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Cigarette smoke is well recognized to cause injury to the airways and the alveolar walls over time. This injury usually requires many years of exposure, suggesting that the lungs may rapidly develop responses that initially protect it from this repetitive injury. Our studies tested the hypotheses that smoke induces an inflammatory response and changes in mRNA profiles that are dependent on sex and the health status of the lung, and that the response of the lungs to smoke differs after 1 day compared to 5 days of exposure. Male and female wildtype (WT) and Scnn1b-transgenic (βENaC) mice, which have chronic bronchitis and emphysematous changes due to dehydrated mucus, were exposed to cigarette smoke or sham air conditions for 1 or 5 days. The inflammatory response and gene expression profiles were analyzed in lung tissue. Overall, the inflammatory response to cigarette smoke was mild, and changes in mediators were more numerous after 1 than 5 days. βENaC mice had more airspace leukocytes than WT mice, and smoke exposure resulted in additional significant alterations. Many genes and gene sets responded similarly at 1 and 5 days: genes involved in oxidative stress responses were upregulated while immune response genes were downregulated. However, certain genes and biological processes were regulated differently after 1 compared to 5 days. Extracellular matrix biology genes and gene sets were upregulated after 1 day but downregulated by 5 days of smoke compared to sham exposure. There was no difference in the transcriptional response to smoke between WT and βENaC mice or between male and female mice at either 1 or 5 days. Taken together, these studies suggest that the lungs rapidly alter gene expression after only one exposure to cigarette smoke, with few additional changes after four additional days of repeated exposure. These changes may contribute to preventing lung damage.

Selecting and implementing a tissue clearing protocol is challenging. Established more than 100 years ago, tissue clearing is still a rapidly evolving field of research. There are currently many published protocols to choose from, and each performs better or worse across a range of key evaluation factors (e.g., speed, cost, tissue stability, fluorescence quenching). Additionally, tissue clearing protocols are often optimized for specific experimental contexts, and applying an existing protocol to a new problem can require a lengthy period of adaptation by trial and error. Although the primary literature and review articles provide a useful starting point for optimization, there is growing recognition that results can vary dramatically with changes to tissue type or antibody used. To help address this issue, we have developed a novel, freely available repository of tissue clearing protocols named T-CLEARE (Tissue CLEAring protocol REpository; https://doryworkspace.org/doryviz). T-CLEARE incorporates community responses to an open survey designed to capture details not commonly found in the scientific literature, including modifications to published protocols required for specific use cases and instances when tissue clearing protocols did not perform well (negative results). The goal of T-CLEARE is to help the community share evaluations and modifications of tissue clearing protocols for various tissue types and potentially identify best-in-class methods for a given application.