This method enables the examination of protein dynamics in vivo within the intact central nervous system to address questions in neuroscience and immunology. The main advantages of this procedure are an excellent motion stability for image acquisition, brief surgical time, reduced operator exposure to gaseous anesthesia, and inexpensive customizable backplates. Visual demonstration of this method is critical as it is difficult to remove the bone without damaging the closely underlying nervous system tissue during the laminectomy steps of the procedure.
Before beginning the procedure, use 3D computer aided design software to create a model to the indicated dimensions. So we've got an object, which is the three dimensional object, that that's what we can start with. What I can then do is we can put settings on that for the printing version.
So if I open Edit this, go into Advanced, open a window that enables me to look at all of these settings. Set the nozzle temperature to 205 degree Celsius, the bed temperature to 45 degrees Celsius, and the printing speed to 45 millimeters per second on a 3D printer. Use a 0.4 millimeter hot ent nozzle and a 0.2 millimeter layer height to print the back plates.
Then assess the printed back plates visually for their structural integrity. Gross structural failures indicate printing defects. When the back plates are ready, place the head of an eight to 12 week old anesthetized mouse on a heat pad between the ear bars of a surgical restrainer and apply ointment to the animal's eyes.
After confirming a lack of response to toe pinch, use a number 11 blade to make a 1.5 centimeter rostral to caudal midline incision over the lower thoracic upper lumbar region and separate the skin from the peritoneum. Use forceps to peel back any remaining transparent connective tissue under the skin to expose the superficial musculature. Displace the muscles with a foam surgical spear along with the remaining deeper muscular of the target vertebra.
To create a seat for the back plate, clear the muscle from the posterior aspect of thoracic 11 and the anterior aspect of thoracic 13. Using forceps to remove the remaining muscle from the tendons as necessary. When all of the muscle has been removed, carefully cut the tendons with forceps until sufficient space for visualizing and manipulating the spinal cord is achieved.
Confirm that the dura mater of the intervertebral space, the semitransparent laminar bone, the central superficial blood vessel under the bone, and the anterior radiating artery are clearly visible. Wet the region with warm artificial cerebral spinal fluid and use a micro-drill to thin the laminar bone using straight strokes parallel to the long axis of the spinal cord. Gently grasping the superficial spinous process with forceps, lift the vertebra.
The bone should lift away easily. Use number four forceps to clear away any bone shards, controlling any bleeding with a surgical spear and gentle steady pressure as necessary. Then rinse the tissue with warm artificial cerebral spinal fluid.
For cover glass implantation, gently apply a three millimeter borosilicate cover glass to the exposed cord. Use a small spatula to apply 39 degree 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 place 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. In this representative experiment, EGFP positive claudin-5 was visualized within the fluorescently labeled tight junctions throughout a vascular plexus.
The clear delineation of the tight junction structures in the Z projection indicates that minimal XY image displacement is produced after a successful laminectomy, window placement, and back plate implantation. While attempting this procedure it's important to remember that this microsurgery is difficult and may take time to master. However once mastered, this surgery can be performed in approximately 30 minutes.
Following this procedure, other methods, like two photon microscopy and in vivo imaging, can be performed to answer additional questions about neurovascular remodeling and other disease processes involving the spinal cord.
