A Randomized Trial On The Changes Of Activities Of Nitric Oxide Synthases In The Carotid Allografts Of Rabbits

PrefaceNowadays, transplantation of vascular autografts and reconstruction of human's vessels, aiming at resuming the normal blood flow and rebuilding the per-fusion of tissues as fast as possible, has been widely undertaken in clinical remedies. But the resources of patients' vessels for autografting are pretty limited practically. It even might be very difficult and would take much time to harvest autograft vessels under some emergent circumstances, especially in those patients who had got multi - organic trauma. It would ask for many a doctor to take part in the harvest of vessels, which would make much less room for operating space and prolong the overall operation time. This would be of much awful disadvantage for salvaging the patients in danger.The aim of this trial is to assess the influence of transplantation of allografts vessels upon the varieties of both the histomorphological pathology and the enzy-mologic activities of nitric oxide synthases (NOSs) of the allografts through establishing an animal model of common carotid transplantation in vivo between two kinds of homozygotic rabbits. We randomizedly took New Zealand rabbit as the donors of allografts, and Janpanese rabbit as the recipients in this trial. En-dothelial constitutive NOS (ecNOS) and inducible NOS (iNOS) in allografts vessels were detected at different levels, namely body fluid (plasma NO) , enzymatic activities (protein) , and gene expression (mRNA). We compared the long - term outcome of allografts vessels without any exposure factors, or correspondingly preconditioned by antibiotic sterilization and preserved under the room temperature, or cryopreserved in liquid nitrogen after being pretreated bytwo antibiotics (Penicillin/Streptomycin) , to those in vivo implantation of auto-grafts. Our goals are to find a relative easier and more practical way of preconditioning and preserving allograft vessels to keep up them in long - term functional unimpeded fettle without early functional failure, to accumulate experiences and creditable evidences which could give some theoretical advice and could help to establish a bank of human allograft vessels in the forthcoming days.Materials and Methods1. 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 (HERMLE Z383K) , PTC -100 PCR amplifier, KODAK ID gel image analyzer, and Type S -500P ultraviolet/visible - light sequential spectrophotome-ter. All RT - PCR reagents, including total RNA extraction kit, cDNA synthesis kit, PCR amplification kit, and marker (ΦX174/Hinc Ⅱ) , 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 iNOS/ecNOS were respectively designed according to the known gene sequences of rabbit iNOS/ecNOS free downloaded from GenBank of National Library of Medicine of USA. The primer sequence fragments of rabbit ecNOS/iNOS were correspondingly 5' - TTC CGC TAC CAG CCA GAC - 3' (ecNOS - U sense) , 5' - CGA GGG ACA CCA CAT CAT AC - 3'( ecNOS - L antisense) , 5' - CAT TGC ATT CAG ATC CCG AAA CG -3' (iNOS -U sense), 5' -GGA AGC CTC TTG TCT TTG ACC -3' (iN-OS - L antisense) . The other detective reagents and detection kits were Medium RPMI 1640 (Gibcol BRL Co. ) , Dimethyl Sulphoxide (DMSO, Merck Co.) , Penicillin (PC, 400 MIU per ampoule) , Streptomycin (SM, 100 MIU per ampoule) , immunohistochemistry detection kits for ecNOS/iNOS, including anti -rabbit lamb serum (multicloning antibodies) , hegoat serum (non - specific) and all - purpose SP detection kit).2. Grouping and Precondition Methods: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 randomizedly selected as donors of common carotid allografts (Group G). Thirty healthy homozygotic Japanese rabbits (male) , body weight from 1.65 kg to 2.50 kg ( mean ± SD, 1. 89 ±0. 19 kg) , were taken as recipients and randomizedly assigned into four groups ( Group A, n = 3, null control; Group B, n =9, the 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 to the surviving days of postoperation (POD) . Grouping criteria: subgroup I: less than POD 7; subgroup Ⅱ: POD 12-15; subgroup Ⅲ; more than POD 15. Three rabbits in the Group A underwent in situ reimplanting 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.3. 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 allografts 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' follow -up. The harvested blood samples (about 5 ml) were centrifuged for 15 minute3 000 revolutions per minute (rpm) under room temperature, the plasma were extracted, infused into plastic tubes pre - syringed with EDTA - 2Na/aprotinin (30 μl 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 were an obstruction, thrombosis, or stenosis in the graft fragment. Harvesting all the autografts/al-lografts, cut off a fragment about 3 mm, fixed it with 4. 0% paraformaldehyde with 0. 1% DEPC for 18-24 hours, embedded with wax after automatically serial dehydration, and slided it every 5 μm for histomorphological stains and immunohistochemistry stains; the rest (around 1 cm) was deposited into a labeled plastic tube with a screwed cap proportionally.4. Observational data;( 1 ) Plasma NO concentration (nitrite reductase) : Manipulated under the instruction of test kit for NO in body liquid. (2) 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 lymphocytes) , if there would aci-dophilic 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. ( 3 ) Activities of iNOS/ecNOS (Immunohistochemistry) : Manipulated under the direction of the immunohistochemistry detection kit for iNOS/ecNOS of rabbits. Single - blindly examined, evaluated and recorded the site, size, and density of granules (nigger - brown staining) of positively stained cells ( cy-tomembrane 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. (4) Expression of iNOS/ecNOS mRNA in the allografts ( reverse transcription polymer-ase chain reaction):Extraction total RNA from allografts and synthesis of cloning DNA (cDNA) were operated under super clean circumstances. The tempera-lures of denaturation, annealing, and primer extension were respectively 94℃ , 61/57 ℃, and 72 ℃ for iNOS/ecNOS, and cycles were 30 and 32. The density of amplification bands of iNOS/ecNOS was standardized by the densitometric data of beta - actin before statistical analysis.5. Statistical Analysis: All the data were expressed as mean ± standard deviation and analyzed by the statistic software SPSS 8. 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.Result1. All the samples of implanted vessels but one were obstructed by white thrombus expanding into the distal end, its proximal end kept up open but with no coming blood flow. Total unobstructed ratio was less than 4 % (1/30). Unobstructed ratio in Group A was significant higher than the other groups. The vascular structural destruction of autografts was the least in all the trial groups. Allografts' structure in Group D was less destroyed much similar to group A and better than those in Group B or C significantly. Allografts in Group B were de-structed the worst in all the observatory groups.2. The diversifications of plasma NO concentrationl: There seemed no significant difference amid Group A(36. 33 ±35. 35μmol/L) , Group C(55. 67 ± 17.44 μmol/L)and Group D(50.22 ± 15. 26μmol/L) ( P >0. 05) . However, the plasma NO concentration of rabbits in Group B(69.22 ±22. 60μmol/L) was significant higher than that in Group A and D( P < 0.05).3. Activities of iNOS/ecNOS: (1) There seemed no expression of iNOS in Group A and D. The ratio and intensity of iNOS expression in Group B were 22.2% and 44.4% correspondingly. Ratio and intensity of iNOS expression in Group C was 44.4% and 77. 8% , which were significantly higher than the other groups( P <0.05). (2) The expression of ecNOS was otherwise much different from what we had seen above. The ratio of expression was respectively 33. 3% ,33. 3% , and 22.2% in Group A, B and D. There was no significant difference between these three groups. The intensity of ecNOS in Group A was 66. 7% , which was significantly higher than the other groups (55.6 % and 33. 3% , respectively in Group B and D) . There seemed not any expression of ecNOS at all in Group C.4. Expression of iNOS/ecNOS mRNA:(1) The expressions of iNOS in Group B (1.20 ±0.06) was significantly higher than that in Group A( 1. 08 ± 0.13), C(1.04±0.13), and D (1.03 ±0.26), all the value of P less than 0.05. (2) But on the contrary, the expression of ecNOS mRNA in Group B (0. 60 ±0. 55) was significantly less than that of Group A(0. 96 ± 0. 06) , C (1.06±0.03) and D(1.06±0.01)( P < 0.05). There seemed no significant difference between Group A, C, and D in both the expression of iNOS and ecNOS at mRNA level.DiscussionThe antigenicity of vessel allografts is the main factor leading to the early functional failure of allografts. The clinical and pathological behavior of graft coronary arteriosclerosis (GCA) is much different in many ways from that of primary arteriosclerosis, but there seem many similar or common mechanisms between them. They are all the results of inflammatory responses, if taking a look from the broad sense of immunology. The episode of coronary arteriosclerosis is the consequence of interactions between inflammatory mediators and vascular en-dothelial cells (VECs). All types of arteriosclerosis behaved some common features concerned about activation of immune system; GCA is just a peculiar example amid them.NO was first researched many decades ago as a sort of contaminate gases. But it got "great reputation" from earlier 1990s after some research evidences shown it is the "hot spot" molecule taking part in many courses of biophysiolo-gy. NO synthases ( NOSs) were extracted from many different cells and the NOSs gene sequences in different species were amplified and submitted by re-searchers day after day.Today, many research works backup the theory that NO is a sort of messenger molecule, effector molecule, and oxygen free radicals (OFRs). It could be "both angel and Satan" because it can arouse explosion of OFRs, which could be magnified into well - known uncontrollable "water-fall" effects. NO could also regulate and modulate both the inflammatory response and rejection.NO is synthesized by hereto three types of NOSs (endothelial constitutive NOS, inducible NOS, and neuronal NOS) , all of which is expressed in VECs. NO production is close related to the activities of these three NOSs.Our trial findings shown that the activities and expressions of iNOS/ecNOS in situ autografts (Group A) varied the least. The vascular structure destructed a little, there's only infiltration of some inflammatory cells inside the wall of autografts. The "lucky dog" of unobstructed graft was observated in Group A. It might be deduced that even though autografts didn't lead to immune rejection, but could undoubtedly cause inflammatory reactions.The histomorphological exhibitions of allografts in Group B, C, D were nearly covered the histopathological manifestations of all the four types of rejection. We found the expression of ecNOS in allografts rinsed by heparin/NS (Group B) was much significant less than autografts (Group A) and allografts in Group C and D. Otherwise, the expression of mRNA of iNOS in allografts of Group B was significant higher than that in Group A, C and D. The vascular structure in Group B destructed the worst in all the three allografts groups. It might result from the eruption of systemic NO because of both the local and somatic NOSs enzymatic system, especially the iNOS was activated by inflammatory reactions and immune rejection caused by their fierce antigenicities. We found the plasma NO level in Group B was much higher than the others'. Consequently, the fragment of allograft was much destructed and behaved structural deformation, fibrinoid necrosis of the allograft vascular structures. We even encountered some white thrombus inside the lumen of allografts, which had been organized and nearly rotted over the walls in the severe cases. There were too much cellular infiltration and hyperaemia in vascular tissues. It showed that theunsalted allograft would be doomed to lead to drastic rejection after being implanted into recipient's body, to activate the iNOS system beyond its in situ milieu, and to lose its function and commit necrosis earlier after being implanted. But the possibility of onset of graft - versus - host disease (GVHD) will be much less because the quantity of heterogeneous cells, MHC - IIA/HLA related antigenicities and antibodies held by allografts were awful limited. But immediately after the recovery of blood flowing, the heterogeneous antigens in allografts VECs would contact with recipient's blood, which is the carrier of both the body liquid immune and cellular immune. It is the ABO blood - type antigens system that acts as the "prime criminal" and triggers the rejection. The ABO blood -type antigens could be expressed in multi - organs and tissues. It might be one of the key reasons related to agglutinin that the combination of antigen/antibody lead to allografts' failure. The eruption of NO would improve the phagocytosis, chemotaxis of Macrophages (MΦ) , and would enhance its ability as antigen presenting cells (APC).We found that there seemed no expression of ecNOS in Group C allografts, the plasma NO concentration level was at relatively normal range, and the vascular structures were also next to normal in the allografts vessels of Group C. But there were too many ruptures, hyperaemia, thrombosis, and organization of thrombus inside the graft vessels. The vitality and the antigenicity of all the cells of allografts, such the donor's blood cells with immunoactivity, especially the VECs, were probably reduced or even completely knocked out through the precondition of antibiotics and or preservation under room temperature, which could be taken as a sort of "hot ischemia". Even though there might no episodes of severe immune rejection, and the massive release of NO after the activation of iNOS caused by the inflammatory reactions and tissues necrosis around the allografts would be much helpful for the repairing and remodeling of allografts, but retarded VECs function would never be "Gospel" because the failure of VECs' barrier would boost the aggregation of platelets, leukocytes, and immunocytes. MΦ and vascular smooth muscle cells (VSMCs/SMC) would penetrate damaged VECs layer and congregate under the VECs, which is the main cause leading to...