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In animals with a unilateral dopaminergic (DA) lesion, there is an imbalance of motor activity. Complete DA lesion can be induced by unilateral intracerebral stereotactic injection of 6-hydroxydopamine in the medial forebrain bundle (MFB). This model is useful in the study of DA replacement therapy. This chapter describes methods to induce a rat Parkinson's disease model with complete unilateral DA lesion.
- Sprague-Dawley rats (200−250 g)
Media and Reagents
- Sterile saline
- 70% Ethanol
- 6-hydroxydopamine (Sigma-Aldrich, Cat. no. H116)
- Apomorphine (Sigma-Aldrich, Cat. no. A4393)
- Amphetamine (Sigma-Aldrich, Cat. no. A1263)
- Stereotactic frame
- Animal balance
- Isoflurane inhalation chamber
- Electric razor
- Tissue forceps
- 10 μL Hamilton syringes and needles
- Dental drill
- Sutures or staples
Make a 2-mg/mL solution of 6-hydroxydopamine (6-OHDA) in saline. Store protected from light up to 12 months at −20°C.
Preparing the Animal
- Weigh a rat and place it into an isoflurane chamber and apply oxygen and isoflurane until the animal is deeply anesthetized.
- Position the rat in a stereotactic frame and fix the plastic tube connected to the anesthesia machine to the nose of the rat using surgical tape. Maintain isoflurane at ~1.5% with an oxygen flow of 2−3 liters/minute.
- Shave the top of the rat's head with an electric razor. Clean the skin with betadine and 70% ethanol.
- Perform a midline incision with a scalpel and identify the bregma at the intersection of the coronal and the sagittal sutures.
- Adjust the incisor bar in the rat until the heights of lambda and bregma skull points are equal.
- Calculate the stereotactic coordinates for injection. For a MFB lesion, coordinates are in reference to the bregma: Anteroposterior (A/P) −2.2 mm; mediolateral (M/L) 1.5 mm.
- Drill a burr hole at the target site using a dental drill.
- Fill a 10-μL Hamilton syringe with 5 μL 6-OHDA solution. Attach the syringe to the holder on the stereotactic frame.
- Lower the needle of the Hamilton syringe so that it is 8 mm from the dura.
- Inject the 6-OHDA solution at a rate of 1 μL/minute.
- Leave the needle in place for 5 minutes and withdraw the tip slowly.
- Close scalp margins with sutures or staples. Remove the rat from the stereotactic frame and place it in its home cage. Put food on the floor of the cage and monitor the animal's weight for 3 days after surgery.
Evaluating the Behavior At 10−14 days after injection of 6-OHDA, the rats that exhibit at least 210 contralateral rotations over 30 minutes when challenged with the dopamine receptor agonist apomorphine (0.2 mg/kg, i.p.), or 630 ipsilateral rotations over 90 minutes when challenged with the DA-releasing substance amphetamine (5 mg/kg, i.p.) are suitable for future study.