Abstract
Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.
Highlights
Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals
To visualize the spatiotemporal dynamics of the colon, we designed a colonic window to span the abdominal cavity without restricting movement (Fig. 1a)
One of the greatest challenges in imaging the colon is minimizing motion artifact caused by intrinsic motor patterns without obstructing gastrointestinal transit
Summary
The vasculature landmarks labeled with Texas Red-Dextran consistently disappeared by the third day, likely due to leaky vasculature following inflammatory challenge These methods demonstrate the colonic window enables tracking of monocyte recruitment and activation in response to colonic inflammation. When electrical stimulation was delivered at 14 Hz, commonly used in FDA-approved therapeutic SNS35,37,50, it evoked calcium responses in 10% of colon myenteric neurons on average in four out of five mice, with at least 25 cells per mouse Intravital imaging through the colonic window revealed Fast Blue-labeling in GCaMP-expressing myenteric neurons, which permitted us to monitor their calcium activity in real-time (Fig. 4f). Fast Blue, 9.8% on average responded to SNS in four mice (Fig. 4h) These data demonstrate the colonic window can be used to monitor enteric neural activity in real time under chronic, survival conditions
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