A Viral Vector Injection into the Rat Substantia Nigra for Neurodegenerative Disease Modeling

Secure an anesthetized rat in a stereotactic frame.

Disinfect the scalp and make a cranial midline incision to expose the skull. 

Identify the bregma and lambda, two reference points on the skull, to determine the injection site.

Prepare a microinjection syringe containing a viral vector suspension.

The viral vector carries a transgene encoding alpha-synuclein, a protein that undergoes misfolding and aggregation in the neurons of the substantia nigra in neurodegenerative diseases.

Position the syringe in the stereotaxic instrument connected to a microinjection pump.

Create a burr hole at the injection site, avoiding brain injury.

Penetrate the dura with a needle and clean any blood with sterile tissue.

Insert the microinjection needle through the burr hole to reach the substantia nigra.

Slowly inject the viral suspension, ensuring even distribution.

Pause briefly to prevent backflow, then slowly withdraw the needle.

Suture the incision.

The model is now ready for further analysis.

Begin, by properly anesthetizing an eight-week-old female Wistar rat according to approved protocols. Check that a surgical plan of anesthesia has been achieved by squeezing each paw and noting an absence of the withdrawal reflex. Next, use a microtransponder implanter to place a microtransponder on the back of the rat. Check that the microtransponder is positioned correctly, and that a readout can be obtained.

Now cut the hair on the scalp of the rat and apply local anesthetic to the scalp and ears. Transfer the rat to a laminar flow hood, and perform the rest of the procedure using aseptic technique. Place the rat in the stereotaxic frame by securing the ear bars, followed by the mouth and nose bar. Cover the body of the rat with a paper blanket to avoid a drop in body temperature. Apply an ocular lubricant to prevent the eyes from drying.

Next, disinfect the scalp according to approved procedures. 1% iodine in 70% isopropanol is used here. After making a small incision in the midline of the scalp, gently scrape away the membranes on the skull, and rinse with saline. After allowing the skull to dry, ensure that bregma and lambda can be clearly seen.

Now, fill a 10 microliter, 30-gauge 20 millimeters microinjection syringe with recombinant AAV, and place it onto the motorized microinjection pump connected to the stereotaxic instrument.

For specific transduction of the dopaminergic neurons of the substantia nigra, recombinant AAV vectors are the first choice because of their high titers and efficiency for transducing dopaminergic neurons.

Test the flow by releasing a drop of AAV. Dispose of any released AAV in a polyvalent cleaning detergent. Visually check that the head is fixed straight in the head frame. Then check that the skull is flat by first moving the tip of the needle to bregma, and measuring the height at this point by lowering the tip of the needle in the dorsoventral direction, until it touches the skull. Repeat the procedure at lambda.

After returning the needle to bregma, move the needle in the anteroposterior and mediolateral direction to the stereotaxic coordinates for microinjection. Write down the coordinates. At the site of injection, measure the height of the skull as before, and ensure that it does not differ more than 0.3 milimeters from the height of bregma.

Then carefully drill a 2-millimeter hole in the skull. Once the hole is drilled, measure the height of the dura to determine the reference from which to apply the dorsoventral coordinate. Penetrate the dura using a 26-gauge needle. Absorb any blood with a sterile tissue, and proceed only after all bleeding has stopped.

Now slowly lower the needle of the preloaded microinjection syringe into the brain to the dorsal ventral coordinate, and pause for one minute. Then inject three microliters of AAV at a rate of 0.25 microliters per minute. After the injection, allow the needle to remain in place for another five minutes to prevent backflow along the needle track before slowly removing it.

Stitch the scalp using coated braided polyester 3.0, and disinfect the skin with 1% iodine in 70% isopropanol, to loosen the nose and mouth bar, and then the two ear bars to gently remove the animal from the stereotactic instrument. At this time, administer analgesia, and if required, an anesthesia reversal agent. Then, place the rat in a clean cage on a heating plate set to 38 degrees Celsius and monitor closely until it wakes up.