This article describes a method for visualizing cerebrospinal fluid (CSF) flow in the brain using fluorescent tracers. The technique involves injecting a tracer into the cisterna magna of an anesthetized mouse and capturing the resulting fluorescence to observe CSF dynamics.
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 Cannula is not taut, and that it has no kinks. Observe the respiratory rate and pink coloration of the mucous membranes to confirm a good oxygenation.
Turn on the microscope camera and the light-emitting diode and start the live mode. Adjust the magnification of the imaging field so that the nasofrontal suture at the top of the field and lambdoid suture at the bottom can clearly be visualized. Once in place, tape the head holder to the macroscope stage, and focus the macroscope on the exposed skull until the focal plane is located on the lateral sides of the parietal bones, posterior to the coronal sutures.
For CSF tracer infusion, set the infusion pump to the appropriate rate and volume, and set the appropriate excitation wavelength and exposure time for the tracer for each channel. Then check that the triggering function on the macroscope is correct before simultaneously initiating the tracer infusion and the imaging.
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