The overall goal of this procedure is to successfully perform a heterotrophic mucosal grafting surgery for delivering compounds into the brain. This is accomplished by first isolating a nasal septum graft from a donor mouse. The second step is to transplant the mucosal membrane from the septum onto a skull defect on a recipient mouse.
Next, the brain is dosed through the graft for a desired period of time. The final step is to analyze the consequence of the brain dosing procedure by using fluorescence, microscopy, or immunohistochemistry to show diffusion of the delivered compounds into the brain.Parenchyma. Utilization of neurop pharmaceuticals is highly limited due to the presence of the blood-brain barrier.
We have developed a technique based on human endoscopic transnasal surgery where we can graph mucosa into a hole in the blood-brain barrier and use this as a conduit for drug delivery directly to the brain. Here, Richie Komen, a postdoctoral student in Han's Lab at Boston University, is gonna demonstrate this technique in a mouse model. Begin this procedure by removing the skin around the nasal region of a euthanized mouse to expose the skull.
Then mark three lines with the pneumatic drill. Two of the lines laterally flank the nasal region, and the third one is in line with the eyes, which connects the two lines perpendicularly. Next, drill down ventrally.
In order to separate the nasal septum from the surrounding tissue, cut the septum free from any tissue adhered to it. Then store it in a sterile saline solution under the microscope, clean the graft and remove any connected tissue. The ideal situation is to successfully remove all the connective tissue without damaging the mucosal membranes present on both sides of the cartilage septum.
After anesthetizing a mouse using standard aseptic mirroring surgical procedures mounted in theological frame, apply ointment to the eyes to prevent dryness. Next, expose the skull and level the head. Then cut a 1.25 millimeter diameter circular hole on the skull using a pneumatic drill.
When targeting the atu, wet the drilled area with sterile saline and use a razor blade to remove the skull. After that, manually remove the dura using the tip of a 30 gauge needle. Subsequently, place the mucosal membrane above the brain surface, taking extreme care to keep the epithelial side facing away from the wound.
Using the tip of a pair of surgical scissors, hold the membrane off the cartilage and onto the skull and brain surface to generously overlap all bony edges of the craniotomy site. Next, cover the graft with a sterile piece of nitrile. This acts to prevent adhesion of the skin to the graft during healing.
Trim excess membrane if necessary, and avoid any motion of the nitrile once it has come into contact with the graft. Then close the skin with suture and let the mouse recover for three to seven days before proceeding to the next step. Make sure appropriate post-surgical treatments are performed and regular analgesics are administered to dose the animal.
After securing the anesthetized mouse in the stereotaxic frame, cut the suture with scissors and remove excess skin around the skull. Remove the nitrile barrier and clean the surface of the skull. Use sterile saline and cotton swabs to clean the area until the graft is visible.
It may be necessary to cut the graft with the razor if it is grown larger than the desired surface area. Next, place the well above the graft so that the edges are in contact with the skull. Then apply cyanoacrylate adhesive at the junction between the well and the skull.
Fill the well with sterile saline and check to make sure there are no leaks. Subsequently, apply cement on the skull to secure the well in place. Afterward, remove the saline from the well with a pipette.
Wash the well several times to verify that adhesive has not leaked in. Then add 50 microliters of fluorescent dextran. Cap the top of the well by using a circular piece of nitrile secured to the top with cyanoacrylate adhesive, and make sure the adhesive does not come in contact with the well contents.
After the desired amount of time has passed, euthanize the animal and remove the implant and the brain. Be careful to keep the graft in place. Once removed, flash, freeze the brain In a solution of isobutane cooled in a dry ice bath, embed the brain in OCT solution and slice at 50 micrometers.
Place the desired slices directly on a microscope slide. Then image the slice using a fluorescent microscope as soon as the OCT solution has dried. This is a fluorescent microscope image of a mouse brain slice following transmucosal dosing.
With 40 kilodalton tetraethyl Rumine conjugated dextran for 24 hours, the mucosal graft is visible on the surface of the brain. Once mastered, this technique should take a total of four hours of surgery time to perform properly. After watching this video, one should be able to isolate a nasal septum and transfer mucosal membrane over a craniotomy site for the purpose of drug delivery to the brain.
