This new technique for real-time mesoscopic imaging of florescent CSF traces through the intact skull of live mice can be used to evaluate glymphatic transport. The main advantage is that this technique enables dynamic measurements of in-vivo intracisternal tracers at a fraction of the cost of other imaging modalities. The procedures required to place the head plate on cisterna magna cannula are simple and minimally invasive but require practice to be performed correctly.
Visual demonstration of the critical steps of this method is required to maximize the image quality and to enable reproducible comparisons between different mice and experimental groups. After confirming a lack of response to toe pinch wet the neck and head with sterile water and shave the moistened fur. Wipe the exposed skin with an alcohol swab to remove any residual hair and place the mouse in a stereotactic frame on top of a temperature controlled pad.
Apply ointment to the animals eyes and clean the exposed skin with a Chlorhexidine swab. After 2 minutes remove the Chlorhexidine with an alcohol wipe and apply an iodone solution. When the iodone has dried inject subcutaneous analgesia into the top of the skull and neck.
Starting at the part of the neck that covers the occipital crust, make a midline cut in the overlying skin continuing rostrally toward the intraorbital line. Extend the incision laterally to the border where the temporal muscle inserts into the skull and remove all of the skin of the fusiform incision to expose both the frontal and parietal bones. Irrigate the skull with sterile saline, and use cotton swabs to clean the surface until it is free of debris and hair.
After inserting the cisterna magna cannula apply a mixture of dental cement and cyanoacrylate glue to the ventral side of the head plate around the border. Place the head plate onto the skull so that the anterior border of the plate aligns with the posterior tip of the nasal bone and the posterior border aligns with the anterior aspect of the interparietal bone making sure that the sagittal suture is centered and straight relative to the window. It is important to make sure that the head plate is secured and that it does not obstruct the field of view of the cisterna magna cannulation.
Use a couple of drops of glue accelerator to fix the position of the head plate and fill any remaining gaps with the cement mixture. Glue the cisterna magna cannula to the head plate and use the head plate to place the mouse into the head holder in a fixed position. 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 mucus membranes to confirm a good oxygenation. Turn on the macroscope 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. During imaging the naso frontal, sagittal, coronal, and lambdoid sutures can be readily identified. Once the CSF tracer has been infused into the cisterna magna the tracer fluorescence is first observed in large pools of subarachnoid CSF at the olfactofrontal cistern and the quadrigeminal cistern near the pineal recess and eventually surrounding the middle cerebral arteries.
CSF tracers then enter their brain along peri vascular spaces of the cortical peal branches of the middle cerebral artery. CSF transport imaging after traumatic brain injury reveals tracer initially at the olfactofrontal cistern but the glymphatic influx along the cortical perivascular spaces is completely abolished on the side of the injury. Quantitative analysis at the in-vivo images demonstrates that the ipsilateral influx area is decreased nearly a third compared to the contralateral hemisphere.
To avoid motion artifacts and the changes in the focal plane remember to correctly secure the head plate and the head plate holder and check the anesthesia level of the animal. After the experiment in-vivo imaging results can be further validated by quantifying the tracer penetration using the actual This technique allows the study of the glymphatic system in a physiological and minimally invasive manner and can help address future questions about CSF hydrodynamics in health and disease.
