It has been 5 years since our team reported the first successful model of orthotopic single lung transplantation in the mouse. There has been great demand for this technique due to the obvious experimental advantages the mouse offers over other large and small animal models of lung transplantation. These include the availability of mouse-specific reagents as well as knockout and transgenic technology. Our laboratory has utilized this mouse model to study both immunological and non-immunological mechanisms of lung transplant physiology while others have focused on models of chronic rejection. It is surprising that despite our initial publication in 2007 only few other laboratories have published data using this model. This is likely due to the technical complexity of the surgical technique and perioperative complications, which can limit recipient survival. As two of the authors (XL and WL) have a combined experience of over 2500 left and right single lung transplants, this review will summarize their experience and delineate tips and tricks necessary for successful transplantation. We will also describe technical advances made since the original description of the model.

Activated T lymphocytes are abundant in the airway during lung allograft rejection. Based on respiratory viral studies, it is the current paradigm that T cells cannot divide in the airway, and that their accumulation in the lumen of the respiratory tract is the exclusive result of recruitment from other sites, such as mediastinal lymph nodes. Here, we show that CD8+ T cell activation and proliferation can occur in the airway after orthotopic lung transplantation. We also demonstrate that airway epithelium expresses major histocompatibility class I predominantly on the apical surface, both in vitro and in vivo, and initiates CD8+ T cell responses in a polarized fashion, favoring luminal activation. Our data identify a unique site for CD8+ T cell activation after lung transplantation, and suggest that attenuating these responses may provide a clinically relevant target.

Background. Signaling pathways that target I-kappa B kinase beta (IKK beta) activation stimulate the expression of nuclear factor (NF)-kappa B-dependent genes and are thus believed to primarily promote inflammation and injury in solid organ grafts.Methods. We examined the role of IKK beta in a mouse model of lung transplantation-mediated ischemia-reperfusion injury using NF-kappa B essential modulator (NEMO)-binding domain (NBD) peptide to pharmacologically inhibit IKK activation. As myeloid cells are primarily responsible for the production of acute inflammatory mediators after lung transplantation, we also investigated the effects of myeloid cell-specific IKK beta gene deletion on acute lung graft injury by transplanting mutant mice.Results. When NBD was administered at a dose that partially inhibits IKK beta activation, we observed attenuated lung graft injury and blunted expression of intragraft proinflammatory mediators. Surprisingly, when the dose of NBD was increased to a level that ablates intragraft IKK beta activation, graft inflammation, and injury were significantly worse compared with recipients treated with control peptide. Similar to lung recipients with pharmacologically ablated IKK beta activity, donor-recipient transplant combinations with a myeloid cell-specific IKK beta gene deletion had marked intragraft inflammation and poor lung function.Conclusions. Our data show maintenance of IKK beta activity is critical for promoting graft homeostasis with important implications for targeting NF-kappa B-dependent signaling pathways for treating acute lung injury.

Activated T lymphocytes are abundant in the airway during lung allograft rejection. Based on respiratory viral studies, it is the current paradigm that T cells cannot divide in the airway, and that their accumulation in the lumen of the respiratory tract is the exclusive result of recruitment from other sites, such as mediastinal lymph nodes. Here, we show that CD8(+) T cell activation and proliferation can occur in the airway after orthotopic lung transplantation. We also demonstrate that airway epithelium expresses major histocompatibility class I predominantly on the apical surface, both in vitro and in vivo, and initiates CD8(+) T cell responses in a polarized fashion, favoring luminal activation. Our data identify a unique site for CD8(+) T cell activation after lung transplantation, and suggest that attenuating these responses may provide a clinically relevant target.

Immune-mediated pulmonary diseases are a significant public health concern. Analysis of leukocyte behavior in the lung is essential for understanding cellular mechanisms that contribute to normal and diseased states. Here, we used two-photon imaging to study neutrophil extravasation from pulmonary vessels and subsequent interstitial migration. We found that the lungs contained a significant pool of tissue-resident neutrophils in the steady state. In response to inflammation produced by bacterial challenge or transplant-mediated, ischemia-reperfusion injury, neutrophils were rapidly recruited from the circulation and patrolled the interstitium and airspaces of the lung. Motile neutrophils often aggregated in dynamic clusters that formed and dispersed over tens of minutes. These clusters were associated with CD115+ F4/80+ Ly6C+ cells that had recently entered the lung. The depletion of blood monocytes with clodronate liposomes reduced neutrophil clustering in the lung, but acted by inhibiting neutrophil transendothelial migration upstream of interstitial migration. Our results suggest that a subset of monocytes serve as key regulators of neutrophil extravasation in the lung and may be an attractive target for the treatment of inflammatory pulmonary diseases.

Objectives: Orthotopic left lung transplantation in the mouse, as recently developed by our laboratory, represents a physiologic model for studies in transplantation biology. However, because of the limited overall respiratory contribution of the murine left lung, left lung transplant recipients remain healthy despite immune-mediated graft necrosis. We sought to develop a lung transplantation model in which animal survival depends on graft function.Methods: Orthotopic vascularized right lung transplantations using cuff techniques were performed in syngeneic and allogeneic strain combinations. Grafts were assessed histologically or functionally by measuring arterial blood gases from 7 to 28 days after transplantation. In a parallel set of experiments, syngeneic and immunosuppressed allogeneic hosts underwent a left pneumonectomy 2 weeks after right lung transplantation, with assessment of graft function 1 week later.Results: We performed 40 right lung transplantations, with a survival rate of 87.5%. Syngeneic grafts remain free of inflammation as far as 28 days after transplantation. On day 7, arterial oxygen levels in syngeneic recipients (481 +/- 90 mm Hg) are equivalent to those in naive mice (503 +/- 59 mm Hg) after left hilar occlusion. Alternatively, allogeneic grafts develop histologic evidence of acute rejection, and arterial oxygen levels are significantly decreased after left hilar clamping (53.3 +/- 10.3 mm Hg). Both syngeneic and immunosuppressed allogeneic right lung recipients tolerate a left pneumonectomy.Conclusions: Right lung transplantation followed by left pneumonectomy represents the first survival model of vascularized lung transplantation in the mouse and will therefore allow for the design of novel studies in experimental lung transplantation. (J Thorac Cardiovasc Surg 2010;139:1637-43)

OBJECTIVE:Endothelial cells express the ectoenzyme ectonucleoside adenosine triphosphate diphosphohydrolase, an apyrase that inhibits vascular inflammation by catalyzing the hydrolysis of adenosine triphosphate and adenosine diphosphate. However, ectonucleoside adenosine triphosphate diphosphohydrolase expression is rapidly lost following oxidative stress, leading to the potential for adenosine triphosphate and related purigenic nucleotides to exacerbate acute solid organ inflammation and injury. We asked if administration of a soluble recombinant apyrase APT102 attenuates lung graft injury in a cold ischemia reperfusion model of rat syngeneic orthotopic lung transplantation.METHODS:Male Fisher 344 donor lungs were cold preserved in a low-potassium dextrose solution in the presence or absence of APT102 for 18 hours prior to transplantation into syngeneic male Fisher 344 recipients. Seven minutes after reperfusion, lung transplant recipients received either a bolus of APT102 or vehicle (saline solution). Four hours after reperfusion, APT102- and saline solution-treated groups were evaluated for lung graft function and inflammation.RESULTS:APT102 significantly reduced lung graft extracellular pools of adenosine triphosphate and adenosine diphosphate, improved oxygenation, and protected against pulmonary edema. Apyrase treatment was associated with attenuated neutrophil graft sequestration and less evidence of tissue inflammation as assessed by myeloperoxidase activity, expression of proinflammatory mediators, and numbers of apoptotic endothelial cells.CONCLUSIONS:Administration of a soluble recombinant apyrase promotes lung function and limits the tissue damage induced by prolonged cold storage, indicating that extracellular purigenic nucleotides play a key role in promoting ischemia-reperfusion injury following lung transplantation.

It is the prevailing view that adaptive immune responses are initiated in secondary lymphoid organs. Studies using alymphoplastic mice have shown that secondary lymphoid organs are essential to initiate allograft rejection of skin, heart, and small bowel. The high immunogenicity of lungs is well recognized and allograft rejection remains a major contributing factor to poor outcomes after lung transplantation. We show in this study that alloreactive T cells are initially primed within lung allografts and not in secondary lymphoid organs following transplantation. In contrast to other organs, lungs are acutely rejected in the absence of secondary lymphoid organs. Two-photon microscopy revealed that recipient T cells cluster predominantly around lung-resident, donor-derived CD11c(+) cells early after engraftment. These findings demonstrate for the first time that alloimmune responses following lung transplantation are initiated in the graft itself and therefore identify a novel, potentially clinically relevant mechanism of lung allograft rejection.

Acute rejection continues to present a major obstacle to successful lung transplantation. Although CD4(+) T lymphocytes are critical for the rejection of some solid organ grafts, the role of CD4(+) T cells in the rejection of lung allografts is largely unknown. In this study, we demonstrate in a novel model of orthotopic vascularized mouse lung transplantation that acute rejection of lung allografts is independent of CD4(+) T cell-mediated allorecognition pathways. CD4(+) T cell-independent rejection occurs in the absence of donor-derived graft-resident hematopoietic APCs. Furthermore, blockade of the CD28/B7 costimulatory pathways attenuates acute lung allograft rejection in the absence of CD4(+) T cells, but does not delay acute rejection when CD4(+) T cells are present. Our results provide new mechanistic insight into the acute rejection of lung allografts and highlight the importance of identifying differences in pathways that regulate the rejection of various organs.

Unlike transplantation of other solid organs, vascularized mouse lung transplantation has only recently been developed. In this protocol, we describe a detailed method for performing a vascularized and aerated mouse orthotopic lung transplant, which to date represents the most physiological mouse model of lung transplantation. The procedure is divided into two separate portions consisting of donor harvest followed by implantation using the cuff technique for bronchovascular anastomoses. After a training period spanning several months, the procedure can be successfully mastered and, in experienced hands, requires ∼90 min to perform. After an initial learning curve, perioperative survival is close to 100%. As the donor hematopoietic cells in the transplanted lung are replaced by those of the host over time, thereby creating a 'chimeric lung,' this model represents a novel research tool for the study of transplantation biology as well as other disease processes, such as malignancies.

Since its introduction several decades ago, solid organ transplantation has had a wide impact as successful therapy for end-stage organ failure. Refinements in surgical techniques have allowed for the expansion of the spectrum of transplantation to replacement of “nontraditional” organs and tissue such as extremity limbs, facial structures, and laryngeal allografts.1Tobin G.R. Breidenbach 3rd, W.C. Pidwell D.J. Ildstad S.T. Ravindra K.V. Transplantation of the hand, face, and composite structures: evolution and current status.Clin Plast Surg. 2007; 34 (ix-x): 271-278Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar Duchenne muscular dystrophy (DMD) is the most common and severe of the human muscular dystrophies, affecting 1 in 3500 live male births. Currently no efficacious therapy is available for this disease, and death generally occurs in the late teens to early twenties from respiratory complications resulting from diaphragmatic dysfunction.2Smith P.E. Calverley P.M. Edwards R.H. Evans G.A. Campbell E.J. Practical problems in the respiratory care of patients with muscular dystrophy.N Engl J Med. 1987; 316: 1197-1205Crossref PubMed Scopus (157) Google Scholar Diaphragm transplantation could thus represent a novel therapy for this disease. As the ultimate success of transplantation hinges on adequate revascularization, the goal of this study was to develop surgical techniques for orthotopic diaphragmatic transplantation. The canine model has been used to develop multiple surgical techniques in thoracic organ transplantation, and the experiments described herein were performed in mongrel hounds weighing 25 to 30 pounds. Similar to human beings, the phrenic arteries in the dog branch directly off the abdominal aorta and are reported to act as the main source of blood supply3Plestis K. Fell S.C. The diaphragm: anatomy, embryology, pathophysiology, and surgery of the phrenic nerve and diaphragm.in: Pearson F. Cooper J. Deslauriers J. Ginsberg R.J. Hiebert G. Thoracic surgery. vol 2. Churchill Livingstone, Philadelphia2002: 1499Google Scholar to the crural and costal diaphragm. To evaluate the possibility of diaphragmatic survival as a free flap based on the phrenic vessels alone, we removed collateral diaphragmatic blood flow by ligating the intercostal branches, internal thoracic arteries, and epigastric and abdominal wall vessels. We then examined regional distribution of blood flow to the diaphragm with multicolored fluorescent microspheres (IMT Labs, Irvine, Calif). This was accomplished by injecting 25 × 106 microspheres of different colors into the left atrium after ligation of collaterals. A reference blood sample was withdrawn from the femoral artery. For evaluation of regional blood flow, the right and left hemidiaphragms were divided into 3 regions designated as zone 1, costal diaphragm furthest from the phrenic blood supply, zone 2, costal diaphragm closest to the phrenic vessels, and zone 3, the crural diaphragm. The number of microbeads in each sample and the reference blood were counted by flow cytometry after tissue digestion and the regional blood flow determined by the following equation: Qi(mL/min)=(fli/flref)⋅R(mL/min), where Qi = flow to the tissue, fli = fluorescence count in each tissue, flref = fluorescence count of the reference blood sample, and R = the reference blood withdrawal rate (Figure E1). Total as well as regional blood flow in the crural and costal diaphragm did not decrease after ligation of these collaterals, supporting our hypothesis that the diaphragm can be transplanted as a free flap based on the phrenic vessels (Figure E1). We then proceeded with orthotopic transplantation. Owing to the anatomic difficulty of transplanting the whole canine diaphragm, it was decided for the purpose of model development to transplant the left hemidiaphragm. The donor graft was harvested by serial ligation of all collateral vasculature except phrenic branches off the aorta and phrenic venous tributaries to the inferior vena cava. The abdominal and chest wall attachments were separated and the costal and crural diaphragm was removed along with a rim of costal margin (Figure 1, A). The graft was topically cooled to 4°C. Initial attempts to implant the donor aortic and vena caval cuffs as interposition grafts in the recipient with a period of complete aortic occlusion resulted in high operative mortality. We were then successful in developing methods of transplantation consisting of dual end-to-side donor-to-recipient suprahepatic inferior vena cava anastomosis using a partially occlusive side-biter clamp (Figure 1, B). Arterial inflow was provided by an end-to-side anastomosis of the donor to the recipient aorta using a similar technique (Figure 1, C). The left hemidiaphragm of the recipient was excised and the transplanted graft inserted orthotopically by securing it to surrounding structures. Statistically identical blood flow was documented in the transplanted left hemidiaphragm compared with the native left hemidiaphragm of control animals 30 minutes after reperfusion (n = 5) (2-tailed t test of probability) (Figure 2).Figure 2After orthotopic transplantation, blood flow was statistically identical to that of the native, unmanipulated diaphragm of control animals in all zones. Black line, Native unmanipulated diaphragm; purple line, transplanted diaphragm) (5 animals represented).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To our knowledge, this is the first demonstration of technical feasibility of diaphragmatic transplantation. We are in the process of establishing a long-term transplantation model that will allow us to study physiology, immunosuppression, and long-term survival and metabolic requirements of a transplanted diaphragm. Methods for reinnervation or direct diaphragmatic pacing will be explored. Inasmuch as a homolog of DMD has been identified in a muscular dystrophic Golden Retriever, the canine model may provide an opportunity for future preclinical large animal trials.

Lung transplantation remains the only therapeutic option for many patients suffering from end-stage pulmonary disease. Long-term success after lung transplantation is severely limited by the development of bronchiolitis obliterans. The murine heterotopic tracheal transplantation model has been widely used for studies investigating pathogenesis of obliterative airway disease and immunosuppressive strategies to prevent its development. Despite its utility, this model employs proximal airway that lacks airflow and is not vascularized. We have developed a novel model of orthotopic vascularized lung transplantation in the mouse, which leads to severe vascular rejection in allogeneic strain combinations. Here we characterize differences in the fate of airway epithelial cells in nonimmunosuppressed heterotopic tracheal and vascularized lung allograft models over 28 days. Up-regulation of growth factors that are thought to be critical for the development of airway fibrosis and interstitial Collagen deposition were similar in both models. However, while loss of airway epithelial cells occurred in the tracheal model, airway epithelium remained intact and fully differentiated in lung allografts, despite profound vascular rejection. Moreover, we demonstrate expression of the antiapoptotic protein Bcl-2 in airway epithelial cells of acutely rejected lung allografts. These findings suggest that in addition to alloimmune responses, other stimuli maybe required for the destruction of airway epithelial cells. Thus, the model of vascularized mouse lung transplantation may provide anew and more physiologic experimental tool to study the interaction between immune and nonimmune mechanisms affecting airway pathology in lung allografts.

AIM To evaluate the hypoglycemic efficacy of insulin liposomes coated by chitosan with different molecular weights and concentrations after oral administration in mice. METHODS Insulin-liposomes were prepared by reversed-phase evaporation. Chitosan coating was carried out by incubation of the liposomal suspensions with the chitosan solution. The hypoglycemic efficacies of chitosan-coated insulin liposomes were investigated by monitoring the blood glucose level using the glucose oxidase method after oral administration to healthy mice. RESULTS In all the insulin liposomes, the insulin liposomes coated by 0.2% chitosan (Mr 1000 kDa) showed a better hypoglycemic efficacy as compared with the other liposomes coated by chitosan. The minimum blood glucose level was 15.1%+/-6.0 % of the initial (n=6). The hypoglycemic efficacy lasted for 4 h after oral administration to mice. CONCLUSION Chitosan-coated liposomes could reduce tryptic digestion on insulin, and enhance enteral absorption of insulin. The molecular weights and concentrations of chitosan had significant effects on hypoglycemic efficacy of chitosan-coated insulin liposomes after oral administration to healthy mice.

采用逆相蒸发法制得胰岛素脂质体,用壳聚糖进行包覆.以小鼠口服该品的降血糖效果作为实验指标,分别应用L16(215)和L8(27)正交实验设计优化壳聚糖包覆胰岛素脂质体的处方和工艺.结果显示最佳处方组成为水相(胰岛素100IU、含1% PVP-K30的pH 7.4磷酸盐缓冲液)、油相(磷脂200 mg、胆固醇25 mg、维生素E 10 mg)、壳聚糖溶液浓度1%.最佳制备工艺为乙醚10 ml、旋转蒸发温度20°C、探头式超声0.5 min后加壳聚糖、包覆温度10°C、时间0.5 h.优化后的工艺制得的制品经大鼠和小鼠口服后,血糖值下降至最低时分别为实验前的39.13%和16.94%.

AIM:To evaluate the hypoglycemic effect of chitosan-coated and sodium alginate-coated insulin liposomes after oral administration in mice.METHODS:Insulin-liposomes were prepared by reverse-phase evaporation. Chitosan and alginate coating was carried out by mixing liposomal suspension with chitosan and sodium alginate solutions, followed by incubation. The particle size and morphology of insulin-liposomes were determined using laser light scattering instrument and transmission electron microscopy (TEM). The entrapment efficiency was analyzed using HPLC and ultracentrifuge. The protection of insulin from peptic and tryptic digestion was studied with HPLC. The hypoglycemic effects of polysaccharide-coated insulin liposomes were investigated using the glucose oxidase method after oral administration in mice.RESULTS:The particle size of uncoated, chitosan-coated and alginate-coated insulin-liposomes was (138 +/- 31) nm, (230 +/- 20) nm and (266 +/- 19) nm, respectively. All insulin-liposomes were of spherical or ellipsoidal shape. The entrapment efficiencies were 81.6%, 73.5% and 68.7%, respectively. Insulin was protected from tryptic digestion by chitosan-coated liposomes and protected from peptic digestion by alginate-coated liposomes. The hypoglycemic effects of insulin-liposomes, coated with 0.1% chitosan and 0.1% sodium alginate, were observed.CONCLUSION:Chitosan-coated and sodium alginate-coated liposomes were shown to reduce peptic or tryptic digestion on insulin, and enhance enteral absorption of insulin.

Objective: A RP - HPLC method was developed for determining the contents of insulin in polysaccha-ride -coated insulin liposomes and the entrapment efficiencies of the liposomes. Methods: Insulin - liposomes were prepared by reversed - phase evaporation and sonication. The entrapment efficiencies of the liposomes and the contents of insulin in the liposomes were analyzed by HPLC and ultracentrifuge. Chromatographic conditions included Lichrospher ODS - C18 (250 mm ×4. 6 mm, 5μm) and mobile phase consisting of a mixture of acetoni-trile - sulfate buffer solution (0. 025 mol · L-1 , pH 2. 0). The flow rate was 1. 0 mL· min-1 . The temperature of column was 40℃ . Detection wavelength was 214 nm. Results: The calibration curve was linear in the range of 2 - 10μg· mL-1. The average recovery, the RSDs of within -day and day -to -day of insulin in blank chitosan - coated liposomes were 94. 65% - 100. 5% , 0. 92% - 1. 6% and 4. 0% - 5. 6% , those in blank alginate -coated liposomes were 96. 12% - 101. 1% , 1.7% -2.4% and 4.0% -4.7% respectively. The entrapment efficiency of chitosan - coated insulin liposomes and the content of insulin in the liposomes were 73. 6% and 0. 682 mg· mL-1 respectively. The entrapment efficiency of alginate - coated insulin liposomes and the content of insulin in the liposomes were 68.7% and 0.713 mg· mL-1 respectively. Conclusions: The method was convenient, sensitive, accurate, reproducible, and suitable for determining the contents of insulin in polysaccharide - coated liposomes and the entrapment efficiencies of the liposomes.