Spinal Cord Window Implantation in a Mouse for In Vivo Imaging

Begin with an anesthetized mouse with a surgically exposed spinal cord.

Place a borosilicate cover glass over the spinal cord to create an optical window for imaging.

Apply warm agarose to the edges of the cover glass, allowing capillary action to draw it underneath to maintain tissue hydration.

Introduce tissue adhesive to the intact bony structures surrounding the site to stabilize the spinal cord window.

Apply dental cement mixed with an accelerant over the adhesive layer, then position a 3D-printed backplate over the cover glass before the cement cures.

Apply additional thin layers of cement to strengthen backplate adhesion.

After the cement has dried, secure the backplate to a forked backplate holder to minimize motion artifacts from respiration and heartbeat.

Add saline to check for leakage and seal with additional cement if necessary.

Finally, transfer the mouse and platform to a two-photon microscope for imaging.

For cover glass implantation, gently apply a 3 millimeter borosilicate cover glass to the exposed cord. Use a small spatula to apply 39 degrees Celsius, warmed 2% agarose to the edge of the glass, and allow capillary action to draw the agarose under the cover glass surface. Apply tissue adhesive to the exposed bony articular processes of the intact adjacent vertebra at the thoracic level 11 and 13 vertebral spines.

Apply additional tissue adhesive in a ring around the laminectomy site, over the adjacent tendon and the transverse process. Next, use a new spatula to apply dental cement mixed with accelerant onto the tissue adhesive layer and place a back plate onto the surgical field centered over the cover glass window.

Applying multiple thin layers of dental cement results in a stronger back plate adhesion, but be sure to place the back plate quickly before the dental cement starts to dry.

After allowing the dental cement to cure for 10 minutes, fill in the interior base and underside of the back plate with additional adhesive.

Applying the dental cement in thin layers can help reduce the risk of cement spreading to the cover glass and agarose, which can compromise the entire protocol.

When the dental cement has dried, apply a forked back plate holder to the appropriate position over the window and screw the back plate into the back plate holder. Then apply saline to the back plate to test for leakage. The animal and the surgical platform can then be transferred to the optical table of a two photon microscope for imaging through the cover glass window.