Rating L-DOPA-induced dyskinesias in the 6-hydroxydopamine rat model of the Parkinson's disease is an important preclinical tool to identify effective antidyskinetic interventions. This model is relatively simple, presents low cost, and has similarities to what happens in the clinic. The procedure will be demonstrated by Danilo Leandro Ribeiro, a PhD student in the laboratory.
Begin the experiment with Sprague-Dawley male rats, weighing 200 to 250 grams. House two to three animals per cage under the standard laboratory conditions with food and water available ad libitum. Before the surgery, administer the norepinephrine transporter inhibitor, imipramine, to the rat intraperitoneally.
After confirming the anesthesia by the lack of response to toe pinch, position the rat in a prone position in the stereotaxic apparatus on top of the heating pad. Once the rat is positioned, use a scalpel to make a one-centimeter-long incision at the region where the microinjection will occur. Then, clean the skull region with cotton swabs and ensure the bregma and lambda are exposed.
Take the medial forebrain bundle, or MFB, stereotaxic coordinates from bregma at 4.3 millimeters anterior, 1.6 millimeters lateral to the right side, and 8.3 millimeters ventral from the durometer. When the coordinates are decided, administer the 6-hydroxydopamine, or 6-OHDA, unilaterally at a rate of 0.4 microliters per minute in the right MFB, with a 50-microliter Hamilton glass syringe. At the end of the surgery, suture the scalp incision and rehydrate the animal with warm, sterile, 0.9%saline solution subcutaneously, at the dose of 10 milliliters per kilogram.
Remove the animal from the stereotaxic frame and place it in a warm recovery cage, while monitoring until consciousness is regained. At four weeks post-lesion, measure the effectiveness of the dopaminergic lesion using a stepping test, by assessing the akinesia of the forelimb contralateral to the lesion. Rats presenting three or fewer adjusting steps with the contralateral forelimb should be included in the study as putative, severely 6-OHDA-lesioned rats.
Four weeks after the 6-OHDA lesions, start the chronic treatment on Monday and record abnormal involuntary movements on Wednesday. To do so, place the rat inside a transparent cylinder with 20 centimeters diameter and 40 centimeters height, and allow it to acclimatize for at least 15 minutes. Ensure that the floor is covered with the bedding material and mirrors are positioned behind the cylinder to observe the animal from all possible angles.
Position a high-resolution video camera to allow the viewing of axial, limb, and orolingual abnormal involuntary movements. The mirrors behind the cylinder will allow tracking abnormal involuntary movements at a 360-degree angle. The camera can be positioned slightly below the plane at a 15-degree angle from animals to observe orolingual abnormal involuntary movements.
When the position is decided, fix the camera directly to the bench. Once the animal is acclimatized for 15 minutes, remove the animal from the cylinder and administer five milligrams per kilogram of freshly prepared L-dopamine or L-DOPA combined with 12.5 milligrams per kilogram of benserazide, subcutaneously. Put the animal back in the cylinder and start a timer to track abnormal involuntary movements.
Use a video camera to record the abnormal involuntary movements for 180 minutes post-injection, at 30-minute intervals, to carry out offline scoring. After the six observation periods, the scores must be given over one to two minute epochs and classified as axial, limb, or orolingual. Ensure not to include normal behaviors.
The treatment with L-DOPA and benserazide should be continued for three weeks, once daily, from Monday to Friday. In the representative time course analysis, the scores applied to axial, limb, and orolingual abnormal involuntary movements over three weeks of chronic L-DOPA administration, are shown. It was noted that L-DOPA-induced, peak-dose dyskinesias occur between 30 to 90 minutes, with a gradual decrease after 120 minutes post-injection.
Additionally, the sum of axial, limb, and orolingual abnormal involuntary movements was presented on individuals, scoring days and weekly over three weeks of chronic L-DOPA administration. The abnormal involuntary movements protocol is usually for the investigation of new therapeutic tools with antidyskinetic translational potential.
