Demyelination takes place in multiple central nervous system diseases. A reliable in vivo drug delivery technique is necessary for remyelinating drug testing. This protocol describes an osmotic pump-based method that allows long-term drug delivery directly into the brain parenchyma and improves the drug bioavailability, with broad application in remyelination research.
Osmotic pump is of great value to the research of myelin regeneration, especially for those drugs with a short half-life, poor blood-brain barrier permeability, and of various peripheral side effects. Osmotic pump can bypass the blood-brain barrier and deliver drugs directly to the corpus callosum, which effectively improves the bioavailability of drugs, especially for some drugs with a short half-life. To begin, secure the head of an anesthetized mouse in the stereotaxic apparatus with a tooth bar and ear bars.
Cover the animal body except for the surgical site. Using a scalpel, make a one centimeter long midsagittal incision of the skin from the base of the neck to in between the eyes to expose the skull. Using a cotton swab containing 30%hydrogen peroxide, gently wipe the surface of the skull to visualize the cranial structures.
Adjust the height of the tooth bar and ear bars to place the lambda point and bregma point at the same height. Place the tip of a 10 microliter and 33 gauge syringe needle gently at the bregma point, and reset the X, Y, and Z coordinates to zero. Move the syringe to the injection site.
Slowly drill a small burr hole through the skull at the injection site without penetrating the dura with a one microliter 26 gauge and 0.45 millimeter syringe needle. Slowly insert the microliter syringe needle into the brain tissue through the hole until a certain depth is reached. Inject 1.5 microliters of 1%lysolecithin at a speed of 0.5 microliters per minute.
Wait for five minutes before pulling out the syringe, and stitch the skin with 5-0 surgical sutures. Place the mouse that has undergone surgery alone in a cage, and monitor the mouse daily after the operation. Attach three depth adjustment spacers to the needle of the brain infusion cannula with tissue adhesive to achieve an injection depth of 1.5 millimeters that is close to the callosum.
Fill the osmotic pump by attaching the syringe needle with a pump package to a one milliliter syringe, and aspirate the drug. Hold the pump in an upright position. Insert the syringe into the opening at the top of the pump and slowly inject the drug without creating an air bubble.
Slowly pull out the syringe as the liquid flows out of the opening. Using scissors or pliers, remove the white flange from the flow regulator. Insert the flow moderator into the pump.
To determine whether there are bubbles in the osmotic pump, weigh the osmotic pump separately, before and after filling. Trim the catheter to the required length according to the size of the animal. Attach the catheter to the brain infusion cannula.
Fill the catheter with drugs using the syringe without introducing air. Connect the catheter to the flow moderator so that it covers about four millimeters of the exposed flow moderator. Immerse the filled pumps in sterile 0.9%saline or PBS at 37 degrees Celsius for at least four to six hours.
Prefer to extend overnight after implementation. Secure the mice on the stereotaxic apparatus again. Open the previously stitched surgical incision and expand the incision up to the shoulder blades.
Separate the skin from the subcutaneous connective tissue at the scapula, using hemostatic pliers or tweezers to open a cavity, and place the osmotic pump into the cavity. With a cotton swab, gently wipe and expose the pin hole on the surface of the skull created when establishing the demyelinisation model. Insert the brain infusion cannula through the pinhole perpendicularly and secure it on the skull with tissue adhesive.
Take off the removable tab above the brain infusion cannula with a pair of scissors. Stitch the incision or attach it with tissue adhesive. Place the animal in a cage alone after the surgery.
After performing implementation DAPI staining revealed that the pinhole in the brain tissue lies just above the white matter, indicating successful implementation of the brain infusion cannula of the osmotic pump. The in situ hybridization experiment was performed with a mature oligodendrocyte marker MAG probe was used to label newly differentiated oligodendrocytes. The results showed that the UM206 treatment yielded more MAG positive cells in the demyelinated region than the control group.
Transmission electron microscopy of the demyelinated region showed that the number of myelinated axons was increased in the UM206 treatment group compared to the control group, suggesting that UM206 induced a higher level of re-myelinisation. When preparing the osmotic pump, be careful not to introduce any bubble into the system.
