochemical staining of TNF in myocytes were much similar to that in allografts. That is, the score in Group B was significant higher than the Group A and D (P＜0.05). But there seems no statistical significance between Group B and C. The score of allografts in Group D was also less than that of unpretreated group.The mRNA expression of TNF: The mRNA expression of TNF of allografts in Group B was significantly up-regulated than Group A (P＜0.05). However, there seemed no significantly statistical difference between any two of the other groups. ConclusionsThe allografts preconditioned by antibiotics and preserved in liquid nitrogen could obtain the best unobstrution ratio and minimal inflammatory response, and prudently suggested utmost the same clinical curative effect as the autografts.Ischemic-reperfusion injury and, or immune response could cause the bursting release of TNF in allografts and overall the recipients some important organs, such as the heart, and lead to pathological damage and dysfunction of allografts.Differential levels of TNF mRNA expression could be found in all the allografts, particularly in those without any pretreatedallografts.