Modeling Parkinson’s Disease in Rats

Introduction

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.

Required Materials

Rats
  • Sprague-Dawley rats (200−250 g)
Media and Reagents
  • Sterile saline
  • Isoflurane
  • Betadine
  • 70% Ethanol
  • 6-hydroxydopamine (Sigma-Aldrich, Cat. no. H116)
  • Apomorphine (Sigma-Aldrich, Cat. no. A4393)
  • Amphetamine (Sigma-Aldrich, Cat. no. A1263)
Special Tools
  • Stereotactic frame
  • Animal balance
  • Isoflurane inhalation chamber
  • Electric razor
  • Scalpel
  • Tissue forceps
  • Scissors
  • 10 μL Hamilton syringes and needles
  • Dental drill
  • Sutures or staples

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Ordering Information

Sku Name Size Price Qty
N7805100 Gibco® Human Astrocytes 1 mL USD 712.00
A1261301 Gibco® Astrocyte Medium 500 mL USD 112.00
17502048 N-2 Supplement (100X) 5 mL USD 70.00
10569010 DMEM, high glucose, GlutaMAX™ Supplement, pyruvate 500 mL USD 26.40
16000077 Fetal Bovine Serum, certified, One Shot™ format, US origin 50 mL USD 48.00
14190144 DPBS, no calcium, no magnesium 500 mL USD 18.35
14040133 DPBS, calcium, magnesium 500 mL USD 18.35
31985062 Opti-MEM® I Reduced Serum Medium 100 mL USD 18.30
13778150 Lipofectamine® RNAiMAX Transfection Reagent 1.5 mL USD 648.72

Preparing Reagents

Preparing 6-hydroxydopamine (6‑OHDA) Solution

Make a 2-mg/mL solution of 6-hydroxydopamine (6-OHDA) in saline. Store protected from light up to 12 months at −20°C.

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Performing the DA Lesion Using a Rat Model

Preparing the Animal

  1. Weigh a rat and place it into an isoflurane chamber and apply oxygen and isoflurane until the animal is deeply anesthetized.

  2. 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.

  3. Shave the top of the rat’s head with an electric razor. Clean the skin with betadine and 70% ethanol.

  4. Perform a midline incision with a scalpel and identify the bregma at the intersection of the coronal and the sagittal sutures.

  5. Adjust the incisor bar in the rat until the heights of lambda and bregma skull points are equal.

  6. 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.

  7. Drill a burr hole at the target site using a dental drill.

Administering 6-OHDA

  1. Fill a 10-μL Hamilton syringe with 5 μL 6-OHDA solution. Attach the syringe to the holder on the stereotactic frame.

  2. Lower the needle of the Hamilton syringe so that it is 8 mm from the dura.

  3. Inject the 6-OHDA solution at a rate of 1 μL/minute.

  4. Leave the needle in place for 5 minutes and withdraw the tip slowly.

  5. 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.
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LT160                    17-Mar-2011