The overall goal of this procedure is to generate a mouse model of Parkinsonism. This is accomplished by first preparing the drugs for the surgery. The next step is to set up the surgical apparatus, then unilateral six hydroxy dopamine lesion surgery is performed following proper postoperative care.
The six hydroxy dopamine lesioned animals can be used for behavioral experiments. Ultimately, the results show a greater than 95%dopamine depletion in the medial four brain bundle unilaterally. The two most important aspects of this procedure that individuals must pay attention to are firstly appropriate postoperative care to ensure survival of the animals.
And secondly, care for preparation of the equipment for infusion of six hydroxy dopamine to ensure delivery of the neurotoxin is complete. To begin this procedure, sterilize the surgical tools in an autoclave. Next, clean all the surfaces and equipment with isopropyl alcohol.
Then prepare the post-surgery by setting up a mouse recovery cage with paper towels on the base. Place the cage under a heating lamp or on top of a heated pad to set up the anesthesia trolley. Ensure that the oxygen inflow is connected and open, and the isof fluorine reservoir is completely full.
After that, set the oxygen flow to 1%and the isof fluorine to two to 4%The next step is to assemble the infusion apparatus. Thread one RN metal nut onto one side of the peak tubing, followed by the peak cup feral. Then the canonical PFA feral orient the ferals so that the cone on the canonical PPFA feral will slip into the mating part of the peak cup.Feral.
Then place the mated peak cup and canonical PFA feral into either end of the dual small hub RN coupler and tighten the RN metal nut on the other end of the dual small hub RN coupler. Insert a 33 gauge injection needle. Thread the needle through one of the RN metal nuts and tighten it.
Ensure the 33 gauge needle is at a 180 degree angle to the dual small hub RN coupler. Then on the other end of the peak tubing thread one of the RN metal nuts followed by a peak cup feral. Then the canonical PFA feral the deeper groove of the second dual small hub RN coupler and tighten the RN metal nut in order to secure the connection.
Next, on the other end of the second small dual RN hub coupler, insert the lure to the small hub RN adapter and to tighten the RN metal nut of the lure in order to secure the connection. The setup of the infusion apparatus is now complete. Another preparatory step is to prime the infusion apparatus.
First, fill the 250 microliter and one microliter Hamilton syringes with sterile water. Ensure that there are no air bubbles. Then attach the 250 microliter syringe to the lure adapter and depress the plunger of the 250 microliter syringe such that water is released through the 33 gauge injection needle on the other end of the tubing.
Continue to push the water through the system while pulling the 250 microliter syringe out of the lure adapter. Leaving a small water bead on the end of the lure adapter depress the plunger of the one microliter syringe until a small water bead is ejected. Next carefully insert the one microliter syringe into the lure adapter.
Make sure that the water bead on the one microliter syringe connects with the water bead on the lure adapter. Then completely insert the one microliter syringe into the lure adapter and be sure that there are no bubbles in the small dual RN hub coupler. Now secure the perfusion apparatus to the stereotaxic frame and the infusion pump clamp the small dual RN hub coupler with the 33 gauge injection needle to the manipulator arm of the stereotaxic frame.
Then clamp the one microliter syringe to the perfusion pump. After that program, the infusion pump to allow an infusion rate of 0.1 microliters per minute, 30 minutes prior to the operation. Weigh the animal and record its weight.
Administer a mixture of 2.5 milligrams per milliliter of dopamine hydrochloride and 0.5 milligrams per milliliter of parline hydrochloride at 10 milliliters per kilogram by intraperitoneal injection to the mouse. Next place a warm heating disc under the ear bars and the incisor bar to maintain the temperature of the animal during surgery and keep the animal at an ideal height to fit the ear and incisor bars of the stereotaxic. Frame 15 minutes following injection of the dopamine hydrochloride and parline hydrochloride mixture.
Place the mouse in a closed and a seizure chamber. Anesthetize the animal by isof fluorine inhalation. The animal is sufficiently anesthetize when it shows no response to hind leg pinch and no blink reflex.
Then shave the top of the mouse's head. At this point, place the mouse in the stereotaxic frame. Place the incisor bar into its mouth and an anesthesia mask over its face.
Then adjust the flow rate of isof fluorine. Apply the topical analgesic lidocaine directly to the skin using cotton wool. Adjust the incisor bar and the ear cups to level the top of the skull.
Then sterilize the skin with Betadine solution. Next, insert the ear cups. The ear cups are inserted correctly when the head is completely flat and cannot be moved in either direction.
After that, cut along the midline of the head using a scalpel blade and retract the skin. Dry the surface of the skull using a gauze pad. Push down the plunger of the one microliter syringe and place the 33 gauge injection needle into the one milliliter tube of 15 milligrams per milliliter.
Six hydroxy dopamine solution covered in tinfoil. Draw the plunger back slowly while keeping the 33 gauge injection needle immersed in the six hydroxy dopamine solution. Next, advance the tip of the injection needle towards B bgma, and to dispense a small amount of six hydroxy dopamine solution in order to form a small bead.
Then slowly lower the injection needle tip torema. When the bead touches bgma, stop advancing the needle and record these coordinates. Retract the injection needle two millimeters in the dorsal direction and move along the sagittal suture in a rostral coddle direction towards Lambda.
When it is on top of Lambda, dispense a small amount of six hydroxy dopamine solution from the injection needle to form a small bead. Then slowly lower the needle tip to lambda. When the bead touches Lambda, stop advancing the needle and record these coordinates.
The medial lateral and dorsal ventral coordinates should be identical for BMA and Lambda. If they're not, adjust the incisor bar accordingly. For the anterior posterior coordinates and the ear bars for the medial lateral coordinates, move the needle to the injection coordinates.
AP minus 1.2 millimeters ml minus 1.1 millimeters relative torema dispense a small amount of solution from the injection needle in order to form a small bead. Lower the small bead onto the skull to mark the location where the hole will be. Bird then retract the needle, burr a hole into the skull using a 25 gauge needle.
Return the injection needle to the injection coordinates and insert the needle to minus five millimeters along the dorsal ventral direction. Next, infuse 0.2 microliters of six hydroxy dopamine or vehicle unilaterally into the median forebrain bundle at a rate of 0.1 microliters per minute. Upon completion of six hydroxy dopamine or vehicle administration, leave the needle in place for another five minutes to allow diffusion from the injection site.
After that, slowly retract the needle, close the incision to the scalp with three sutures, then deliver one milliliter of lactated ringer solution subcutaneously. Lastly, remove the animal from the stereotaxic frame and place it in the recovery cage until consciousness is regained. 15 to 21 days following six hydroxy dopamine lesion surgery.
When the amount of cell death caused by the neurotoxin has reached completion, spontaneous 360 degree rotations can be measured to assess the success of six hydroxy dopamine lesion. In this behavioral assessment, mice with six hydroxy dopamine induced dopamine depletion greater than 95%show significantly more IP subversive rotation than mice with partial six hydroxy dopamine and shammed operated lesion. By correlating the SAL dopamine levels with the percentage of spontaneous IP subversive rotation in each animal, mice, which exhibited 70%or more IP subversive rotations with six hydroxy dopamine lesion are found to have lost greater than 95%Sal dopamine.
After watching this video, you should have a good understanding of how to effectively perform a stereotaxic and profusion controlled six hydroxy dopamine lesion in mice. This will lead to a greater than 95%level of dopamine depletion. Also, you should understand how to perform behavioral assessment of Parkinsonism in mice.
