In Vivo Imaging of Cerebrospinal Fluid Transport in a Mouse Brain Using Fluorescence Macroscopy

Begin with an anesthetized mouse with a surgically exposed skull positioned under a fluorescence macroscope.

The head is stabilized using a head plate over the skull, creating a cranial window.

A cannula containing a fluorescent tracer is inserted into the cisterna magna, or CM, of the brain.

The CM serves as a reservoir for cerebrospinal fluid, or CSF, which circulates through the subarachnoid space surrounding the brain.

Using visible light, focus on the skull.

Inject the fluorescent tracer into the CM, then switch to fluorescence imaging.

Illuminate using light of a specific wavelength to excite the tracer.

The tracer absorbs this light and emits fluorescent signals.

The fluorescent tracers travel with the CSF, enabling visualization of its flow through the subarachnoid space into the perivascular spaces surrounding cerebral arteries and penetrating

arterioles, entering the brain tissue.

Capture the fluorescent signal over time to visualize CSF flow.

Carefully place the mouse and the infusion pump attached to the cannula on a cart for transport to the macroscope, and place the head holder on the stage of the macroscope. Make sure that the line from the syringe pump to the Cisterna Magna C