This article describes a method for inducing Parkinson's disease-like symptoms in rats through the targeted destruction of dopaminergic neurons in the medial forebrain bundle (MFB). The procedure involves the use of the neurotoxin 6-hydroxydopamine (6-OHDA) and the antidepressant imipramine to ensure selective uptake by dopaminergic neurons.
Take a rat and intraperitoneally inject the drug imipramine. Anesthetize the rat, shave its head, and secure it in a stereotaxic frame.
Administer antibiotics intramuscularly to prevent infections.
Disinfect the head and incise to expose the skull. Identify the bregma and the lambda to locate the medial forebrain bundle or MFB.
The MFB comprises neurons connecting the midbrain to the forebrain, including the nigrostriatal dopaminergic neurons that control motor coordination.
Drill a hole, position a microsyringe containing 6-hydroxydopamine or 6-OHDA, a neurotoxin, and inject it into the MFB.
Keep the needle in place to prevent neurotoxin backflow, then withdraw it.
Disinfect and suture the incision. Allow the rat to recover.
The previously injected imipramine blocks transporters in non-dopaminergic neurons, ensuring selective 6-OHDA uptake by dopaminergic neurons.
6-OHDA produces reactive oxygen species, causing oxidative damage and neuronal death.
The loss of dopaminergic neurons disrupts motor function, mimicking the motor function loss in Parkinson's disease.
Begin by weighing the animals to monitor weight changes in the days following the surgery. After determining the dose of imipramine to be administered, inject the drug intraperitoneally using a 1-millimeter syringe equipped with a 27-gauge needle.
Once the fur from the head region is shaved, position the rat head over the incisor bar and fix the bar 3.3 millimeters below the intraoral line. Position the ear bars one side at a time. Position the incisor bar and the ear bars so that the top of the skull remains straight and parallel to the surface. Adjust the nose clamp and test that the head is firm and does not move to either side.
Sterilize the incision site with povidone-iodine. Using a 1-millimeter syringe equipped with a 23-gauge needle, administer the poly-antibiotic suspension intramuscularly. After checking the rat's state of deep anesthesia, using a scalpel, make an approximately 1.5-centimeter-long incision at the site of microinjection. Clean the skull region with cotton swabs and cotton buds until the bregma and lambda can be seen, and then mark them with a fine pen.
Check the dorsal-ventral coordinates of bregma and lambda, and if they are different, readjust the rat's head in the stereotaxic apparatus. Then, note down the AP and ML coordinates of the bregma and write MFB, as described in the text manuscript.
Mark the trepanation region with a fine pen and with the drill slowly pierce the skull, avoiding any injury to the dura mater. Next, position the microinjection needle on the dura mater. After noting the dorsal-ventral coordinates, gently rupture the dura mater by inserting the needle to the dorsal-ventral coordinate of the MFB.
Operate the microinjection pump to release the six-hydroxydopamine solution into the MFB. When the microinjection is finished, check the Hamilton syringe to see if 4 milliliters of six-hydroxydopamine have been injected.
After 10 minutes of injection, remove the microinjection needle slowly and sterilize the incision region with povidone-iodine. Then, suture the incision region with three to four surgical knots. At last, remove the rat from the stereotaxic apparatus, and place it in a clean box for recovery on the thermal blanket.
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