Imaging-Guided Bioreactor for De-Epithelialization and Long-Term Cultivation of Ex Vivo Rat Trachea

Recent synergistic advances in organ-on-chip and tissue engineering technologies offer opportunities to create in vitro-grown tissue or organ constructs that can faithfully recapitulate their in vivo counterparts. Such in vitro tissue or organ constructs can be utilized in multiple applications, including rapid drug screening, high-fidelity disease modeling, and precision medicine. Here, we report an imaging-guided bioreactor that allows in situ monitoring of the lumen of ex vivo airway tissues during controlled in vitro tissue manipulation and cultivation of isolated rat trachea. Using this platform, we demonstrated selective removal of the rat tracheal epithelium (i.e., de-epithelialization) without disrupting the underlying subepithelial cells and extracellular matrix. Through different tissue evaluation assays, such as immunofluorescent staining, DNA/protein quantification, and electron beam microscopy, we showed that the epithelium of the tracheal lumen can be effectively removed with negligible disruption in the underlying tissue layers, such as cartilage and blood vessel. Notably, using a custom-built micro-optical imaging device integrated with the bioreactor, the trachea lumen was visualized at the cellular level in real time, and removal of the endogenous epithelium and distribution of locally delivered exogenous cells were demonstrated in situ. Moreover, the de-epithelialized trachea supported on the bioreactor allowed attachment and growth of exogenous cells seeded topically on its denuded tissue surface. Collectively, the results suggest that our imaging-enabled rat trachea bioreactor and selective cell replacement method can facilitate creating of bioengineered in vitro airway tissue that can be used in different biomedical applications.

Oxidative Stress Response of PM2.5 and Mixed Toxic Gases to Rat Trachea and Lung Tissue

Background：We performed this study to explore the inflammation and oxidative stress responses of rat trachea and lung caused by PM2.5 and mixed toxic gas.Materials and methods：Eighty-four Wistar rats were randomly assigned to receive exposure to PM2.5 and toxic gases containing carbon monoxide (CO), nitrogen dioxide (NO2), sulfur dioxide (SO2) or saline. Microflora in rat respiratory tract were investigated through testing secretion taken from posterior pharyngeal wall on Day 1, 7 and 30. Trachea and lung tissues were processed for scanning electron microscopic (SEM) and transmission electron microscopic (TEM) examinations. Cytokines in rat bronchoalveolar lavage fluid (BALF), serum and lung tissues were analyzed via enzyme-linked immunosorbent assay and real-time quantitative PCR.Results：Eight aerobes and seven anaerobes were firstly detected after exposure (p<0.01, respectively). Most of them are the pathogenic bacteria or opportunistic pathogen. SEM and TEM observations suggested that both normal structure of trachea and lung tissue were injured after exposure to PM2.5 with mixed toxic gas and the injure degree was concentration- and time-dependent. Interleukin 4 (IL-4), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression in the BALF and lung tissue markedly increased on Day 1 and 7 (p<0.01, respectively). Interferon γ (IFN-γ) did not show significant change after exposure. On Day 30, all the detected cytokines decreased and even disappeared. All detected cytokines in the serum had no significant difference before and after exposure (p>0.05, respectively).Conclusion：PM2.5 with mixed toxic gas can lead to dysbacteriosis in respiratory tract and destroy the normal structure of trachea and lung tissue. The injure degree was concentration- and time-dependent. Local inflammatory response instead of systemic reaction may be predominant in the response of rat respiratory tract to PM2.5 with mixed toxic gas.

Pharmacological Evaluation of the Bronchorelaxant Effect of Waltheria indica L. (Malvaceae) Extracts on Rat Trachea

Waltheria indica L. (Malvaceae) is a plant used in Burkina Faso for the treatment of various ailments including asthma. The aim of the study was to evaluate the pharmacological relaxant effect of the leafy stem extracts of Waltheria indica and thereby verify claim of use in treating asthma. Aqueous decoction and hydroalcoholic extracts obtained from the powdered leafy stems were screened for the presence of some phytoconstituents. The in vitro relaxant effect of the two extracts was evaluated on acetylcholine- (ACh 10−5 M) and potassium chloride- (KCl 6 × 10−2 M) induced contractions on rat-isolated tracheal preparations. To examine whether the potassium (K+) channels are involved in the relaxant effect, glibenclamide, an ATP-sensitive potassium channel inhibitor, was used. Moreover, to assess the safety of the extracts, acute oral toxicity was carried out on mice. The phytochemical screening revealed the presence of alkaloids, flavonoids, saponins, steroids, triterpenoids, tannins, and coumarins in the hydroalcoholic extract. Tannins, steroids, triterpenoids, and coumarins were not detected in the aqueous decoction. With respective EC50 values of 1.517 ± 0.002 mg/mL and 1.433 ± 0.001 mg/mL on ACh-and KCl-provoked contractions, the hydroalcoholic extract was found more potent in relaxing the isolated rat tracheal preparations compared to the aqueous decoction. In the presence of glibenclamide, the relaxant effect of the hydroalcoholic extract (EC50 = 0.191 ± 0.002 mg/mL) increased and was higher than that of the aqueous decoction. At dose of 5000 mg/kg of body weight, the extracts did not produce deaths or any significant changes in the general behavior of mice. The results suggest that different mechanisms including modulation of calcium and potassium channels, particularly the ATP-sensitive K+ channels, could be involved in the relaxation effect. These findings could justify the traditional use of W. indica in the management of asthma.

Effect of Boswellia serrata on Rat Trachea Contractility In Vitro

Background: Boswellia serrata (family Burseraceae) has been traditionally used for the treatment of a wide variety of diseases as arthritis, inflammatory bowel diseases, and airway diseases. However, the direct bronchodilator efficacy of Boswellia serrata hasn’t been explored yet. Objective: We aimed at the present study to evaluate the direct effect of Boswellia serrata extract (BSE) on isolated rat tracheal preparations precontracted with either Acetylcholine (ACh) or potassium chloride (KCl). Methods: Tracheal rings were prepared from male Wistar rats (200-250 g). BSE (1-200 μg/ml) was added to tracheal strips precontracted with either ACh or KCl and the response was observed. We also investigated the consequences of epithelial denudation, indomethacin, and N-Nitro-L-arginine on the relaxant effect of BSE as compared to that of the β-adrenoceptor agonist isoprenaline, or the bitter taste receptor (TAS2R) agonist denatonium benzoate. Finally, the possible additive effects of BSE to isoprenaline or denatonium-induced relaxation were evaluated. Results: By using a set of serial dosing and washout experiments with tracheal rings, results showed that exposure to BSE resulted into a significant and concentration-dependent inhibitory effect on airway smooth muscle contractions precontracted with either ACh or KCl. Epithelial denudation, indomethacin, or N-Nitro-L-arginine had no significant effect on the obtained relaxation. Furthermore, BSE potentiated the relaxant effect of isoprenaline on rat trachea. Conclusion: BSE exerts a direct concentration-dependent relaxant effect on precontracted tracheal strips. These results could contribute towards validation of the traditional use of BSE in the treatment of airway diseases.