Reducing Variability in Lung Slice Analysis
Airway constriction is a hallmark of many respiratory conditions, including asthma, but measuring it accurately in lung tissue has long been a complex and labor-intensive task. The level of airway constriction in thin slices of lung tissue can vary dramatically, making it difficult to determine how many airways need to be analyzed to obtain reliable data. A recent publication by Boucher et al (2024) streamlines this process of quantifying airway constriction in Precision Cut Lung Slices (PCLS) by using the new physioLens automated solution for physiology and image analysis. This new solution enables researchers to conduct high-throughput screening of airway constriction with unprecedented precision and efficiency.
Challenge of variability in slices
Traditionally, studying airway constriction in lung slices involves tedious, labor-intensive processes, combined with the significant variability in how different airways respond to stimuli like methacholine. This variability presents a significant challenge: how can one reliably determine the degree of airway constriction in a mouse when responses can range from negligible to complete closure within the same tissue? This inconsistency poses a significant challenge for researchers trying to draw reliable conclusions from their data. Traditional methods require a substantial amount of manual labor to analyze enough airways to account for this variability, making these experiments both time-consuming and resource intensive.
The physioLens addresses this challenge by automating the experimental intervention and image analysis processes, drastically increasing the throughput and accuracy of these experiments. The device was put to the test in a study involving lung slices from male BALB/c mice, some with experimental asthma and others without. These lung tissues were inflated using two different methods—through the trachea or via trans-parenchymal injections with agarose—before being exposed to methacholine, a compound used to induce airway constriction.
The automation provided by the device allowed for the analysis of an average of 45 airways per mouse across 32 mice. The study found that the mean maximal constriction was 37.4 ± 32.0%. Importantly, the method of inflating the lungs did not affect the response to methacholine, but the presence of experimental asthma did, shifting the methacholine concentration–response curve and indicating decreased sensitivity.
Determining Optimal Sample Size
One of the key findings of the study was the determination of the optimal number of airways that need to be analyzed to obtain a reliable measure of constriction for a single mouse. Through random sampling simulations, it was predicted that approximately 35 airways per mouse are needed to obtain a reliable mean of maximal constriction. For standard deviation (SD) and coefficient of variation (CoV), 16 and 29 airways were required, respectively.
This discovery is particularly significant because it provides a guideline for future studies, helping researchers to plan their experiments more efficiently. By knowing the minimum number of airways that need to be analyzed, scientists can save time and resources while still obtaining accurate and reliable data.
The Implications of Experimental Asthma
Interestingly, while the maximal constriction was not significantly different between mice with and without experimental asthma, the sensitivity to methacholine was notably decreased in the asthmatic mice. This finding suggests that the primary effect of asthma in this model is not on the extent of airway narrowing, but rather on the responsiveness of the airways to constrictive stimuli. This aligns with a growing body of literature indicating that hyperresponsiveness in asthma may be more related to airway narrowing heterogeneity and closure rather than defects in smooth muscle contraction.
No Impact of Inflation Technique on Outcomes
A critical aspect of the study was to compare the two lung inflation techniques used. The results were encouraging; neither technique significantly influenced the airway constriction outcomes or the effect of experimental asthma on methacholine response. This finding is particularly valuable as it suggests that data obtained from animal models can be reliably compared with human tissue samples, where different inflation techniques might be employed.
Conclusion
The physioLens PCLS solution marks a significant advancement in respiratory research, offering a reliable, automated method for studying airway constriction in lung slices. By determining the optimal sample sizes needed to achieve accurate results, this study provides a valuable framework for future research. The ability to conduct high-throughput, precise measurements will certainly accelerate our understanding of airway constriction dynamics and pave the way for new therapeutic strategies in treating respiratory conditions like asthma.
Reference
- Boucher, M., et al. (2024). High throughput screening of airway constriction in mouse lung slices. Scientific Reports, 14: 20133
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