齧歯中枢神経系への薬物の直接脳室内デリバリー

The overall goal of the following experiment is to directly administer drugs such as antisense oligonucleotides, as seen in this protocol into the rodent central nervous system by means of the lateral ventricle. This is achieved by two different methods. The first involves surgically implanting a metal catheter connected to an zet osmotic pump into the right lateral ventricle of the mouse.

Once the pump has finished delivering the drug, the pump can either be completely removed, which allows the cessation of treatment or replaced, which allows the continuation of active treatment. The second method to deliver drug relies on a single bolus injection into the right lateral ventricle. A set amount of drug can be rapidly flushed into the entire central nervous system using this method to demonstrate successful delivery of antisense oligonucleotides to the entire rodent central nervous system.

As is demonstrated in this protocol, mRNA and protein levels in several different brain regions following infusion of oligo should be measured. Though this method can provide insight into how to directly administer antisense oligonucleotides to the central nervous system. It can also be applied to other drugs, including antibodies, gene therapy, vectors, and small molecules.

All surgical procedures must be performed under sterile conditions using sterile equipment and solutions, and according to institution and government guidelines. Details can be found in the text protocol and the institutional iacuc guidelines. After anesthetizing the mouse in an induction chamber with isof fluorine, remove the mouse and perform a toe pinch to ensure that the mouse is completely unconscious working.

Quickly shave the hair from the top of the shoulder up to in between the eyes. Then carefully and gently place the anesthetized mouse on the stereo attacks. Push the nose cone over the nose and direct the gas flow to the stereo attacks.

To keep the mouse anesthetized, secure the head with the ear bars. Leveling ear bars are used here though to target the lateral ventricle, a perfectly level skull is not necessary due to the large size of the ventricle. Turn the isof fluorine level down to 2%for maintenance of anesthesia dab eye ointment.

Onto each eye, wipe the top of the head and neck with a cotton swab dipped in 95%Ethanol, followed by a cotton swab dipped in iodine to sterilize the surgical area. Also, place a sterile drape over the mouse. Ensure that the body temperature of the mouse remains at 37 degrees Celsius.

A rectal probe feedback, temperature controlled heating system is used here. Then proceed to implantation of the zat pump. Select the appropriate pump according to the required number of days of infusion, and prepare the pump according to the instructions in the text portion of the protocol.

After making an incision from the base of the neck to the area between the eyes, insert blunted scissors with the curve facing up underneath the skin at the base of the neck smoothly. Slide the scissors back towards the left hind limb and open them slightly to form a subcutaneous pocket. Wipe the skull clean with sterile cotton swabs, followed by a cotton swab dipped in hydrogen peroxide.

To enhance bgma, use the curved hemostat to carefully take a pump from the conical tube, ensuring that the pump does not touch the sides of the conical tube. Hold the base of the pump with forceps and push the flow modulator in the rest of the way. With the curved hemostat stat, use a curved hemostat to hold the pump where the flow modulator and tubing meet.

Insert the pump under the skin at the base of the neck and carefully push it back toward the left hind limb as far as it will go without resistance. Be careful to not let the catheter touch anything with the curved hemostat. Grab the cannula at the groove where the top meets the pedestal.

Move the cannula driver into position and secure into place. Then place a single drop of super glue on the base of the cannula and push the top of the cannula into the driver and position it so that the tubing is pointed straight back. Working Quickly, touch the catheter tip to bgma and zero the coordinates on the digital display while holding the skin out of the way.

With the curved hemostat, raise the catheter and move it 1.1 millimeters laterally to the right and 0.5 millimeters in the posterior direction. As indicated on this diagram, drive the catheter quickly through the skull until the cannula base is securely pressed against the skull. The catheter can be driven directly through the skull and mice due to the relative thin skull.

Pull any skin that has glue on it away from the skull. Then with the cannula driver holding the cannula in place, wait one to two minutes for the glue to fully dry. Next, hold the cannula in place with the curved hemostat while raising the driver.

Slowly release the hemostat to ensure that the cannula is properly secured to the skull. Use a cotton swab to press down on the top of the cannula. Fit the sterile rat bone clippers into the groove between the top and base of the cannula and with the clippers level.

To avoid detaching the cannula from the skull clip off the top of the cannula while still pressing down with the cotton swab. Using the five aught nylon suture, regular hemostat and forceps suture along the full opening, paying extra attention to the area over the cannula. Apply a dab of antibiotic ointment over the head and neck.

Unscrew the ear bars and loosen the nose cone. Remove the mouse from the stereo attacks and place on a warming pad for recovery. Monitor the mice daily after the surgery to check for pain or discomfort and infections and treat as necessary with analgesics such as carprofen according to an approved protocol.

To remove a spent pump, carefully pull the pump out of a 1.5 centimeter incision made perpendicular to the pump junction on the back of the mouse without significantly pulling or pushing on the tubing. Next, clip the tubing approximately 0.5 centimeters above the flow modulator and touch the super glue to the tubing to seal it shut. Wait one to two minutes for the glue to dry.

Place the tubing back into the incision and suture the incision closed dab antibiotic ointment onto the sutured skin and place the mouse on a heated recovery pad. If changing the pump, have the fresh pump ready along with a 10 centimeter dish filled with 95%ethanol. Use forceps to grasp the new pump.

Then dip the fingers of the other hand into 95%ethanol and take hold of the pump. Discard the new pump flow modulator and carefully pull off the old pump from its flow modulator. Attach to the tubing and cannula As soon as the old pump is off, slowly slide the new pump on ensuring that the flow modulator never touches the outside of the mouse.

Reinsert the new pump through the incision back into the subcutaneous pocket and suture the skin after suturing the skin as before dab antibiotic ointment onto the wound and place the mouse onto a recovery pad. Again, monitor the mice daily after the surgery to check for pain or discomfort and infections and administer analgesics as needed to administer an intracerebral ventricular bolus. A scalp incision is made as before, and the skull of the animal is wiped clean and treated with hydrogen peroxide.

Then move the syringe with drug into place. Orient the beveled portion of the needle towards the posterior and secure into place. Lower the needle until it touches bgma.

Zero all of the coordinates, and then move the needle laterally to the right 1.0 millimeters and anterior 0.3 millimeters. Slowly drive the needle through the skull until the top of the needle bevel is flushed with the top of the skull. Again, this can be done due to the thinness of the mouse skull zero.

The Z coordinate and lower the needle to minus 3.0 millimeters at a rate of one millimeter per second. Wait two to three minutes for the brain to seal around the needle. Deliver the full 10 microliters of antisense oligonucleotide at a rate of one microliter per second.

Wait two to three minutes. A one microliter per second infusion rate is used for infusing oligonucleotides into the ventricle. Though this rate may differ between different drugs, hold a cotton swab against the skull at the base of the needle.

Raise the needle at a rate of one millimeter per second. As soon as the needle is out of the skull, roll the cotton swab over the needle hole and hold for one minute to prevent any drug from leaking out. After suturing the skin closed and applying antibiotic ointment, place the mouse on a heated recovery pad.

Depending on the oligo concentration, it may take 20 minutes to a couple hours to fully recover. Again, monitor the mice daily after the surgery to check for pain or discomfort and infections and administer analgesics as needed. This image shows a perfused brain immediately after administration of fast green dye to test the zet osmotic pump insertion coordinates for administration to the ventricle.

As demonstrated in this video, if the catheter is successfully placed in the ventricle, the dye will be distributed throughout the mouse ventricular system as seen here. This image shows a perfused brain shortly after administration of fast green dye to test the intra cerebro ventricular bolus injection coordinates and procedure. If the needle is successfully placed in the ventricle, the dye will again be distributed throughout the mouse ventricular system.

The following images show results from an antisense oligonucleotide experiment to reduce rat, so D one in vivo antisense. So D one oligonucleotides were infused for 28 days into the right lateral ventricle of normal rats at 100 micrograms per day. This image shows that endogenous OD one mRNA levels from brain and spinal cord are reduced.

Following OD one oligo infusion as measured by Q-R-T-P-C-R, this image shows that SOD one protein levels are also reduced. Following OD one antisense oligonucleotide infusion here, protein levels for tubulin and OD one are quantified in different regions of the brain following infusion showing that the so D one oligo is specific to so D one knockdown. In addition, oligos against presenilin one or GSK three beta were infused for two weeks into the right lateral ventricle of non transgenic mice and mRNA levels assessed.

This result showcases the wide array of targets that can be modulated using antisense oligonucleotides. Once mastered these techniques, both the pump and plantation and the bolus injection can be done in 10 minutes if they're performed properly.