| Part1:Establishment and modification on rat model of left lung orthotopic transplantationBackgrounds:Lung transplantation is an established therapy for a variety of end-stage pulmonary diseases. According to the report from The Registry of the International Society for Heart and Lung Transplantation in2010, more than2700lung and heart-lung transplantations were performed annually. But their outcomes are far from satisfied when comparing with those of other solid organs. The overall survival rate after transplantation is currently79%at1year and about52%at5years. Furthermore, most immune and non-immune mechanisms that complicate the following pathophysiological processes remain poorly understood. Accordingly, many experimental lung transplantation models were employed to develop novel preventative and therapeutic strategies. Orthotopic lung transplantation in animal is a procedure that simulates human lung transplantation, following which, rejection, infection and ischemia/reperfusion processes, can offer much information 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 prosmising candidates for the advantages of availability of inbred strains and potentiality of low cost since the first practice in1971, especially after Mizuta’s cuff technique and a variety of its modifications were introduced in1989and later. Objective:Optimize the previously established rat model of orthotopic single lung transplantation 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 recipients received orotracheal intubation. To prevent the potential injury on vocal cords during this procedure,16G angiocatheter was selected.2, the cuff tail was cut off, so that any part of the cuff body could be hold during cuff inserting. In addition, a recess of0.2mm was made at one end on cuff body. Ligation at the recess guaranteed the firm fixation and prevented the cuff detachment.3, the left lung of recipient was preserved and retracted outside the incision for hilum exposition. It was removed until the graft was planted.4, in recipient procedure, it was unnecessary to isolate the pulmonary artery, the pulmonary vein and the main bronchus thoroughly. Conservative dissection in hilum was performed to make two tunnels between these three structures for anastomosis. Soft tissues on their surface were preserved to antagonize tension and protect stomas.5, the bronchus, the pulmonary vein and the pulmonary artery were anastomosed in turns.6, the model adopted the aforementioned modified procedures was performed by a single surgeon without the aid from extra collaborator and special instruments.Results:80rats received orotracheal intubation and77of them succeed, with the success rate of96.25%. The other3were failure and changed into tracheotomy. A total of39orthotopic left lung transplantation was smoothly performed and the other1was ceased because of animal suffocation during operation. Blood pressure of recipient was stable during the whole operation procedure (100±15mmHg), except for two transient hypotensions. The two transient hypotensions were observed at the beginning of mechanical ventilation which gradually back to normal pressure and the period of hilum blocking which recovered at the point of reperfusion. Electrocardiogram excluded arrhythmia during the whole operation. All the cuff insertions succeed in one attempt with no 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 of respiratory failure8.5hours after operation and another one died on postoperative day10because of weakness.Conclusion:Orthotopic lung transplantation in rat model can be favorably preformed by modified cuff technique. The modified procedure demonstrated many advantages, such as easy graft implanting, short operation time, less complications and high reproducibility. Improvements adopted in this work are applicable in rat model of lung transplantation. Part2Pathological change in allograft following orthotopic rat lung transplantationBackgrounds:Transbronchial biopsy is considered as the gold standard for the diagnosis of rejection after lung transplantation. Its results contribute a decisive factor to the treatment of planning regarding augmentation or adjustment in immunosuppressive medication for suspended allograft rejection. Patients with pathologically confirmed rejection often demonstrate good response to immunotherapy. But interestingly, some patients without pathological evidence of rejection could also benefit from the experimental treatment. Their resolution or strong improvement of symptoms under corticosteroid therapy clinically indicates the existence of rejection. The reason for these phenomenons is unclear and the potential existence of undiagnosed rejection maybe relate to some extent. Accordingly, finding a reliable method to correctly diagnose rejection following lung transplantation is of great importance. By far, perivascular mononuclear infiltration in biopsies is considered as the characteristics element of acute lung rejection and adopted as one of the diagnostic criteria by transplant centers worldwide. On this basis, different grades 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 this classification in rejection diagnosis and high variability of rejection grading was observed in the pathologic interpretation of transbronchial biopsy. Even in the same center, the concordance rate of grading from different pathologists was fair to moderate. This highlights the inaccuracies and embarrassment during the diagnosing process. Allograft rejection is a complicated pathophysiological process during which the recruitment and activation of T lymphocytes cause the injury and dysfunction of transplant. Therefore, qualitation and quantitation of lymphocytes in lung graft could be employed for rejection surveillance and treatment. Considering that the minimum HE staining offers limited information for rejection grading, some special staining such as histochemical, immunohistochemical and in situ hybridization could be performed to facililate the interpretation.Objectives:Study the pathological changes in allograft following rat lung transplantation and evaluate the application of immunohistochemistry in the diagnosis of rejection.Methods:90rats received allogeneic or isogeneic lung transplantation were sacrificed at different postoperative time according to the experiment design. Parts of their lung tissues were fixed, embedded, sectioned and stained with H.E. or immunohistochemistry for pathological examination.Results:Besides4cases of lung transplantation failed due to various reasons,37out of76allografts manifested complete atelectasis or destroyed. Lung injury happened mostly in the outbred or stock transplant (p=0.006) and increased with postoperative time(p=0.017). By contrast, 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 lesions disappeared on postoperative day7and there was significant difference of lung injury score between 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 and became intensive with the time while the inbred graft looked like the normal lung with perfect ventilation and perfusion. Different degrees of typical rejection which were similar with those in clinical lung transplantation could be seen on slides of grafts under microscope. They were corresponding to human A, B, C, and D type of rejection and their subtypes. In addition, immunohistochemically stained lymphocytes greatly facilitated the diagnosis and grading of rejection. Some rejection grading were accordingly modified and the concordance rate between two diagnoses was71.95%with an overall kw of0.648(p=0.000). With the increase of rejection intensity, the lymphocytic infiltration in lung interstitium became severer. The number of lymphocyte in graft of active group outweighed that of stable group (p＜0.01)Conclusions:Postoperative rejection following orthotopic rat lung transplantation could mimic that of human. Immunohistochemistry could facilitate diagnosing and grading of lung rejection. Part3:CD8+Tregs on the regulation of the acute rejection of rat lung graft and their mechanism of immune toleranceBackgrounds:Emerging data suggest the survival benefit of lung transplantation for selected patients with end-stage lung disease. According to the2010report of the International 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) which manifests progressive fibrotic destruction of the small airways greatly effects the postoperative life quality of recipients and is identified as the leading cause of late mortalities after lung transplantation for allograft dysfunction. Statistically, nearly half of the patients who receive lung transplantation and survive3months are inclined to have this complication and ample evidence has showed the high relevance between OB and acute rejection. Three or more episodes or one episode of mild acute rejection is associated with OB. What’s more, some think that the OB is the outcome of serials of recurrent acute rejections. Therefore, prevention, attenuation and effective treatment of acute rejection following lung transplantation is of important clinical significance. By far, immunosuppression remains the mainstay of therapy for acute rejection following lung transplantation. 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 the outcome. Tregs are population of T lymphocyte with regulatory characteristic of self-tolerance and immune homeostasis. They have the advantages of maintenance of immune tolerance to transplanted tissues but potential to preserve intact immune responses against pathogens. Therefore, using Tregs as therapeutic strategy is becoming promising choice for postoperative management. By far, different subset of Tregs had been identified and a plethora of data indicates their importance in experimental and clinical transplantation. But which Treg is associated with tolerance of lung graft and potential for the clinical treatment is under investigated.Objectives:To explore the CD8+Tregs on the regulation of the acute rejection of rat lung graft and their mechanism of immune tolerance.Methods:Different groups of adult male Lewis rats received isogenetic or allogenetic left lung transplantation and sham operation respectively. They were sacrificed on scheduled postoperative 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, local lymph nodes, spleen and bronchoalveolar lavage fluid were acquired by centrifugation, lysis or trituration and stained with fluorochrome conjugated antibodies. Their phenotype and the expression of Foxp3were analyzed by flow cytometry.Results:Typical acute rejections of different degree could be observed on lung allografts while the lung of isograft or control manifested nearly normal.Western blot showed increased expression of Foxp3in lung transplants. They were higher in isografts than those in 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 ratio of CD4/CD8was less than1and kept decreasing with the intensity of acute rejection. A population of CD8+CD25+Foxp3+T lymphocytes were identified either in blood or lung tissue.They were more in isografts than those in allografts. T lymphocytes could divided into CD45RC+and CD45RC-subgroups according to the expression intensity of CD45RC and the ratio of CD45RC+/CD45RC-in isograft and control group were lower than those in allograft group. In addition, The ratio CD8+CD45RC Treg to CD8+and CD3+T cell in lung tissue or local lymph nodes of control and isograft group were higher than those of allograft group, but there were little differences in blood and spleen between these three groups. Further study showed downward trends of CD8+CD45RC-Treg with the prolonging of postoperative day and the intensity of acute rejection. Finally, the ratio of CD8+CD45RC-to CD3+in bronchoalveolar lavage fluid from isograft group was higher than that from allograft group.Conclusions:There are at least two subsets of CD8+Tregs with immunomodulatory effects in Lewis rats, namely CD8+CD25+Foxp3+Treg and CD8+CD45RC-Treg. They are involved in the process of lung protection via direct or indirect inhibiting mechanism and related to the tolerance to lung transplant... |
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