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The effects of oxygen concentration on cell death, anti-oxidant transcription, acute inflammation, and cell proliferation in precision-cut lung slices.

ABSTRACT: Although animal models are often used in drug research, alternative experimental models are becoming more popular as they reduce animal use and suffering. Of particular interest are precision-cut lung slices, which refer to explants - with a reproducible thickness and diameter - that can be cultured ex vivo. Because lung slices (partially) reflect functional and structural features of whole tissue, they are often applied in the field of immunology, pharmacology, toxicology, and virology. Nevertheless, previous research failed to adequately address concerns with respect to the viability of lung slices. For instance, the effect of oxygen concentration on lung slice viability has never been thoroughly investigated. Therefore, the main goal of this study was to investigate the effect of oxygen concentration (20 vs. 80% O2) on the degree of cell death, anti-oxidant transcription, acute inflammation, and cell proliferation in lung slices. According to the results, slices incubated at 20% O2 displayed less cell death, anti-oxidant transcription, and acute inflammation, as well as more cell proliferation, demonstrating that these slices were considerably more viable than slices cultured at 80% O2. These findings expand our knowledge on lung slices and their use as an alternative experimental model in drug research.

SUBMITTER: Ruigrok MJR

PROVIDER: S-EPMC6838147 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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The effects of oxygen concentration on cell death, anti-oxidant transcription, acute inflammation, and cell proliferation in precision-cut lung slices.

Ruigrok Mitchel J R MJR Tomar Jasmine J Frijlink Henderik W HW Melgert Barbro N BN Hinrichs Wouter L J WLJ Olinga Peter P

Scientific reports 20191107 1

Although animal models are often used in drug research, alternative experimental models are becoming more popular as they reduce animal use and suffering. Of particular interest are precision-cut lung slices, which refer to explants - with a reproducible thickness and diameter - that can be cultured ex vivo. Because lung slices (partially) reflect functional and structural features of whole tissue, they are often applied in the field of immunology, pharmacology, toxicology, and virology. Neverth ...[more]

PMID: 31700101

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Project description:BackgroundRespiratory diseases represent a global health burden. Because research on therapeutic strategies of airway diseases is essential, the technique of precision-cut lung slices (PCLS) has been developed and widely studied. PCLS are an alternative ex vivo model and have the potential to replace and reduce in vivo animal models. So far, the majority of studies was conducted with short-term cultivated PCLS (≤ 72 h). As there is large interest in research of chronic diseases and chronic toxicity, feasibility of cultivating human PCLS long-term over 2 weeks and recently over 4 weeks was investigated by another research group with successful results. Our aim was to establish a model of long-term cultivated rat PCLS over a period of 29 days.MethodsRat PCLS were cultured for 29 days and analysed regarding viability, histopathology, reactivity and gene expression at different time points during cultivation.ResultsCultivation of rat PCLS over a 29-day time period was successful with sustained viability. Furthermore, the ability of bronchoconstriction was maintained between 13 and 25 days, depending on the mediator. However, reduced relaxation, altered sensitivity and increased respiratory tone were observed. Regarding transcription, alteration in gene expression pattern of the investigated target genes was ascertained during long-term cultivation with mixed results. Furthermore, the preparation of PCLS seems to influence messenger ribonucleic acid (mRNA) expression of most target genes. Moreover, the addition of fetal bovine serum (FBS) to the culture medium did not improve viability of PCLS. In contrast to medium without FBS, FBS seems to affect measurements and resulted in marked cellular changes of metaplastic and/or regenerative origin.ConclusionsOverall, a model of long-term cultivated rat PCLS which stays viable for 29 days and reactive for at least 13 days could be established. Before long-term cultivated PCLS can be used for in-depth study of chronic diseases and chronic toxicity, further investigations have to be made.

Project description:BackgroundAsthma exacerbations evoke emergency room visits, progressive loss of lung function and increased mortality. Environmental and industrial toxicants exacerbate asthma, although the underlying mechanisms are unknown. We assessed whether 3 distinct toxicants, salicylic acid (SA), toluene diisocyanate (TDI), and 1-chloro-2,4-dinitrobenzene (DNCB) induced airway hyperresponsiveness (AHR) through modulating excitation-contraction coupling in human airway smooth muscle (HASM) cells. The toxicants include a non-sensitizing irritant (SA), respiratory sensitizer (TDI) and dermal sensitizer (DNCB), respectively. We hypothesized that these toxicants induce AHR by modulating excitation-contraction (EC) coupling in airway smooth muscle (ASM) cells.MethodsCarbachol-induced bronchoconstriction was measured in precision-cut human lung slices (hPCLS) following exposure to SA, TDI, DNCB or vehicle. Culture supernatants of hPCLS were screened for mediator release. In HASM cells treated with the toxicants, surrogate readouts of EC coupling were measured by phosphorylated myosin light chain (pMLC) and agonist-induced Ca2+ mobilization ([Ca2+]i). In addition, Nrf-2-dependent antioxidant response was determined by NAD(P) H quinone oxidoreductase 1 (NQO1) expression in HASM cells.ResultsIn hPCLS, SA, but not TDI or DNCB, potentiated carbachol-induced bronchoconstriction. The toxicants had little effect on release of inflammatory mediators, including IL-6, IL-8 and eotaxin from hPCLS. In HASM cells, TDI amplified carbachol-induced MLC phosphorylation. The toxicants also had little effect on agonist-induced [Ca2+]i. CONCLUSION: SA, a non-sensitizing irritant, amplifies agonist-induced bronchoconstriction in hPCLS via mechanisms independent of inflammation and Ca2+ homeostasis in HASM cells. The sensitizers TDI and DNCB, had little effect on bronchoconstriction or inflammatory mediator release in hPCLS.ImplicationsOur findings suggest that non-sensitizing irritant salicylic acid may evoke AHR and exacerbate symptoms in susceptible individuals or in those with underlying lung disease.

Dataset Information

The effects of oxygen concentration on cell death, anti-oxidant transcription, acute inflammation, and cell proliferation in precision-cut lung slices.

Publications

The effects of oxygen concentration on cell death, anti-oxidant transcription, acute inflammation, and cell proliferation in precision-cut lung slices.

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