The overall goal of this procedure is to create a window view of spinal cords and adult mice to study experimental autoimmune encephalomyelitis by longitudinal intravital two photon microscopy. This is accomplished by first implanting a spinal clamp to support the vertebrae for surgery and imaging. The next steps are to expose the spinal cord and prepare the lateral edges of the vertebrae for supporting the glass window.
This is followed by implanting the window with a thin layer of silicone elastomer between the window and the dura. The final step several weeks after the implantation is to induce EAE in the implanted mice. Ultimately, results can show the spatiotemporal changes in cell distributions and cellular interactions in the spinal cords of EAE mice using two photon microscopy.
Visual demonstration of this method is critical. The method can be difficult to learn because it's time sensitive and requires a high degree of precision. Begin by anesthetizing the adult mouse per surgery and applying eye ointment shave.
Its back approximately two centimeters wide by five centimeters long, centered on the thoracic hump and cleaned the exposed skin using iodine soap, followed by iodine solution. Using tissue scissors, cut the dorsal skin longitudinally about two centimeters over T 11 to L one. Next, using a scalpel, make bilateral longitudinal incisions between the spinous and transverse processes from T 11 to L one.
Suspend the mouse in the spinal fork stereotaxic by first clamping the top right spinal fork into position. Second, grasp T 11 using addin forceps and position the T 11 transverse process over the fixed spinal fork using the same procedure position L one within the spinal fork. Now using a number 23 scalpel, gently scrape away the muscles between the spinous and transverse processes from T 11 to L one so that the lemonade are clearly visible.
For a better view of the pedicles of T 11, cut the tendon projecting roly from the tip of the transverse process. Rinse the modified staples with supplemented PBS. Then insert the tips of the staples lateral to the vertebral pedicles of T 11 and L one.
Adjusting the distance between the tips so that they're lightly touching the pedicles Glue, the staples in place with a small drop of cyanoacrylate at each insertion point, and remove the mouse from the spinal forks. Now apply a thin layer of cyanoacrylate to the cut edges of the skin and exposed muscles around the cleared vertebrae. Be careful to not glue the vertebrae.
Bend the tips of a modified paperclip so that they enter under the protruding sections of the implanted staples and rest parallel to the long axis of the isolated vertebral column and leave in this position. Starting at the skin muscle interface, apply several layers of dental cement to cover the entire opening except the exposed vertebrae being careful to implant the paperclip securely within the dental cement. After five to 15 minutes, the dental cement will cure.
Then attach the mouse to a device that can securely grasp the exposed portion of the paperclip without causing stress to the clip or chamber. Using forceps, remove the muscle and other soft tissue between the spinous processes and laminate of the exposed vertebrae. Now using the bone removal drill, then the laminate of T 12 and T 13 bilaterally.
Then proceed to remove the dorsal bone. Grasp the spinous process with blunt forceps and lift, taking care to apply no downward force towards the spinal cord. If the bone does not easily come away, continue to thin the lamina until the spine is processed, can be easily removed.
Using the bone removal drill, continue to shape the lateral edges of the vertebrae to form a support structure. For the window. Gently remove all of the small bone fragments touching the dura matter.
Using blunt forceps, be sure to maintain the humidity of the dura matter. Using PBS, choose one of the prepared windows that is slightly wider than the laminectomy opening and test how well it rests on exposed bone. Ideally, the window will rest bilaterally on both vertebrae so that the middle of the window touches the dura matter.
The window should not, however, compress the spinal cord, even when pushing the window towards the spinal cord. If the window is unbalanced or rests above the dura, further shape the lateral edges of the vertebrae. Once satisfied with the laminectomy, place a small piece of tissue paper soaked in PBS onto the spinal cord to prevent drying of the dura and set the mouse aside.
Soak the window in the cleaning solution for several seconds. Then use a piece of microscope lens tissue to wipe the window dry with a surgical microscope. Verify that the window is clean.
Otherwise, repeat the cleaning process. Then keep the window in a clean area ready to implant. Applying the silicone elastomer to the spinal cord followed by placing of the window, must be done quickly, ideally in less than 15 seconds.
It's very important to have your workspace arranged so that all your materials are easily accessible. First on the mouse, remove the tissue paper and gently remove any debris from the dura using blunt forceps and allow the dura to dry until slightly tacky. Now, quickly fill the silicone mixer tube with silicone at less than 27 degrees Celsius extrude silicone onto a clean slide until there are no bubbles emerging.
Then immediately administer a small bead of silicone along the midline of the exposed spinal cord. Pick up the clean window using blunt forceps and place over the silicone. Using the forceps.
Apply a small amount of pressure to the window so that it spreads the silicone to the edges of the window and hold in place for about a minute. Slowly release pressure from the window. If the window rises when the pressure is released, then maintain pressure on the window to hold it in place.
Next, apply cyanoacrylate, rostral and coddle of the window. Use a toothpick, guide the cyanoacrylate to the edges if necessary. Then guide the glue along the lateral edges of the window to cover all of the surrounding muscles.
Only cover the outer edges of the window or optical clarity will be compromised. Insufficient glue will result in air bubbles between the silicone and window to protect the cyanoacrylates and increase structural integrity of the chamber. Apply dental cement to all regions covered with cyanoacrylate.
As with the cyanoacrylate, start at the rostral and coddle edges and guide the dental cement along the edges of the window. First, inject dexamethasone and ril over the pelvic regions every two days for 10 days. Inject dexamethasone and ril to inject a mouse with an implanted window.
First, lightly sedated with iso fluorine in an induction chamber. Do not scruff mice with implanted windows to take images lightly anesthetize the mouse and attach it firmly to a clip holder by the exposed portion of the implanted paperclip using the implantation method. Most windows were clear at the time of EAE induction and remains clear throughout EAE progression.
Two photon imaging was feasible up to 12 times over 36 days. In comparing EAE clinical progression in animals with and without implanted windows, EAE mice had a slight delay in the onset of clinical signs. However, following onset, there was no difference in the overall progression or peak clinical score.
The timeline of axon degeneration typified by EAE was examined in the spinal cord over the course of EAE progression by intra vital two photon microscopy. There were many uninjured axons as well as axons in different stages of degeneration, such as axons with focal swelling. Fragmented axons were also observed.
The average number of uninjured axons at multiple time points relative to the onset of clinical signs was counted in five. Mice counts were made within the same region from session to session by orienting to vascular markers within regions of interest. The number of axons with focal swellings were quantified as were the fragmented axons.
The number of damaged axons increased with the onset of EAE clinical signs and axon deterioration progressed from swellings to fragmentation at later time. Points Once mastered the window implantation surgery can be done in about an hour and a half if it's done properly. So there are a couple of things that we should note about today's surgery.
First, there was a bit of compression of the central vein during window implantation. This is what I would consider the maximum amount of compression allowable before axon damage would occur. Second, there were some bubbles located under the window in the quicks seal after placement.
A couple of these bubbles were spherical and located within the quicks seal, whereas one was located between the germ outter and the quick seal. After about 15 minutes. This second bubble was resorbed and really won't affect imaging whatsoever.
The other two bubbles were rostral and coddle in the visual field, and so we can also still get very nice images in between these two locations.
