連続IV注入は、脳卒中誘発脳浮腫を研究するためにマウスにおけるアルギニンバソプレシン受容体拮抗コニバプタンのための選択の治療のルートであります

The overall goal of this pre-clinical study, is to create a successful mouse model of Conivaptan administration after stroke, to help improve the design of future research studies in the stroke field. This method can help answer key questions in the stroke research field, such as, what is the importance of the delivery route to the beneficial effects of Conivaptan on stroke recovery in mice. The main advantage of this technique, is that it will assist researchers in the modelling studies of Conivaptan and it's effects on stroke-evoked brain edema.

At least two hours before the surgery, cut a 12 millimeter piece of filament from a 7-0 nylon suture, then mix two parts dental resin with one part hardener, and quickly dip the filament into the mixture, until approximately four millimeters of the filament have been coated. Immediately verify that the filament tip is covered with a smooth coat of dental resin. Then, air-dry the filament for two hours.

When the resin is ready, place an anesthetized mouse onto a heating pad on the operating table, and confirm a lack of response to toe-pinch. Remove the hair on the front and back of the neck with clippers, spray the neck with rubbing alcohol, and apply ointment to both eyes. To continuously monitor body temperature during the surgery, use a rectal temperature probe connected to a digital display.

Place the mouse in the supine position under a dissecting microscope, and if necessary, position a piece of rolled gauze underneath the neck to arch it. Then disinfect the neck with a Povidone Iodine 10%scrub solution. Then, cover the animal with a sterile drape, and use a size 10 surgical blade to make a mid-line incision along the neck.

Using a 6-0 silk suture, temporarily ligate the left common carotid artery, and follow this with a tie on the left external carotid artery. Place a microvascular clip on the internal carotid artery, and use microvascular scissors to cut a small hole on the external carotid artery. After inserting the dental resin-coated filament into the opening, advance the filament cephalically while removing the clip until resistance is felt.

Place a temporary tie onto the internal carotid artery to secure the filament in position, and close the skin with a silk suture. Then infiltrate the wound on the front of the neck with 0.2 millimeters of 0.5%Bupivacaine and transfer the mouse to a recovery chamber with monitoring until sternal recumbance is maintained. For Neurological Deficit Scoring, lift the mouse by the tail, and place the animal on a flat surface for two to three minutes.

Score the animal's behavior according to the text, excluding mice that exhibit a Neurological Deficit Score lower than two, due to an insufficient occlusion of the middle cerebral artery. To remove the filament, re-sterilize the incision with a 10%Povidone Iodine scrub solution, and cut and remove the suture to re-open the surgical wound. Then, under a dissecting microscope, place a microvascular clip on the internal carotid artery, and untie the internal carotid arterial suture.

Hold the clip open to slide out the filament. Then immediately close the clip again, and suture the external carotid artery proximal to the cut. When the external carotid artery ligature is secure, remove the clip and untie the suture around the common carotid artery to restore the blood flow to the brain.

Immediately after removing the common carotid artery suture, disinfect the back of the neck with a 10%Povidone Iodine scrub solution, and use a size 10 surgical scalpel blade to make a one centimeter incision. Then re-sterilize the front of the neck, and re-open the surgical wound. Now tunnel a piece of 0.94 millimeter outer-diameter tubing, nested within a piece of 3.18 millimeter outer-diameter tubing under the skin of the animal from the back to the front of the neck, and exteriorize the tubing when it reaches the front incision.

Remove the subcutaneous adipose tissue from the left side of the front of the neck about one centimeter laterally from the mid-line. Then locate the left jugular vein under the dissecting microscope, and place two ties along the vein 5 millimeters apart, slightly stretching the vessel. Next, use microvascular scissors to make a small hole on the jugular vein between the two ties, and insert the tip of the inner tubing five millimeters deep caudally toward the heart.

Secure the tubing with both ties to the jugular vein, and use a 3-0 silk suture to close the skin on the front of the neck. Then secure the tubing to the skin on the back of the neck with a suture and connect the tubing to a micro-infusion intravenous pump through a swivel. Infiltrate the skin incisions on the front and the back of the neck with 0.2 millimeters of 0.5%Bupivacaine to prevent post-operative pain, and allow the animal to awaken in the recovery chamber with monitoring.

When the animal is recumbent, set the infusion rate for a continuous 48 hour intravenous treatment of Conivaptan or vehicle, at 1.5 millimeters per kilogram per hour, and start the infusion. Alternatively, administer 0.2 milligrams of Conivaptan and 1.44 milliliters of 5%Dextrose twice intraperitoneally, daily for two days, followed by a brief observation of the animals after each injection. Middle cerebral arterial occlusion in mice, produces an infarct volume in the ipsilateral hemisphere at 48 hours with approximately 50%of the hemisphere affected as visualized by tetrazolium chloride staining.

The evaluation of the brain water content at 48 hours after a 60 minute middle cerebral arterial occlusion induction, reveals that intravenous Conivaptan administration significantly reduces the brain edema, in both the ipsilateral and contralateral hemispheres. In contrast, daily intraperitoneal Conivaptan treatment by a 0.2 millimeters bolus fails to produce similar positive effects on the stroke-evoked brain edema. Once mastered, this technique can be completed in two hours, if it is performed properly.

While attempting this procedure, it's important to remember to avoid excessive blood loss in the experimental animal because it can affect your study result and lead to increased mortality. Following this procedure, other studies like evaluating the blood brain barrier disruption can be performed to answer additional questions about the involvement of inflammation in post-ischemic brain edema. After the development, this technique paved the way for researchers in the field of neuroscience, to explore the importance of route of drug administration for clinical studies.

After watching this video, you should have a good understanding of how to surgically create ischemic conditions in mice and stow an intravenous catheter into the jugular vein and administer Conivaptan by continuous IV infusion in mice. Don't forget that working with live animals can be extremely hazardous and that precautions such as wearing surgical gloves and masks should always be taken while performing this procedure.