OBJECTIVE: To establish a new method for in vitro observation of ciliary activity of the tracheal membrane in rabbit under a common light microscope. METHODS: Nine healthy adult rabbits were used. Two equal sized cervical trachea flaps were removed after the rabbits were anesthetized by urethane (4 mg/kg). The removed trachea flap was randomly assigned to the ephedrine (Eph) and DMEM control group, respectively. The observed trachea flap was placed in a small flat-bottomed glass container with its membranous side upward in DMEM culture medium solution (DMEM control group) or in 0.5% ephedrine solution prepared with DMEM (Eph group). One drop of 1% methylthioninium stained autologous blood cells was added into the glass container as the tracer, and the trachea flap was observed under a common light microscope (400 x). The latter was attached with a digital camera linking to an image manipulation system, the computed dynamic image analyser. The velocity of the tracer cell movement worked as the indicator for ciliary activity and was automatically determined by the digital image manipulation system. The variation coefficient (VC) was used as the indicator of the cell movement velocity. RESULTS: There was no significant difference among the VC at different time points in the DMEM control group. VC of the Eph group decreased regularly with the time point. CONCLUSION A thin layer of flowing fluid was found on the surface of the tracheal mucociliary blanket which is driven by the activity of the mucociliary system. The new method of using the tracer to evaluate the ciliary activity of mammalia tracheal membrane in vitro is reliable and stable. It is practical and valuable in the in vitro observation and evaluation of ciliary activity of the tracheal membrane.
Animals ; Cell Movement ; Cilia ; In Vitro Techniques ; Microscopy ; Mucociliary Clearance ; Mucous Membrane ; cytology ; Rabbits ; Trachea ; cytology

Little is known about how gap junctions are involved in the respiratory-related or other types of physiological neuronal activity since physiologically active gap junction currents (GJCs) have never been characterized from single respiratory-related neurons or from single neurons of any other types. In the present study we hypothesized that GJCs could be selectively revealed from single neurons by elimination of transmembrane electrochemical gradients in voltage patch-clamp recording, and this hypothesis was tested in single inspiratory tracheal preganglionic vagal motor neurons (I-TPVMs). The results showed that GJCs were selectively revealed in all I-TPVMs when the transmembrane electrochemical gradients were eliminated in voltage patch-clamp recording, and were rhythmically activated by central inspiratory activity. Therefore, this method may be used as a fast way to detect GJCs within spontaneously active neuronal networks.
Gap Junctions ; physiology ; Motor Neurons ; physiology ; Patch-Clamp Techniques ; Trachea ; cytology

Vitrification is a promising alternative to tissue preservation, which will greatly avoid the ice-crystal formation and circumvent the hazardous effects associated with ice formation during the entire procedures. In this study, we evaluate the effect of vitreous cryopreservation of rabbit trachea by comparing the vitrification procedure with the conventional computer-programmed slow freezing approaches. Harvested tracheae were tailored and divided into groups and cryopreserved by vitrification and programmed freezing, respectively. The morphology and ultrastructure of the thawed tracheal fragments were examined by using HE staining, TUNEL test, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to assess the integrity of the tracheal fragments. The morphological studies demonstrated both cryopreservation procedure retain the interity of trachea, and that epithelium mucosae, cilia and cartilage cells were in good shape. Compared with slow freezing methods, vitrification was less detrimental to cartilage cells and had a higher survival rate of chondrocytes and coverage of epithelium and cilia. Therefore, vitrification method is a more satisfactory method to preserve trachea, the survival of chondrocytes in situ in cartilage tissue is adequate, and respiratory epithelium is soundly preserved.
Animals ; Cryopreservation ; methods ; Cryoprotective Agents ; chemistry ; Microscopy, Electron, Scanning ; Rabbits ; Tissue Preservation ; methods ; Trachea ; cytology ; ultrastructure

OBJECTIVETo observe the homing and differentiation of marrow-derived mesenchymal stem cells (MSC) transplanted intravenously in smoke inhalation injured rabbits.

METHODSThirty-two New Zealand big ear rabbits were divided into normal control group (NC), inhalation injury group (II), normal control + MSC treatment group (NM), and MSC treatment group (MT) according to the random number table, with 8 rabbits in each group. Rabbits in NC group were injected with 10 mL phosphate buffered saline (PBS) via ear marginal vein. Rabbits in NM group were injected with 10 mL PBS containing the third generation MSC labeled by BrdU (1 × 10(7) per 10 mL PBS) via ear marginal vein. Severe smoke inhalation injury model was reproduced in the other two groups, among them rabbits in II group were treated as rabbits in NC group, rabbits in MT group treated as rabbits in NM group. On the 7th and 28th day post treatment (PTD), lung tissue and trachea tissue were harvested from four groups for observation on injury with HE staining. Homing of MSC in injured tissue was observed with immunohistochemistry staining. The differentiation of MSC into functional cells was observed with immunohistochemical double staining of combining nuclear marker BrdU with lung (trachea) membrane-specific marker aquaporin-5 (AQP-5), alkaline phosphatase (AKP), CD34, and cytokeratin respectively.

RESULTS(1) MSC homing in lung and trachea tissue was observed in MT group on PTD 7, which was not observed in NM group. (2) AQP-5, AKP, and CD34 positive MSC were observed in lung tissue in MT group on PTD 28, while cytokeratin positive MSC was not observed in trachea tissue. No positively marked MSC was observed in NM group. (3) Injury in lung and trachea was less severe in MT group than in II group; and the proliferation of fibroblasts was less in MT group.

CONCLUSIONSIntravenous injection of MSC to rabbits with smoke inhalation injury can migrate to lung and trachea tissue at obviously inflammatory site, and differentiate into alveolar epithelial cells typeI and II, and pulmonary vascular endothelial cells, which may participate in the process of tissue repair in smoke inhalation injury.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; Epithelial Cells ; cytology ; Lung ; cytology ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Pulmonary Alveoli ; cytology ; Rabbits ; Smoke Inhalation Injury ; pathology ; Trachea ; cytology

BACKGROUNDRe-epithelialization has remained a major obstacle in both tracheal and lung transplantations. This study examines the realization of re-epithelialization by epithelial inoculation in a rat heterotopic tracheal transplantation model.

METHODSThe original epithelia of tracheas from donor Wistar rats were removed and the tracheas were then inoculated with 10(6)/ml in vitro cultured epithelial cells of the Spraque-Dawley (SD) rat phenotype. These allo-tracheas were then heterotopically transplanted into SD rats. After 28 days, the allo-trachea tissues were recovered and assessed for epithelial morphology and cellular differentiation using immunohistochemical analysis. An additional experimental group was used to compare the outcomes of re-epithelialization in immunosuppressed animals.

RESULTSHistological examination showed that allografts with epithelial inoculation maintained patent tracheal lumens, which were obliterated in controls. Recipient immunosuppression facilitated the formation of an integrated ciliated epithelial layer, further demonstrated by the presence of a dense cilia population, a well-developed plasma membrane, and readily recognizable intercellular junctions. Epithelial cellular differentiation markers such as cytokeratin 14 and 18, and cystic fibrosis transmembrane conductance regulator (CFTR) were all positive in allografts under immunosuppression.

CONCLUSIONConcurrent recipient-derived epithelial inoculation with immunosuppression can result in complete re-epithelialization with the recipient phenotype and suppress the luminal obliteration process in heterotopic transplantations.

Allografts ; cytology ; Animals ; Bronchiolitis Obliterans ; surgery ; Epithelial Cells ; cytology ; Female ; Male ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Trachea ; cytology ; transplantation ; Transplantation, Heterotopic

Animals ; Aquaporin 3 ; analysis ; Cell Separation ; Fluorouracil ; pharmacology ; Humans ; Lung ; cytology ; Membrane Proteins ; analysis ; Octamer Transcription Factor-3 ; genetics ; Stem Cells ; cytology ; Trachea ; cytology

OBJECTIVETo explore the feasibility of constructing tissue engineered trachea-like cartilage graft in vitro by using bone marrow stromal cells (BMSCs) sheet and PLGA internal support.

METHODSRabbit BMSCs were expanded and induced by transforming growth factor-1 to improve chondrocyte phenotype of BMSCs. BMSCs sheets were obtained by continuous culture and wrapped the PGLA scaffold in the shape of cylinder. The constructs were incubated in spinner flask for 8 weeks and cartilage formation was investigated by gross inspection, histology, glycosaminoglycan and mechanical strength content.

RESULTSAfter in vitro culture, cartilage like tissue in cylindrical shape had been regenerated successfully. Stiff, shiny, pearly opalescence tissues were observed. Histological analysis showed engineered trachea cartilage consisted of evenly spaced lacunae embedded in matrix, cells stationed in the lacunae could be noticed clearly. Safranin-O staining on the sections showed homogenous and positive red staining, which demonstrated that the engineered tissue was rich in proteoglycans.

CONCLUSIONSBased on the cell sheet and internal support strategy, trachea-like cartilage in cylindrical shape could be successfully fabricated which provided a highly effective cartilage graft substitute and could be useful in many situations of trachea-cartilage loss encountered in clinical practice.

Animals ; Biocompatible Materials ; Bone Marrow Cells ; cytology ; Cartilage ; Feasibility Studies ; Female ; Lactic Acid ; Male ; Polyglycolic Acid ; Rabbits ; Stromal Cells ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds ; Trachea ; surgery

Since most of the respiratory epithelial cell lines are descended from neoplastic tissues or have been fused with neoplastic cells when being selected to a cell line, their biological behaviors are far different from normal respiratory epithelial cells. Aiming at better reflecting the biological properties of epithelial cells under respiratory pathological conditions, our study probed into a new isolation technique and culture method of mice respiratory epithelial cell. Pronase was applied for cell isolation from mouse trachea, and a modified medium with collagen-coated plate for primary culture. The ciliary movement can be observed under microscope, which demonstrates that the original biological functions of the tracheal epithelial cell have been reserved with our method. The research presents the efficacy, convenience and reliability of the separation technique and culture method established by this study, and laying preferable condition for cell models of respiratory diseases. Meanwhile, this method may be used for other animal models.
Animals ; Cell Separation ; methods ; Culture Media ; Epithelial Cells ; cytology ; Female ; Male ; Mice ; Mice, Inbred BALB C ; Primary Cell Culture ; methods ; Pronase ; pharmacology ; Trachea ; cytology

The effects of retinoic acid on the beta-catenin/TCF pathway in cultured porcine tracheobronchial epithelial cells (TBEC) were investigated. After TBEC were treated with retinoic acid at various concentrations, mRNA and protein changes of beta-catenin in cytoplasm, nucleus and whole cell of the TBEC were observed by immunocytochemical stain, RT-PCR and Western blotting. And the changes of the target gene cyclinD1 of beta-catenin/TCF pathway were also observed. It was found that there was no significant difference in beta-cat mRNA level after retinoic acid treatment. However, the expression of beta-catenin in the whole cell and cytoplasm was elevated with the increase of retinoic acid concentration (P<0. 01). The nuclear protein beta-catenin and target gene cyclinD1 of beta-catenin/TCF pathway was decreased (P<0.05). It was indicated that retinoic acid could increase beta-catenin level of the whole cell protein and decrease nuclear beta-catenin, downregulating beta-cat/TCF signaling activity and reducing target gene cyclinD1 protein level. As a result, retinoic acid can downregulate beta-catenin/TCF pathway in porcine tracheobronchial epithelial cell, suggesting that retinoic acid can inhibit the proliferation and accelerate differentiation of tracheobronchial epithelial cells.
Animals ; Bronchi ; cytology ; metabolism ; Cells, Cultured ; Cyclin D1 ; biosynthesis ; genetics ; Epithelial Cells ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Swine ; TCF Transcription Factors ; biosynthesis ; genetics ; Trachea ; cytology ; metabolism ; Tretinoin ; pharmacology ; beta Catenin ; biosynthesis ; genetics

OBJECTIVETo assess the severity and reversibility of the chronic toxicity of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and the dose-effect relationship.

METHODSA total of 100 Sprague-Dawley rats (equal numbers of male and female) were randomly divided into five groups (20 rats in each group): four groups were treated with rhG-CSFa at 500, 100, 10, 1 µg/kg, respectively, and one group was treated with vehicle only to serve as the control. The rats were received subcutaneous injections of rhG-CSFa or vehicle daily for 13 weeks. During the course of the chronic toxicity study, the physical status, body weight, and food consumption were monitored. Half of the rats in each group (n = 10) were sacrificed after the last rhG-CSFa administration, and the other half were sacrificed at five weeks after the last rhG-CSFa administration. Urinalyses, blood biochemistry, hematological analysis, histopathological examination, and immunological tests were performed for each of the rats.

RESULTSThe hematological analyses revealed that the mean white blood cells count, neutrophils count, and neutrophils percentage were increased in male rats at the dose of 10 µg/kg or higher, and these were related with the biological activity of rhG-CSFa. Some small abnormalities were observed in the spleen of a few rats when used highest dose (500 µg/kg, a dosage of 200 folds higher than the normal clinical dosage), but these abnormalities were recovered within 5-week recovery period. No other rhG-CSFa-related abnormalities were observed in this chronic toxicity study.

CONCLUSIONNo significant toxicity and immunogenicity are observed with rhG-CSFa administration to rats in the chronic toxicity studies.

Animals ; Bilirubin ; urine ; Blood Chemical Analysis ; Dose-Response Relationship, Drug ; Female ; Granulocyte Colony-Stimulating Factor ; genetics ; toxicity ; Humans ; Lung ; cytology ; drug effects ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; Spleen ; cytology ; drug effects ; Trachea ; cytology ; drug effects