Modification On Rat Model Of Left Lung Orthotopic Transplantation And The Immunomodulatory Effects Of CD8~+Tregs On Lung Transplant

Backgrounds: Lung transplantation is an established therapy for a variety of end-stagepulmonary diseases. According to the report from The Registry of the International Societyfor Heart and Lung Transplantation in2010, more than2700lung and heart-lungtransplantations were performed annually. But their outcomes are far from satisfied whencomparing with those of other solid organs. The overall survival rate after transplantation iscurrently79%at1year and about52%at5years. Furthermore, most immune andnon-immune mechanisms that complicate the following pathophysiological processesremain poorly understood. Accordingly, many experimental lung transplantation modelswere employed to develop novel preventative and therapeutic strategies. Orthotopic lungtransplantation in animal is a procedure that simulates human lung transplantation,following which, rejection，infection and ischemia/reperfusion processes, can offer muchinformation for transplantation research. By far, many animals models such as dog, pig,goat and rat have been employed for the research, but rat is one of the most prosmisingcandidates for the advantages of availability of inbred strains and potentiality of low costsince the first practice in1971，especially after Mizuta’s cuff technique and a variety of itsmodifications were introduced in1989and later. Objective: Optimize the previously established rat model of orthotopic single lungtransplantation with cuff technique.Methods:40LTs were performed with a new modified cuff technique in the experiments.The modifications embodied in the following procedures:1, both donors and recipientsreceived orotracheal intubation. To prevent the potential injury on vocal cords during thisprocedure,16G angiocatheter was selected.2, the cuff tail was cut off, so that any part ofthe cuff body could be hold during cuff inserting. In addition, a recess of0.2mm was madeat one end on cuff body. Ligation at the recess guaranteed the firm fixation and preventedthe cuff detachment.3, the left lung of recipient was preserved and retracted outside theincision for hilum exposition. It was removed until the graft was planted.4, in recipientprocedure, it was unnecessary to isolate the pulmonary artery, the pulmonary vein and themain bronchus thoroughly. Conservative dissection in hilum was performed to make twotunnels between these three structures for anastomosis. Soft tissues on their surface werepreserved to antagonize tension and protect stomas.5, the bronchus, the pulmonary veinand the pulmonary artery were anastomosed in turns.6, the model adopted theaforementioned modified procedures was performed by a single surgeon without the aidfrom extra collaborator and special instruments.Results:80rats received orotracheal intubation and77of them succeed, with the successrate of96.25%. The other3were failure and changed into tracheotomy. A total of39orthotopic left lung transplantation was smoothly performed and the other1was ceasedbecause of animal suffocation during operation. Blood pressure of recipient was stableduring the whole operation procedure (100±15mmHg), except for two transienthypotensions. The two transient hypotensions were observed at the beginning ofmechanical ventilation which gradually back to normal pressure and the period of hilumblocking which recovered at the point of reperfusion. Electrocardiogram excludedarrhythmia during the whole operation. All the cuff insertions succeed in one attempt withno twisting, laceration or leakage happened. Grafts were well inflated with perfect blood perfusion and the mean operative time was63.4±16.1minutes. One animal was died ofrespiratory failure8.5hours after operation and another one died on postoperative day10because of weakness.Conclusion: Orthotopic lung transplantation in rat model can be favorably preformed bymodified cuff technique. The modified procedure demonstrated many advantages, such aseasy graft implanting, short operation time，less complications and high reproducibility.Improvements adopted in this work are applicable in rat model of lung transplantation. Backgrounds: Transbronchial biopsy is considered as the gold standard for the diagnosisof rejection after lung transplantation. Its results contribute a decisive factor to thetreatment of planning regarding augmentation or adjustment in immunosuppressivemedication for suspended allograft rejection. Patients with pathologically confirmedrejection often demonstrate good response to immunotherapy. But interestingly, somepatients without pathological evidence of rejection could also benefit from the experimentaltreatment. Their resolution or strong improvement of symptoms under corticosteroidtherapy clinically indicates the existence of rejection. The reason for these phenomenons isunclear and the potential existence of undiagnosed rejection maybe relate to some extent.Accordingly, finding a reliable method to correctly diagnose rejection following lungtransplantation is of great importance. By far, perivascular mononuclear infiltration inbiopsies is considered as the characteristics element of acute lung rejection and adopted asone of the diagnostic criteria by transplant centers worldwide. On this basis, differentgrades of rejection are demarcated according to their intensity and extension. Unfortunately, the pathologic interpretation to such grades is challenging and sometimes problematic.Several factors could subjectively or objectively influence the application of thisclassification in rejection diagnosis and high variability of rejection grading was observedin the pathologic interpretation of transbronchial biopsy. Even in the same center, theconcordance rate of grading from different pathologists was fair to moderate. Thishighlights the inaccuracies and embarrassment during the diagnosing process. Allograftrejection is a complicated pathophysiological process during which the recruitment andactivation of T lymphocytes cause the injury and dysfunction of transplant. Therefore,qualitation and quantitation of lymphocytes in lung graft could be employed for rejectionsurveillance and treatment. Considering that the minimum HE staining offers limitedinformation for rejection grading, some special staining such as histochemical,immunohistochemical and in situ hybridization could be performed to facililate theinterpretation.Objectives: Study the pathological changes in allograft following rat lung transplantationand evaluate the application of immunohistochemistry in the diagnosis of rejection.Methods:90rats received allogeneic or isogeneic lung transplantation were sacrificed atdifferent postoperative time according to the experiment design. Parts of their lung tissueswere fixed, embedded, sectioned and stained with H.E. or immunohistochemistry forpathological examination.Results: Besides4cases of lung transplantation failed due to various reasons,37out of76allografts manifested complete atelectasis or destroyed. Lung injury happened mostly in theoutbred or stock transplant （p=0.006）and increased with postoperative time(p=0.017). Bycontrast, atelectasis was seldom found in the inbred recipient and was independent of time（p=0.926）. H.E staining of inbred graft section showed lung injury of alveolar exudation,septa edema and interstitial infiltration on postoperative day3. However, those lesionsdisappeared on postoperative day7and there was significant difference of lung injury scorebetween these two days（3.57±0.98vs0.86±0.69, p=0.000）. Focal or fused atelectasis and hemorrhagic, oedema or exudative lesions were seen in non-inbred lung transplant andbecame intensive with the time while the inbred graft looked like the normal lung withperfect ventilation and perfusion. Different degrees of typical rejection which were similarwith those in clinical lung transplantation could be seen on slides of grafts undermicroscope. They were corresponding to human A, B, C, and D type of rejection and theirsubtypes. In addition, immunohistochemically stained lymphocytes greatly facilitated thediagnosis and grading of rejection. Some rejection grading were accordingly modified andthe concordance rate between two diagnoses was71.95%with an overall kwof0.648(p=0.000). With the increase of rejection intensity, the lymphocytic infiltration in lunginterstitium became severer. The number of lymphocyte in graft of active groupoutweighed that of stable group（p﹤0.01）.Conclusions: Postoperative rejection following orthotopic rat lung transplantation couldmimic that of human. Immunohistochemistry could facilitate diagnosing and grading oflung rejection. Backgrounds: Emerging data suggest the survival benefit of lung transplantation forselected patients with end-stage lung disease. According to the2010report of theInternational Registry of the International Society for Heart and Lung Transplantation,more than2700cases of this procedure were annually performed worldwide and the1,3,5and10-year survival rate of recipients after surgery were79%,63%,52%and29%respectively. Of note, the first postoperative year is the most critical period, within which,nearly21%of patients died of perioperative accidents, underlying disease and most importantly, the acute rejection of graft. In addition, obliterative bronchiolitis （OB）whichmanifests progressive fibrotic destruction of the small airways greatly effects thepostoperative life quality of recipients and is identified as the leading cause of latemortalities after lung transplantation for allograft dysfunction. Statistically, nearly half ofthe patients who receive lung transplantation and survive3months are inclined to have thiscomplication and ample evidence has showed the high relevance between OB and acuterejection. Three or more episodes or one episode of mild acute rejection is associated withOB. What’s more, some think that the OB is the outcome of serials of recurrent acuterejections. Therefore, prevention, attenuation and effective treatment of acute rejectionfollowing lung transplantation is of important clinical significance. By far,immunosuppression remains the mainstay of therapy for acute rejection following lungtransplantation. Although the combinations of calcineurin inhibitor, antimetabolite,corticosteroid and mTOR inhibitors greatly improve the prognosis of lung transplantation,their relatively poor efficacy, side effects and related complications greatly challenge theoutcome. Tregs are population of T lymphocyte with regulatory characteristic ofself-tolerance and immune homeostasis. They have the advantages of maintenance ofimmune tolerance to transplanted tissues but potential to preserve intact immune responsesagainst pathogens. Therefore, using Tregs as therapeutic strategy is becoming promisingchoice for postoperative management. By far, different subset of Tregs had been identifiedand a plethora of data indicates their importance in experimental and clinicaltransplantation. But which Treg is associated with tolerance of lung graft and potential forthe clinical treatment is under investigated.Objectives: To explore the CD8+Tregs on the regulation of the acute rejection of rat lunggraft and their mechanism of immune tolerance.Methods: Different groups of adult male Lewis rats received isogenetic or allogenetic leftlung transplantation and sham operation respectively. They were sacrificed on scheduledpostoperative day according to the experimental design. HE staining was employed to evaluate the pathological changes in graft or control lung and their expressions of Foxp3and FR4were compared by western blot. Lymphocytes in peripheral blood, graft, locallymph nodes, spleen and bronchoalveolar lavage fluid were acquired by centrifugation,lysis or trituration and stained with fluorochrome conjugated antibodies. Their phenotypeand the expression of Foxp3were analyzed by flow cytometry.Results: Typical acute rejections of different degree could be observed on lung allograftswhile the lung of isograft or control manifested nearly normal.Western blot showedincreased expression of Foxp3in lung transplants. They were higher in isografts than thosein allografts of A1or A2rejection but similar as those in allografts of A3or A4rejection.Lymphocytes, mainly CD8+T cells, increased postoperatively in blood and lung. The ratioof CD4/CD8was less than1and kept decreasing with the intensity of acute rejection. Apopulation of CD8+C D25+Foxp3+T lymphocytes were identified either in blood or lungtissue.They were more in isografts than those in allografts. T lymphocytes could dividedinto CD45RC+and CD45RC-subgroups according to the expression intensity of CD45RCand the ratio of CD45RC+/CD45RC-in isograft and control group were lower than thosein allograft group. In addition, The ratio CD8+C D45RC-T reg to CD8+and CD3+T cell inlung tissue or local lymph nodes of control and isograft group were higher than those ofallograft group, but there were little differences in blood and spleen between these threegroups. Further study showed downward trends of CD8+CD45RC-Treg with theprolonging of postoperative day and the intensity of acute rejection. Finally, the ratio ofCD8+CD45RC-to CD3+in bronchoalveolar lavage fluid from isograft group was higherthan that from allograft group.Conclusions: There are at least two subsets of CD8+Tregs with immunomodulatory effectsin Lewis rats, namely CD8+C D25+F oxp3+T reg and CD8+CD45RC-Treg. They are involvedin the process of lung protection via direct or indirect inhibiting mechanism and related tothe tolerance to lung transplant...