組換えアデノ随伴ウイルスベクターの定位注射を介して、パーキンソン病のα-シヌクレインベースのラットモデルの開発

The overall goal of this procedure is to develop a viral vector based animal model for Parkinson's Disease via Stereotactic Injection in the central nervous system. This method can be used to develop normal animal models which allow for pre-clinical drug testing and can be beneficial in studying the molecular mechanism of Parkinson's Disease as well as many other neurodegenerative disorders. The main advantage of this technique is that it can be used for specific targeting of brain regions.

High trenching brain expression can be achieved and that it can be used to create animal models in specific animal strains and species. The different final vector systems have been developed. The choice of the vector system depends on the size of the trenching you want to express, the duration of the gene expression, the sounds that you want to target, and bio-safety issues.

Demonstrating the procedure will be Annelies Aertgeerts a technician from our laboratory. Begin by properly anesthetizing an eight week old female Wistar rat according to approved protocols. Check that a surgical plane 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. One percent iodine in 70%isopropyl is used here.

After making a small incision in the mid-line 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 clearly be seen. Now, fill a 10 micro-liter, 30 gauge 20 milometer microinjection-syringe with recombinant AAV and place it onto the motorized micro-injection pump connected to the stereotaxic instrument.

For specific transaction of the Dopaminergic neurons of the substantia nigra, recombinant AV 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 dorsal-ventral direction until in touches the skull. Repeat the procedure at lambda.

After returning the needle to bregma, move the needle in the anterior posterior and medial lateral 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 from more than 0.3mm from the height of bregma.

Then carefully drill a two milimeter hole in the skull. Once the hole is drilled, measure the height of the dura to determine the reference from which to apply the dorsal-ventral coordinate. Penetrate the dura using a 26 gauge needle.

Absorb any blood with the sterile tissue, and proceed only after all bleeding has stopped. Now slowly lower the needle of the pre-loaded microinjection syringe into the brain to the dorsal-ventral coordinate. And pause for one minute.

Then inject three micro-liters of AAV at a rate of 0.25 micro-liters per minute. After the injection, allow the needle to remain in place for another five minutes to prevent back flow along the needle track before slowly removing it. Stitch the scalp using coded braided polyester 3.0 and disinfect the skin with one percent iodine and 70%isopropyl.

To loosen the nose and mouth bar, and then the two ear bars, to gently remove the animal from the stereotaxic 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.

Following surgery, the kinetics of neurodegeneration can be studied in the whole animal using behavioral tests and PET imaging and after sacrifice, using immunohistochemical techniques. The cylinder test to evaluate spontaneous forelimb use, was performed at various time points following microinjection of recombinant AAV expressing A53T Mutation alpha synuclein into the substantia nigra. From three weeks after injection, the significant motor impairment was seen in rats that received the median dose.

At four weeks after injection, a 50%decrease in spontaneous contralateral forepaw use was observed. Whereas the control eGFP injected animals showed no asymmetry in forepaw use. Rats that received a higher dose of recombinant AAV 2/7A53T alpha synuclein, showed a more pronounced impairment of forepaw use at 29 days after injection.

To prove that the observed motor impairment was dopamine dependent, a single dose of L-doba was administered. When the cylinder test was repeated 45 minutes after L-dopa treatment, a full recovery of the forepaw use in the recombinant AAV 2/7A53T alpha synuclein injected animals, was observed. To follow up the kinetics of nigrostriatal dopaminergic neurodegeneration non-invasively over time, in individual animals, dopamine transported binding was quantified using small-animal positron emission tomography with F-18FECT as the radioligand.

DAT binding significantly decreased in the ipsilateral caudate budiman of recombinant AAV 2/7A53T alpha synuclein injected rats over time. After 32 days, a decrease in DAT binding of up to 85%was observed. As the positive control, injection of the neurotoxin 6 OHDA in the SN induced 90%loss of DAT binding within seven days.

Brain sections from rats microinjected with recombinant AAV 2/7A53T alpha synuclein over time were immunostained for tyrosine hydroxylase a marker of dopaminergic neurons. This figure demonstrates the progressive loss of dopaminergic neurons over a 29 day period after microinjection. Once mastered, this technique can be done in 45 minutes.

While attempting this procedure, it is important to use high quality final vector matches. Following this procedure, other techniques, like non-invasive PET imaging, behavior analysis, and Immunohistochemical analysis can be performed in order to determine the level of neurodegeneration and neuropathology. After watching this video, you should have a good idea of how to perform a viral vector injection into the substantia nigra in order to develop viral vector based animal models for Parkinson's Disease.

Don't forget that working with viral vectors can be hazardous, and that precautions such as working under a laminar flow and proper waste decontamination should always be taken following this procedure.