Tissue optical clearing, three-dimensional imaging, and computer morphometry in whole mouse lungs and human airways

Area of Science:

• Pulmonary Biology
• Neuroscience
• Microscopy and Imaging

Background:

• Complete identification of airway nerves in adult mouse lungs has been challenging due to their patchy distribution and small size.
• Existing methods are insufficient for visualizing the intricate three-dimensional (3D) neural networks within lung tissue.

Purpose of the Study:

• To develop and validate a novel method for comprehensive 3D imaging and analysis of lung innervation.
• To enable detailed quantification of nerve populations and their relationship with airway structures.

Main Methods:

• Utilized tissue clearing techniques to achieve whole-lung imaging.
• Developed a method combining fluorescent casting and custom morphometry software for analyzing nerve images within 3D tissue compartments.
• Applied the method to quantify visceral pleural nerves in mice and intrinsic airway ganglia in human lung tissue.

Main Results:

• Successfully acquired the first complete 3D image of lung innervation in adult mice.
• Demonstrated the ability to pair nerve image analysis with 3D tissue compartment identification and airway morphometry.
• Validated the method's utility in human tissues for imaging full-thickness innervation and quantifying sparse neural elements.

Conclusions:

• The developed tissue clearing and imaging method provides unprecedented visualization of lung neural architecture.
• This approach overcomes previous limitations in studying sparse and complex neural networks in the lung.
• The open-source software and methodology offer a powerful tool for future research in pulmonary neurobiology and related diseases.