Ke Jiang

Improvements of surgical techniques in a rat model of an orthotopic single lung transplant

BackgroundRats are widely used in modeling orthotopic lung transplantation. Recently the introduction of the cuff technique has greatly facilitated the anastomosing procedure used during the transplant. However, the procedure is still associated with several drawbacks including twisting of blood vessels, tissue injury and the extensive time required for the procedure. This study was performed to optimize the model of rat lung transplantation (LT) with the cuff technique.MethodsA total of 42 adult Lewis rats received orthotopic LT with our newly modified procedures. The modified procedures were based on the traditional procedure and incorporated improvements involving orotracheal intubation; a cuff without a tail; conservative dissection in the hilum; preservation of the left lung during anastomosis; successive anatomizing of the bronchus, the pulmonary vein, and the pulmonary artery; and one operator.ResultsTransplants were performed in 42 rats with a successful rate of 95.23% (40/42). The mean duration for the complete procedure was 82.93 ± 14.56 minutes. All anastomoses were completed in one attempt without vessel laceration, twisting or angulation. In our study, two animals died within three days and one animal died ten days after the operation. All grafts were well inflated with robust blood perfusion and functioned normally as demonstrated by blood gas analysis.ConclusionsWe have developed a modified orthotopic LT technique that can be easily performed while overcoming major drawbacks. The modified technique has many advantages, such as easy graft implanting, shortened operation time, fewer complications and high reproducibility.

Heme oxygenase-1 expression in rats with acute lung rejection and implication

This study investigated the expression of hemeoxygenase-1 (HO-1) in rats with acute lung rejection and its implication. A valid rat orthotopic left lung transplantation model (SD rat→Wistar rat) was established by using an improved three-cuff anastomosis technique. The rats were divided into control group, CoPP (HO-1 inducer)-treated group and ZnPP (HO-1 inhibitor)- treated group. The severity of acute rejection was graded on the basis of the morphologic changes of the lung samples stained with HE. The expression of HO-1 protein in lung tissue was detected by using immunohistochemistry and Western blot, and HO-1 mRNA activity was assayed by RT-PCR. The results showed that the expression of HO-1 protein was significantly increased with the acute rejection grading in rats (P<0.01). As compared with control and ZnPP-treated groups, the severity of acute rejection was not alleviated and the grade not reduced significantly in CoPP-treated group (P>0.05). It was concluded that HO-1 protein might be involved in the pathological process of post-graft acute rejection. The expression of HO-1 protein was increased gradually with aggravation of acute rejection, and HO-1 protein might be used as an index to monitor acute rejection after lung transplantation.SummaryThis study investigated the expression of hemeoxygenase-1 (HO-1) in rats with acute lung rejection and its implication. A valid rat orthotopic left lung transplantation model (SD rat→Wistar rat) was established by using an improved three-cuff anastomosis technique. The rats were divided into control group, CoPP (HO-1 inducer)-treated group and ZnPP (HO-1 inhibitor)- treated group. The severity of acute rejection was graded on the basis of the morphologic changes of the lung samples stained with HE. The expression of HO-1 protein in lung tissue was detected by using immunohistochemistry and Western blot, and HO-1 mRNA activity was assayed by RT-PCR. The results showed that the expression of HO-1 protein was significantly increased with the acute rejection grading in rats (P<0.01). As compared with control and ZnPP-treated groups, the severity of acute rejection was not alleviated and the grade not reduced significantly in CoPP-treated group (P>0.05). It was concluded that HO-1 protein might be involved in the pathological process of post-graft acute rejection. The expression of HO-1 protein was increased gradually with aggravation of acute rejection, and HO-1 protein might be used as an index to monitor acute rejection after lung transplantation.

Multiple calcifying fibrous pseudotumor of the bilateral pleura.

Calcifying fibrous pseudotumor is a rare lesion characterized histologically by hypocellular hyalinized collagenous tissue with calcifications and patchy lymphocytes infiltration. Occurring most often in children and young adults, calcifying fibrous pseudotumor is a clinically benign lesion that can form over a broad anatomic distribution, including in subcutaneous and deep soft tissues, but is rarely found in the pleura. The cause and mechanisms of pathogenesis of calcifying fibrous pseudotumor are unknown. In this article, we describe a case of a 44-year-old woman with multiple calcifying fibrous pseudotumor disseminated in the bilateral pleura that was pathologically diagnosed. We discuss the differential diagnosis with other benign or malignant soft tissue diseases and also review the recent literature on this rare benign entity. Complete resection of all disseminated lesions was possible with followed thoracotomy. Although multiple lesions may prevent the complete resection and calcifying fibrous pseudotumor of the pleura is considered as benign lesion, complete surgical resection of all lesions seems to be the best therapy for calcifying fibrous pseudotumor of the pleura to reduce additional dissemination and local recurrence.

Orthotopic and heterotopic tracheal transplantation model in studying obliterative bronchiolitis.

BACKGROUND Several animal models have been established to investigate the mechanisms of obliterative bronchiolitis after lung transplantation. In this study, we compared three prevalent murine models of obliterative bronchiolitis in terms of several basic pathologic changes in a relatively short span of time after transplantation. METHODS Each of the recipient mice simultaneously received orthotopic, intra-omental and subcutaneous tracheal transplantation in both syngeneic and allogeneic settings. No immunosuppressive treatment was administered. Tracheal grafts were harvested on Day 14, 21 and 28 after transplantation for histological and immunohistochemical analyses. RESULTS Syngeneic tracheal grafts from different transplant sites retained normal histologic structures, while their corresponding allografts demonstrated more occlusion of the airway lumen as well as more infiltration of CD4(+)/CD8(+) mononuclear cells and myofibroblasts, but less regenerative epithelium and neovascularized vessels at indicated times (P<0.05). Compared with two heterotopic allografts, orthotopic allografts had less occlusion of the tracheal lumen as well as less infiltration of CD4(+)/CD8(+) mononuclear cells and myofibroblasts, but more regenerative epithelium and neovascularized vessels (P<0.05). CONCLUSIONS Orthotopic tracheal transplantation in mice can be considered as a model to study early stages of obliterative bronchiolitis, and heterotopic tracheal transplantation can be a model for late stages of obliterative bronchiolitis.

Interleukin-35 on B cell and T cell induction and regulation

Interleukin (IL)-35 is a relatively newly discovered member of IL-12 cytokine family that is unique in that it is a dimer formed by two subunits. The review documents the structure, secretion and signal transduction of IL-35, the regulation effect of IL-35 on B cells and T cells as well as the adoptive transfer of IL-35+ regulatory B cells (Breg), therapeutic prospects of recombinant IL-35 (rIL-35) and IL-35 regulation role in various diseases. B-cell regulation expands the regulatory range of IL-35 and alters the view that IL-10 is the chief immune mechanism for Breg cells which secrete IL-35. IL-35 induces Breg cells, which then can induce Treg cells. IL-35 also plays an immunomodulatory role in the human body.

Short-term inhalation of nitric oxide inhibits activations of toll-like receptor 2 and 4 in the lung after ischemia-reperfusion injury in mice

In order to investigate the effects of different terms of inhaled nitric oxide (NO) preconditioning with low concentration on the activations of Toll-like receptor 2 and 4 (TLR2/4) in the lung ischemia-reperfusion (IR) injury in mice, we divided the male C57BL mice into five groups: sham (S) group, IR group, NO 1-min preconditioning group (15 ppm NO inhalation for 1 min before ischemia, NO 1-min), NO 10-min preconditioning group (15 ppm NO inhalation for 10 min before ischemia, NO 10-min), NO 60-min preconditioning group (15 ppm NO inhalation for 60 min before ischemia, NO 60-min). The changes of partial pressure of oxygen in artery (PaO2), left lung wet-to-dry weight ratio (W/D), and myeloperoxidase (MPO) in the injured lung were measured in every group at 6th h of reperfusion after 60 min of left lung ischemia. The changes of TLR2/4 activations and plasma TNF-α were measured in this procedure in additional mice. As compared with IR group, PaO2 increased, MPO and W/D decreased evidently after reperfusion in NO 10-min group. The changes in NO 60-min group were similar to those in NO 10-min group. There was no difference between NO 1-min and IR group. In NO inhalation group, the expressions levels of TLR2/4 mRNA and proteins were diminished, TNF-α concentrations were decreased, and the lung injuries were ameliorated effectively. We concluded that short term inhalation of NO protected lung IR injury. But the protective effect of NO was not increased with extension of inhaled NO. Inhaled NO could inhibit the activations of TLR2/4 in the lung after IR injury. TLR signal pathway might contribute to the effect of protection with NO in this model.SummaryIn order to investigate the effects of different terms of inhaled nitric oxide (NO) preconditioning with low concentration on the activations of Toll-like receptor 2 and 4 (TLR2/4) in the lung ischemia-reperfusion (IR) injury in mice, we divided the male C57BL mice into five groups: sham (S) group, IR group, NO 1-min preconditioning group (15 ppm NO inhalation for 1 min before ischemia, NO 1-min), NO 10-min preconditioning group (15 ppm NO inhalation for 10 min before ischemia, NO 10-min), NO 60-min preconditioning group (15 ppm NO inhalation for 60 min before ischemia, NO 60-min). The changes of partial pressure of oxygen in artery (PaO2), left lung wet-to-dry weight ratio (W/D), and myeloperoxidase (MPO) in the injured lung were measured in every group at 6th h of reperfusion after 60 min of left lung ischemia. The changes of TLR2/4 activations and plasma TNF-α were measured in this procedure in additional mice. As compared with IR group, PaO2 increased, MPO and W/D decreased evidently after reperfusion in NO 10-min group. The changes in NO 60-min group were similar to those in NO 10-min group. There was no difference between NO 1-min and IR group. In NO inhalation group, the expressions levels of TLR2/4 mRNA and proteins were diminished, TNF-α concentrations were decreased, and the lung injuries were ameliorated effectively. We concluded that short term inhalation of NO protected lung IR injury. But the protective effect of NO was not increased with extension of inhaled NO. Inhaled NO could inhibit the activations of TLR2/4 in the lung after IR injury. TLR signal pathway might contribute to the effect of protection with NO in this model.

Differentiation character of adult mesenchymal stem cells and transfection of MSCs with lentiviral vectors

SummaryThis study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lentiviral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and transfection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Aggracan was positive in chondrogenic lineages and the expression of aggracan and type collagen II was significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lentiviral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and transfection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Aggracan was positive in chondrogenic lineages and the expression of aggracan and type collagen II was significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.

Increased expression of Tim-3 and its ligand Galectin-9 in rat allografts during acute rejection episodes

The aim of the study is to elucidate the profiles of T-cell immunoglobulin and mucin domain-3 (Tim-3) and its ligand Galecin-9 in acute pulmonary rejection by using a rat model of lung transplantation. Left lung grafts retrieved from Lewis or Fisher 344 rats were orthotopically transplanted into Lewis recipients without any immunosuppressions; the grafts were harvested at day 3, 7 or 10 after transplantation. The grade of acute rejection was histopathologically evaluated. Tim-3, Galectin-9, immune antigen and related cytokines expression were assessed with immunological techniques and real-time polymerase chain reaction (RT-PCR), respectively. Then, our results showed that Tim-3 and its ligand Galectin-9 were markedly up-regulated at protein and mRNA levels in allografts compared with syngrafts. Meanwhile, the decreased CD4/CD8 ratio was associated with acute rejection occurring and Tim-3 expression on CD4(+) and CD8(+) T cells in allografts was increased. Therefore, our study firstly described that enhanced Tim-3 and its ligand Galectin-9 in allografts might play an important role in the pathogenesis of rat lung transplant rejection, implying new valuable markers for detecting acute allograft rejection.

14-3-3ζ promotes lung cancer cell invasion by increasing the Snail protein expression through atypical protein kinase C (aPKC)/NF-κB signaling.

14-3-3ζ has been identified as a putative oncogene in several cancers, including non-small cell lung cancer (NSCLC). However, the mechanisms underlying its functions remain undefined. In this study, we show that overexpression of 14-3-3ζ was frequently detected in lung adenocarcinoma (LuAC) tissues and was significantly associated with lymph node metastasis and poor outcome. Functional studies demonstrated that 14-3-3ζ promoted migration and invasion in A549 cells, both of which were effectively inhibited when 14-3-3ζ was silenced with short hairpin RNA (shRNA). Furthermore, 14-3-3ζ-mediated invasion of cancer cells was found to upregulate Snail through the activation of atypical protein kinase C (aPKC). Activation of aPKCζ mediates this effect by stimulating NF-κB signaling. Our results identify a specific pathway by which 14-3-3ζ induces tumor invasion and provide insight into potential therapeutic approaches to target 14-3-3ζ-associated lung adenocarcinoma.

Improvements of the surgical technique on the established mouse model of orthotopic single lung transplantation.

Background A wide range of knockout and transgenic murine models for the study of nonimmune and immune mechanisms in lung transplants are available nowadays, but the microsurgical techniques are difficult to learn. We describe methods to simplify techniques and facilitate learning. Methods Traditional procedures were implemented to perform lung transplants in 30 cases (group 1). Improved techniques which included cuff without tail, broadening of the cuff diameter for bronchus, establishment of one tunnel between three structures, innovative technology of the vascular anastomosis and placement of the chest tube post-operation were used to perform lung transplants in 30 cases (group 2). Results The improved techniques considerably shorten operative times (96.75±6.16 min and 85.32±6.98 min in groups 1 and 2, respectively). The survival rates in the recipient animals were 86.7% and 96.7% in groups 1 and 2, respectively. Chest X-rays and macroscopic changes of transplanted recipients showed that grafts were well inflated on postoperative day 30. There was no significant difference of the arterial oxygen tension (PaO2) between two groups (115.9±7.11 mm Hg and 116.3±6.87 mm Hg in groups 1 and 2, respectively). Histologically, no lung injury was seen in grafts. Conclusions We described the modified procedures of orthotopic left lung transplants in mice, which could shorten operative time and increase survival rate.