Using lightsheet microscopy to investigate the initial lymphatic network in the murine knee joints

Area of Science:

• Biomedical Engineering
• Anatomy
• Immunology

Background:

• Studying joint lymphatic vasculature in mice is challenging due to anatomical variations.
• Previous 2D studies indicated increased lymphatic capillaries in post-traumatic osteoarthritis (PTOA) murine joints.
• 2D histology is limited by sampling bias for variable systems like lymphatic vasculature.

Purpose of the Study:

• To hypothesize that lightsheet microscopy can reveal the detailed structure of the initial lymphatic network.
• To establish and validate a lightsheet imaging protocol for analyzing the initial lymphatic network in mouse knee joints.

Main Methods:

• Developed a lightsheet imaging protocol for mouse knee joints.
• Utilized lymphatic endothelial marker LYVE1 for vessel identification.
• Quantified four outcome parameters to analyze the initial lymphatic network.
• Validated the protocol in PTOA and sham-operated mouse knee joints.

Main Results:

• Post-traumatic osteoarthritis (PTOA) joints exhibited significantly less mean branch length (58.91±6.50 μm) and mean branch diameter (12.25±0.80 μm) compared to sham joints.
• PTOA joints showed significantly more total branching points (378±239) and a higher total initial lymphatic network volume (51247±14239 μm³) than sham joints.
• Statistical significance was observed for all measured parameters (p <0.0001 to p =0.037).

Conclusions:

• Lightsheet imaging is a powerful tool for studying the murine joint lymphatic system and arthritis.
• The study confirmed that PTOA joints have increased branching with smaller and shorter lymphatic vessels.
• This 3D imaging approach overcomes limitations of 2D histology for analyzing complex lymphatic structures.