Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

In this video article four Methods of drug delivery to the central nervous System are demonstrated. The first three methods are intravenous injection, intraperitoneal injection, and oral gavage. All three of these methods result in systemic delivery of the drug.

The final method is a local drug infusion into the brain of the mouse. For this method, in vivo, optical imaging is used to verify proper infusion. The three systemic delivery techniques are standard procedures that are widely used in preclinical settings.

The local administration technique is a bit more challenging, but allows for direct administration of agent to the brain bypassing the blood brain barrier. These methods can help answer key question. In new oncology, such as the efficacy and in distribution of Nobel therapeutics, Though these methods can provide insight into the novel treatment of brain tumors.

These methods can also be applied to other diseases of the central nervous system, such as neurodegenerative and psychiatric disorders. Using a heat lamp to dilate the tail vein warmer mouse for five to 10 minutes, carefully monitor the mouse at all times to avoid hypothermia while warming the mouse. Load the drug into a 28 gauge insulin syringe.

No more than 1%of the mouse's body weight in volume should be injected at once. After five to 10 minutes have passed. To transfer the mouse to a holding device in the mouse tail, there are four visible vessels.

The dorsal and lateral vessels are veins. The ventral vessel is an artery to access the lateral tail vein, hold the tail of the mouse at the most distal point and rotate it 90 degrees so that the vein is facing upwards. Clean the injection sites with an alcohol swab.

Next, insert the prepared syringe bevel site up into the vein. If the needle is properly placed in the vein, it should move freely and without pressure. Slowly inject the drug with even pressure over five to 10 seconds.

If the tail becomes swollen, stop injecting as this indicates that the needle is no longer in the vein. After the injection apply gentle pressure to the injection site until the bleeding stops. This normally takes 30 to 60 seconds.

Monitor the mouse for five to 10 minutes to ensure that there is no further bleeding. Prepare for this procedure by loading a syringe with a 28 gauge needle with the drug. Remove the mouse from its cage by the tail and place it onto a textured surface, such as a cage lid, so that the mouse has something to grip.

Allow the mouse to stretch its body. Next, grasp the skin on the back of the mouse taking care to lightly pinch as much skin as possible between the thumb and index and middle fingers. Turn the mouse over so that the abdomen of the mouse is facing upwards.

If the mouse can freely move its head, release the grip and try again. So as to avoid being bitten during the injection, hold the mouse slightly inverted to help move the organs away from the injection site. Insert the prepared syringe needle at a 30 degree angle into the lower left quadrant of the mouse to ensure that the needle is in the intraperitoneal space.

Pull back on the syringe plunger. If no fluid is aspirated into the syringe, then inject the syringe contents with an even pressure over one to five seconds. Release the mouse.

Monitor the mouse for five to 10 minutes after the injection to ensure that it returns to normal activity levels. Begin oral gavage by determining how far the needle can be inserted into the mouth of the mouse. Hold an 18 G ball tip curved gavage needle at the last rib.

Mark the length using a permanent marker. Weigh the mouse to determine the injection volume. The maximum volume that can be delivered by oral gavage is 10 milliliters per kilogram of body weight.

Then load the needle. Next, restrain the mouse using the same grip as shown for the intraperitoneal injection. Insert the gavage needle into the mouth over the tongue and advance the needle through the pharynx to avoid puncturing the esophagus.

Do not insert the needle past the stopping mark. The needle should proceed smoothly without any pressure. With the needle in place, slowly depress the plunger.

Remove the needle at the same angle that it was inserted. Do not attempt to inject larger volumes than recommended. As this may result in reflux and incomplete drug delivery.

Place the mouse back in its cage and monitor it for five to 10 minutes. Paying close attention to any signs of labored breathing, which may indicate that fluid has entered the lungs. Repeat this procedure as necessary.

Up to three doses can be given in a 24 hour period since a reflux resistant CED cannula for rodent is not yet commercially available. Construct a cannula consisting of three parts. A 100 micron diameter piece of silica tubing through which the infuse eight flows.

A rigid metal needle for structural support and flexible Teflon tubing for loading the infuse.Eight. To prepare the surgical area, spray all surfaces with 2%chlorhexidine disinfectant. Then cover the surfaces with absorbent drapes.

The following supplies should be placed in the surgical area. Choose more petro dishes, one containing 3%hydrogen peroxide and one containing 2%Chlorhexidine sterile GREs and cotton swabs. Sterile disposable number 21 scalpels autoclave, mouse skin stapler, staples and staple remover.

Sterile 20 gauge needles heating pad to maintain mouse body temperature, a mouse stereotaxic frame, and a controlled rate syringe pump. To set up the cannula, attach a one milliliter syringe to the Teflon tubing. Using a set of plastic syringe adapters, affix the cannula to the stereotaxic frame so that it's perpendicular to the surgical surface.

Using a one milliliter syringe, disinfect the cannula by running 70%ethanol through it. Repeat this process using sterile saline. Then check for any leaks around the cannular joints.

Pull back on the syringe so that a small air bubble is drawn into the cannula. This air bubble will separate the infuse eight from the saline. Next back load the infuse eight.

Prime the pump by briefly running the cannula. Swap the skin of an anesthetized mouse with sterile gore dipped in 2%Chlorhexidine, apply ophthalmic ointment to maintain adequate eye moisture during the procedure. Using a sterile scalpel, create a sagittal incision along the center of the skull, approximately 1.5 centimeters long.

Clean the skull's surface using a cotton swab soaked in a 3%hydrogen peroxide solution. The suture lines of the skull should be apparent at this point. If not gently swab the skull with a fresh cotton swab soaked in 3%hydrogen peroxide solution.

Identify the bgma, then measure two millimeters to the right and one millimeter posterior to locate the infusion site. Using a sterile 20 gauge needle, gently create a hole in the skull at this point by twisting the needle against the skull. Avoid forcing the needle downward against the skull.

At this point, transfer the mouse to the stereotaxic frame and begin administering inhaled anesthetic. At a low level. Carefully monitor the mouse for changes in respiration rate, adjusting the anesthetic accordingly.

So this next step is probably the most difficult aspect of the entire method. When placing the cannula, be sure to center it over the hole. To do this, check the cannula placement from all angles.

Next, position the cannula over the skull hole and then lower three millimeters below the skull surface. Begin the infusion by turning on the syringe pump using the rates and durations shown here at the end of the infusion. Slowly remove the cannula and swap the skull with 3%hydrogen peroxide.

Apply sterile bone wax to the hole using forceps. Draw the skin together and staple to close. Administer buprenorphine subcutaneously for postoperative pain relief.

Place the mouse in a cage on a heating pad to avoid hypothermia. Do not house the recovering mouse with other active mice. Monitor the mouse postoperatively until it regains consciousness and mobility.

Due to the length of the procedure, it may take up to an hour for the mouse to regain full activity. If imaging will be performed, it is best to wait two to three hours to perform imaging of fluorescent labeled infuse.Eight. Following CED administration, position the anesthetized mouse dorsal site up in an imaging station using an appropriate filter setting for the fluorescence that is being imaged.

Acquire an image for CED. A successful infusion should show most of the material in the skull near the infusion site. A lack of adverse reaction to administration of therapy is an important indicator of successful injection.

The following are indicators of improper injections for tail injections, change in the appearance of size or color of the tail, a bubble or blister in the tail, a indicating subcutaneous rather than intravenous delivery. For intraperitoneal injections. A bump on the skin or discoloration of the abdomen may indicate subcutaneous injection or damage to internal structures.

In oral gavage, a mouse with labored breathing or coughing may indicate that fluid was injected into the lungs rather than into the stomach. For CED, neurologic function is important for assessing successful administration of therapy. A mouse that is exhibiting seizures or hemiparesis may have received improper infusion.

Here in vivo imaging was performed on a mouse with a successful CED infusion shown on the left, and an unsuccessful infusion shown on the right. Note that in the successful infusion, the imaging signal is localized to the brain. While in the unsuccessful infusion, the signal has spread down the neuro axis.

As you've seen, the most challenging aspect of this procedure involves the proper selection of route of administration. When delivering drugs to the central nervous system, it's important to consider the pharmacologic property of the drugs in order to select the best load of administration. So that's it.

I hope you found this presentation useful, and good luck with your experiments.