Rat Trachea Research Articles

The Effect of Electronic Cigarettes (E-Cigarettes) and Conventional Smoke on the Trachea and Heart of Male White Rats

Electronic cigarettes (e-cigarette) smoker was increasing from 0.3% in 2011 to 2.5% in 2015. E-cigarettes claimed to be safe for smoker, but subsequent research has shown that e-cigarette smoke also contains harmful ingredients beside nicotine, like carbon monoxide (CO). These contents presumed to increase the goblet cells in the trachea and can cause myocardial ischemia in the left ventricle of the heart which leads to necrosis. This study was conducted to determine the effect of e-cigarette and conventional cigarette smoke on tracheal goblet cells and left ventricular necrosis of the heart of male white rats (Rattus norvegicus strain wistar). Experimental study with post-test control only group design. 16 rats were divided into 4 groups: normal group, conventional cigarette group (kretek) with 2.3 mg nicotine, 3 mg nicotine e-cigarette, and 0 mg nicotine e-cigarette. Trachea and heart of rats were prepared using HE-staining and observed with a light microscope with 400x magnification. All data were analyzed using One-way ANOVA, Kruskall Wallis and Post-Hoc tests. Cigarette smoke had a significant effect on tracheal goblet cell (ANOVA p=0.000) and left ventricular necrosis of the heart (Kruskal Wallis p=0.002). Post-Hoc test showed a significant difference in the number of goblet cells and left ventricular necrosis of the heart (p (-- removed HTML --) 0.005). This is due to the toxic materials such as carbon monoxide, propylene glycol, formaldehyde, nitrosamines, and other carcinogenic substances. E-cigarette and conventional cigarettes smoke could affect increasing of tracheal goblet cells and left ventricular necrosis of rat.

IL-11 drives the phenotypic transformation of tracheal epithelial cells and fibroblasts to enhance abnormal repair after tracheal injury

Tracheal stenosis (TS) is a multifactorial and heterogeneous disease that can easily lead to respiratory failure and even death. Interleukin-11 (IL-11) has recently received increased attention as a fibrogenic factor, but its function in TS is uncertain. This study aimed to investigate the role of IL-11 in TS regulation based on clinical samples from patients with TS and a rat model of TS produced by nylon brush scraping. Using lentiviral vectors expressing shRNA (lentivirus-shRNA) targeting the IL-11 receptor (IL-11Rα), we lowered IL-11Rα levels in the rat trachea. Histological and immunostaining methods were used to evaluate the effects of IL-11Rα knockdown on tracheal injury, molecular phenotype, and fibrosis in TS rats. We show that IL-11 was significantly elevated in circulating serum and granulation tissue in patients with TS. In vitro, TGFβ1 dose-dependently stimulated IL-11 secretion from human tracheal epithelial cells (Beas-2b) and primary rat tracheal fibroblasts (PRTF). IL-11 transformed the epithelial cell phenotype to the mesenchymal cell phenotype by activating the β-catenin pathway. Furthermore, IL-11 activated the atypical ERK signaling pathway, stimulated fibroblasts proliferation, and transformed fibroblasts into alpha-smooth muscle actin (α-SMA) positive myofibroblasts. IL-11-neutralizing antibodies (IL-11NAb) or ERK inhibitors (U0126) inhibited IL-11 activity and downregulated fibrotic responses involving TGFβ/SMAD signaling. In vivo, IL-11Rα knockdown rats showed unobstructed tracheal lumen, relatively intact epithelial structure, and significantly reduced granulation tissue proliferation and collagen fiber deposition. Our findings confirm that IL-11 may be a target for future drug prevention and treatment of tracheal stenosis.

A Tracheal Aspirate-derived Airway Basal Cell Model Reveals a Proinflammatory Epithelial Defect in Congenital Diaphragmatic Hernia.

Rationale: Congenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood. Objectives: To establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing. Methods: We developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from patients with CDH and preterm and term non-CDH control subjects were derived and analyzed by bulk RNA sequencing, assay for transposase accessible chromatin with sequencing, and air-liquid interface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH. Measurements and Main Results: Transcriptomic and epigenetic profiling of CDH and control BSCs reveals a proinflammatory signature that is manifested by hyperactive nuclear factor kappa B and independent of severity and hernia size. In addition, CDH BSCs exhibit defective epithelial differentiation invitro that recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, blockade of nuclear factor kappa B hyperactivity normalizes epithelial differentiation phenotypes of human CDH BSCs invitro and in nitrofen rat tracheas invivo. Conclusions: Our findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

GATA6 triggers fibroblast activation and tracheal fibrosis through the Wnt/β-catenin pathway

Tracheal fibrosis is a key abnormal repair process leading to fatal stenosis, characterized by excessive fibroblast activation and extracellular matrix (ECM) deposition. GATA6, a zinc finger-containing transcription factor, is involved in fibroblast activation, while its role in tracheal fibrosis remains obscure. The present study investigated the potential role of GATA6 as a novel regulator of tracheal fibrosis. It was found that GATA6 and α-smooth muscle actin (α-SMA) were obviously increased in tracheal fibrotic granulations and in TGFβ1-treated primary tracheal fibroblasts. GATA6 silencing inhibited TGFβ1-stimulated fibroblast proliferation and ECM synthesis, promoted cell apoptosis, and inactivated Wnt/β-catenin pathway, whereas GATA6 overexpression showed the reverse effects. SKL2001, an agonist of Wnt/β-catenin signaling, restored collagen1a1 and α-SMA expression which was suppressed by GATA6 silencing. Furthermore, in vivo, knockdown of GATA6 ameliorated tracheal fibrosis, as manifested by reduced tracheal stenosis and ECM deposition. GATA6 inhibition in rat tracheas also impaired granulation proliferation, increased apoptosis, and inactivated Wnt/β-catenin pathway. In conclusion, our findings indicate that GATA6 triggers fibroblast activation, cell proliferation, and apoptosis resistance in tracheal fibrosis via the Wnt/β-catenin signaling pathway. Targeting GATA6 may represent a promising therapeutic approach for tracheal fibrosis.

Astaxanthin alleviates fine particulate matter (PM2.5)-induced lung injury in rats by suppressing ferroptosis and apoptosis.

Objectives: Fine particulate matter (PM2.5), a small molecule particulate pollutant, can reach the lungs via respiration and cause lung damage. Currently, effective strategies and measures are lacking to prevent and treat the pulmonary toxicity of PM2.5. Astaxanthin (ASX), a natural xanthophyll carotenoid, has attracted attention due to its unique biological activity. Our research aims to probe into the prevention and treatment of ASX on PM2.5-induced lung injury and clarify its potential mechanism. Methods: Sprague-Dawley (SD) rats were given olive oil and different concentrations of ASX orally daily for 21 days. PM2.5 suspension was instilled into the trachea of rats every two days for one week to successfully develop the PM2.5 exposure model in the PM2.5-exposed and ASX-treated groups of rats. The bronchoalveolar lavage fluid (BALF) was collected, and the content of lung injury-related markers was detected. Histomorphological changes and expression of markers associated with oxidative stress, inflammation, iron death, and apoptosis were detected in lung tissue. Results: PM2.5 exposure can cause changes in lung histochemistry and increase the expression levels of TP, AKP, ALB, and LDH in the BALF. Simultaneously, inflammatory responses and oxidative stress were promoted in rat lung tissue after exposure to particulate matter. Additionally, ASX preconditioning can alleviate histomorphological changes, oxidative stress, and inflammation caused by PM2.5 and reduce PM2.5-related ferroptosis and apoptosis. Conclusion: ASX preconditioning can alleviate lung injury after PM2.5 exposure by inhibiting ferroptosis and apoptosis.

Distribution of proteins for synaptic release in nerve endings associated with the trachealis muscle of rats.

The immunohistochemical localization of proteins for synaptic release was examined in smooth muscle-associated sensory nerve endings using whole-mount preparations of the rat trachea. Plant-like smooth muscle-associated nerve endings with immunoreactivity for Na+-K+-ATPase, α3-subunit were identified in the trachealis muscle. VGLUT1, synapsin1, t-SNARE proteins (SNAP25 and syntaxin1), v-SNARE proteins (VAMP1 and VAMP2), and a presynaptic active zone-related protein (piccolo) were detected in the terminal parts of these endings. These results suggest that smooth muscle-associated nerve endings secrete glutamate to modulate sensorimotor functions in the lung deflation reflex.

Successful tracheal regeneration using biofabricated autologous analogues without artificial supports

Tracheas have a tubular structure consisting of cartilage rings continuously joined by a connective tissue membrane comprising a capillary network for tissue survival. Several tissue engineering efforts have been devoted to the design of scaffolds to produce complex structures. In this study, we successfully fabricated an artificial materials-free autologous tracheal analogue with engraftment ability by combining in vitro cell self-aggregation technique and in-body tissue architecture. The cartilage rings prepared by aggregating chondrocytes on designated culture grooves that induce cell self-aggregation were alternately connected to the connective tissues to form tubular tracheal analogues by subcutaneous embedding as in-body tissue architecture. The tracheal analogues allogeneically implanted into the rat trachea matured into native-like tracheal tissue by covering of luminal surfaces by the ciliated epithelium with mucus-producing goblet cells within eight months after implantation, while maintaining their structural integrity. Such autologous tracheal analogues would provide a foundation for further clinical research on the application of tissue-engineered tracheas to ensure their long-term functionality.

The comparative effects of erythromycin and amikacin on acute respiratory Pseudomonas aeruginosa infection.

One of the most common causes of pneumonia is Pseudomonas aeruginosa (P. aeruginosa). As with other microbial pathogens, this bacterium tends to develop resistance to various antibiotics. Amikacin and erythromycin, which are from the aminoglycoside and macrolide antibiotic families, are used to treat respiratory infections caused by P. aeruginosa. This study explored whether amikacin, erythromycin or a combination of both works better against P. aeruginosa acute lung infection. For this study, 32 rats were used. The trachea of rats was exposed aseptically and their lung was infected with P. aeruginosa through trachea. Then, according to the group, they received amikacin, erythromycin or a combination of both for 1 week. Finally, they were euthanised on the 3rd and 7th days post-infection. The macroscopic and microscopic evaluations of the lungs, kidney and liver were performed. The right lung was collected for in vivo bacteriological analysis. The amikacin group (A group) had a statistically significantly lower macroscopic and microscopic scores than the other groups (p < 0.05). In vivo bacteriological test revealed that the A group had significantly lower lung bacterial load (p < 0.05). In summary, it was concluded that amikacin could help alleviate the respiratory infection caused by P. aeruginosa solely, and it was more effective than erythromycin.

Knockdown of SOX9 alleviates tracheal fibrosis through the Wnt/β-catenin signaling pathway.

Trachealfibrosis is an important cause of tracheal stenosis without effective treatments, and new drug targets need to be developed. The role of SOX9 in the injury and repair of the trachea is unknown; this study aims to investigate the role of SOX9 in the regulation of tracheal fibrosis based on clinical samples from patients with tracheal injury and a model of tracheal fibrosis produced by tracheal brushing in rats. The results showed that the expressions of SOX9 and mesenchymal and ECM-related indicators were increased in the injury and fibrosis of the trachea in patients and rats. Serum SOX9 levels exhibited a sensitivity of 83.87% and specificity of 90% in distinguishing patients with tracheal fibrosis from healthy volunteers when the cut‑off value was 13.24ng/ml. Knockdown SOX9 can markedly inhibit granulation tissue proliferation, reduce inflammation and ECM deposition, promote epithelial regeneration and granulation tissue apoptosis, and attenuate the tracheal fibrosis after injury. Additionally, RNA sequencing showed that the proliferation, migration, and ECM deposition of tracheal granulation tissue were related to the activation of Wnt pathway, activation of the β-catenin, and p-GSK3β after injury can be inhibited by the knockdown of SOX9. In summary, SOX9 is upregulated in tracheas fibrosis and may be a novel factor to promote tracheal fibrosis progression. Inhibiting SOX9 may be used to prevent and treat tracheal fibrosis in the future. KEY MESSAGE : The expression of SOX9 is upregulated the process of injury and repair of the tracheal fibrosis. Knocking down SOX9 can attenuate tracheal fibrosis after injury by inhibiting inflammation response, granulation tissue proliferation, ECM deposition, and promoting granulation tissue apoptosis. The Wnt/β-catenin-SOX9 axis is activated during tracheal injury and fibrosis, and inhibition of SOX9 can partially alleviate tracheal fibrosis. SOX9 may act as a new diagnostic and therapeutic target in patients with tracheal fibrosis in the future.

Comparison of Physical Perturbation Devices for Enhancing Lentiviral Vector-Mediated Gene Transfer to the Airway Epithelium.

Natural airway defenses currently impede the efficacy of viral vector-mediated airway gene therapy. Conditioning airways before vector delivery can disrupt these barriers, improving viral vector access to target receptors and airway stem cells. This study aimed to assess and quantify the in vivo histological and gene transfer effects of physical perturbation devices to identify effective conditioning approaches. A range of flexible wire baskets with varying configurations, a Brush, biopsy forceps, and a balloon catheter were examined. We first evaluated the histological effects of physical perturbation devices in rat tracheas that were excised 10 min after conditioning. Based on the histological findings, a selection of devices was used to condition rat tracheas in vivo before delivering a lentiviral vector containing the LacZ reporter gene. After 7 days, excised tracheas were X-gal processed and examined en face to quantify the area of LacZ staining. Histological observations 10 min after conditioning found that physical perturbation dislodged cells from the basement membrane to varying degrees, with some producing significant levels of epithelial cell removal. When a subset of devices was assessed for their ability to enhance gene transfer, only the NGage® wire basket (Cook Medical) produced a significant increase in the proportion of X-gal-stained area when compared with unconditioned tracheas (eightfold, p = 0.00025). These results suggest that a range of factors contribute to perturbation-enhanced gene transfer. Overall, this study supports existing evidence that physical perturbation can assist airway gene transfer and will help to identify the characteristics of an effective device for airway gene therapy.

Rosa damascena Miller essential oil relaxes rat trachea via KV channels, KATP channels, and BKCa channels

Aim/ObjectiveThis study aimed to investigate the effects of Rosa damascena Miller essential oil on rat tracheal smooth muscle contractility and the hypothesis that voltage-gated potassium (KV) channels, ATP-sensitive potassium (KATP) channels, and large-conductance calcium-activated potassium (BKCa) channels may have roles in these effects. MethodsIsometric contraction-relaxation responses of tracheal rings were measured with an isolated tissue bath model. The steady contraction was induced with both 10−5 M ACh and 60 mM KCl, and then the concentration-dependent responses of rose oil (0.1–100 µg/mL) were examined. The time-matched control (double distilled water) group was also formed. To evaluate the role of KV, KATP, and BKCa channels, tracheal rings were incubated with 4-AP (KV channel blocker), glibenclamide (KATP channel blocker), TEA (BKCa channel blocker), and iberiotoxin (selective BKCa channel blocker). Also, a vehicle control group was formed for dimethyl sulfoxide (DMSO). ResultsRose oil exerted the relaxant effects in tracheal rings pre-contracted with both ACh and KCl at concentrations of 1, 10, and 100 µg/mL (p < 0.05). Besides, KV channel blocker 4-AP, KATP channel blocker glibenclamide, and BKCa channel blockers TEA and iberiotoxin incubations significantly inhibited the rose oil-induced relaxation responses (p < 0.05). However, incubation of tissues with DMSO, glibenclamide solvent, for 10 min did not cause a significant change in the relaxation responses to rose oil (p > 0.05). ConclusionsIn conclusion, the first physiological findings were obtained regarding the functional relaxant effects of rose essential oil in rat trachea. The findings showed that rose oil induces bronchorelaxation in a concentration-dependent manner. Besides, this study is the first to report that rose oil-mediated bronchodilation responses are associated with the activity of KV, KATP, and BKCa channels. These results suggest that rose oil might be a useful agent in the treatment of abnormal bronchoconstriction-related diseases such as asthma and chronic obstructive pulmonary disease.

Irisin Relaxes Rat Trachea via KV Channels, KATP Channels, and BKCa Channels.

This study aimed to investigate the effects of irisin on rat tracheal smooth muscle contraction-relaxation responses and the roles of voltage-gated potassium (KV) channels, ATP-sensitive potassium (KATP) channels, and large-conductance calcium-activated potassium (BKCa) channels in these effects. Isometric contraction and relaxation responses of tracheal segments were measured using the tissue bath method. Submaximal contractions were induced by ACh (10-5 M) or KCl (60 mM), and then concentration-response curves of irisin (10-9 to 10-6 M) were obtained. For the temporal control, a double-distilled water group was formed. ACh and irisin were added to the baths after tracheal segments were incubated with 4-AP (KV channel blocker), glibenclamide (KATP channel blocker), TEA, and iberiotoxin (BKCa channel blockers) to assess the role of K+ channels. In addition, a vehicle group was performed for the solvent dimethyl sulfoxide (DMSO). Irisin exhibited the relaxant effects in tracheal segments precontracted with both ACh and KCl at concentrations of 10-8-10-6 M (p<0.05). Besides, incubations of 4-AP, glibenclamide, TEA, and iberiotoxin significantly inhibited the irisin-mediated relaxation (p<0.05), whereas DMSO incubation did not modulate irisin responses (p>0.05). In conclusion, the first physiological results on the relaxant effects of irisin in rat trachea were obtained. Our findings demonstrated that irisin mediates concentration-dependent relaxation in rat tracheas. Moreover, the present study reported for the first time that irisin-induced bronchorelaxation is associated with the activity of the K+ channels.

Improved in-vivo airway gene transfer via magnetic-guidance, with protocol development informed by synchrotron imaging

Gene vectors to treat cystic fibrosis lung disease should be targeted to the conducting airways, as peripheral lung transduction does not offer therapeutic benefit. Viral transduction efficiency is directly related to the vector residence time. However, delivered fluids such as gene vectors naturally spread to the alveoli during inspiration, and therapeutic particles of any form are rapidly cleared via mucociliary transit. Extending gene vector residence time within the conducting airways is important, but hard to achieve. Gene vector conjugated magnetic particles that can be guided to the conducting airway surfaces could improve regional targeting. Due to the challenges of in-vivo visualisation, the behaviour of such small magnetic particles on the airway surface in the presence of an applied magnetic field is poorly understood. The aim of this study was to use synchrotron imaging to visualise the in-vivo motion of a range of magnetic particles in the trachea of anaesthetised rats to examine the dynamics and patterns of individual and bulk particle behaviour in-vivo. We also then assessed whether lentiviral-magnetic particle delivery in the presence of a magnetic field increases transduction efficiency in the rat trachea. Synchrotron X-ray imaging revealed the behaviour of magnetic particles in stationary and moving magnetic fields, both in-vitro and in-vivo. Particles could not easily be dragged along the live airway surface with the magnet, but during delivery deposition was focussed within the field of view where the magnetic field was the strongest. Transduction efficiency was also improved six-fold when the lentiviral-magnetic particles were delivered in the presence of a magnetic field. Together these results show that lentiviral-magnetic particles and magnetic fields may be a valuable approach for improving gene vector targeting and increasing transduction levels in the conducting airways in-vivo.

Muc5b Contributes to Mucus Abnormality in Rat Models of Cystic Fibrosis.

Cystic fibrosis (CF) airway disease is characterized by excessive and accumulative mucus in the airways. Mucociliary clearance becomes defective as mucus secretions become hyperconcentrated and viscosity increases. The CFTR-knockout (KO) rat has been previously shown to progressively develop delayed mucociliary transport, secondary to increased viscoelasticity of airway secretions. The humanized-G551D CFTR rat model has demonstrated that abnormal mucociliary clearance and hyperviscosity is reversed by ivacaftor treatment. In this study, we sought to identify the components of mucus that changes as the rat ages to contribute to these abnormalities. We found that Muc5b concentrations, and to a lesser extent Muc5ac, in the airway were increased in the KO rat compared to WT, and that Muc5b concentration was directly related to the viscosity of the mucus. Additionally, we found that methacholine administration to the airway exacerbates these characteristics of disease in the KO, but not WT rat trachea. Lastly we determined that at 6 months of age, CF rats had mucus that was adherent to the airway epithelium, a process that is reversed by ivacaftor therapy in the hG551D rat. Overall, these data indicate that accumulation of Muc5b initiates the muco-obstructive process in the CF lung prior to infection.

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue.

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation andculture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

Neurotoxicity Study with Titanium Dioxide Nanoparticles on Rat

Numerous studies have shown titanium dioxide nanoparticles (TiO2 NPs) could present a risk or potential risk to humans and other living organisms in certain conditions via inhalation and skin contact. Dermal exposure has limited adverse effects and the possible risks for exogenous inhaled nanoparticles migrating to the brain through the olfactory nerve is still under research. To study the in vivo and in vitro neurotoxicity of brain tissue in rats induced by TiO2 NPs. For in vitro study, rat astrocytes were exposed to TiO2 NPs with three different diameters (10, 50 and 200 nm) at five concentrations levels. Cellular morphology and sulfur rhodamine B (SRB) were carried out to evaluate the viability of particle-treated cells after 72 hours exposure. For in vivo study, suspensions of test material above mentioned were injected into tracheas of Wistar rats at dose of 0.1, 1.0 and 10.0 mg·kg-1 in three groups, respectively. After 72 hours of exposure, the concentration of TiO2 NPs in brain tissue and the levels of IL-1β, TNF-α and IL-10 in brain homogenate were measured, while the cell morphology induced by TiO2 NPs was observed by light microscopy and transmission electron microscopy. TiO2 NPs can significantly affect the growth and morphology of rat astrocytes and inhibit the proliferation of astrocytes, which was positively related to dose-effect and size-dependent response. Pathological observations indicated that TiO2 NPs could penetrate the blood-brain barrier, leading to blood-brain barrier damage in rats, brain tissue necrosis, mitochondrial swelling and apoptosis while the non-nanoscale TiO2 particles showed no significant toxicity in the central nervous system cells.

Taurine Alleviates LPS-Induced Acute Lung Injury by Suppressing TLR-4/NF-κB Pathway.

Taurine has the function of immune regulation, relieving acute and chronic inflammation caused by various agents, and maintaining cell homeostasis. This investigation focused on the protective functions of taurine targeting acute lung injury (ALI) induced by LPS. Sixty male SD rats aged 6-7weeks were segregated at random: blank control group (C group), taurine control group (T group), ALI model group (LPS group), and taurine prevention groups (LPST1, LPST, LPST3 Groups). C group and LPS group were given normal drinking water, while T group and LPST group were given 2% taurine in drinking water. LPST1 group was given 1% taurine in drinking water while. LPST3 group was given 3% taurine in drinking water. On the 14th and 28th day, LPS group and LPST1, LPST, and LPST3 groups were subjected to injection of LPS (25 mg/kg) into the trachea of rats. Serum, peripheral blood, lung tissue, and bronchoalveolar lavage fluid (BALF) were collected at 6h post-LPS injection. The wet/dry ratio (W/D) of lung was measured by hot drying method. The population of white blood cells and the abundance of inflammatory-related cells within peripheral blood were counted by an automatic blood cell analyzer. The population of white blood cells within BALF was counted by a white blood cell counting plate combined with Swiss Giemsa staining, while the proportion of related white blood cells was calculated. BCA reagent was used to determine the protein concentration in BALF. The levels of pro-inflammatory factors (IL-1 β, IL-6, IL-18, TNF - α), anti-inflammation factors (IL-10, IL-4), and taurine within serum and lung tissue were detected by ELISA. Lung structural tissue alterations were observed through HE staining techniques. Myeloperoxidase (MPO) activities within lung tissue were detected through colorimetry. Protein expression levels of TLR4, MyD88, NF-κ Bp65, NF-κ Bp-p65, MCP-1, together with CD68 within lung tissue, were analyzed by Western blot (WB) and immunohistochemistry (IHC). The taurine pretreatment group contained significantly reduced W/D, MPO activity, and the number of inflammatory cells in BALF induced by LPS. In addition, compared with ALI model group, the taurine pretreatment group contained significantly reduced levels of pro-inflammatory factors in lung tissue, increased levels of anti-inflammatory factors, and decreased expression levels of key proteins in TLR-4/NF-κ B pathway. Taurine can protect rats from ALI by inhibiting the activation of neutrophils, macrophages, and TLR-4/NF-κ B signaling pathway.

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 partial 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, 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 localized cell replacement method can facilitate creation of bioengineered in vitro airway tissue that can be used in different biomedical applications.

Effect of Src tyrosine kinase on a rat model of asthma

Src tyrosine kinase is a protein encoded by the Src gene. The present study aimed to determine the role of Src protein kinase in asthma using small interfering RNA (siRNA) technology. Several Src siRNAs were designed and the most effective siRNA pair was selected. A rat model of asthma was established using ovalbumin, and the rats were treated with Src siRNA, empty vector or phosphate-buffered saline (PBS). A non-asthmatic control group was also established. The rats were clinically observed and Src mRNA and protein levels were measured by reverse transcription-quantitative PCR and western blot analysis, respectively. Pathological observation of the lung tissue, counting of white blood cells (WBCs) and eosinophils (EOSs) and analysis of the concentrations of IL-5, IL-33 and IFN-γ in the bronchoalveolar lavage fluid were performed. The expression levels of Src mRNA in the control, PBS, empty vector and siRNA groups were 110±30.7x103, 253±55.4x103, 254±41.3x103 and 180±50.9x103, respectively. Histochemical analysis of the lung tissue of rats in the siRNA group exhibited a relatively complete lung structure and little damage to the alveolar cavity. Src protein expression and IL-5, IL-33 levels, WBC and EOS levels were positively correlated with Src mRNA expression, while the IFN-γ concentration was negatively correlated with Src mRNA expression. These results indicate that Src knockdown inhibits the release of tracheal inflammatory factors and significantly alleviates asthma in rats. In conclusion, the present study utilized a gene transfer technique to interfere with the expression of Src in rats, which decreased the levels of IL-5, IL-33, WBCs and EOSs and increased the level of IFN-γ; these changes effectively ameliorated the condition of the trachea in asthmatic rats.

Homogeneous Distribution of Exogenous Cells onto De-epithelialized Rat Trachea via Instillation of Cell-Loaded Hydrogel.

Injured or diseased airway epithelium due to repeated environmental insults or genetic mutations can lead to a functional decline of the lung and incurable lung diseases. Bioengineered airway tissue constructs can facilitate in vitro investigation of human lung diseases and accelerate the development of effective therapeutics. Here, we report robust tissue manipulation modalities that allow: (i) selective removal of the endogenous epithelium of in vitro cultured airway tissues and (ii) spatially uniform distribution and prolonged cultivation of exogenous cells that are implanted topically onto the denuded airway lumen. Results obtained highlight that our approach to airway tissue manipulation can facilitate controlled removal of the airway epithelium and subsequent homogeneous distribution of newly implanted cells. This study can contribute to the creation of innovative tissue engineering methodologies that can facilitate the treatment of lung diseases, such as cystic fibrosis, primary ciliary dyskinesia, and chronic obstructive pulmonary disease.