Lentiviral-Induced Striatal Pathology as a Preclinical Model of Polyglutamine Spinocerebellar Ataxias

This study reports the development of a lintiviral mouse model for rare neurodegenerative diseases, specifically spinocerellar ataxias. This protocol develops mouse models with some advantages compared with conventional trangenic mouse models. Namely, they are faster, they are cost affordable, and they allow a specific transaction in specific brain regions.

This is suitable for mechanistic studies and for the development of therapeutic strategies. To begin, position the anesthetized C57BL6J mouse on a clean desk and confirm the depth of anesthesia by assessing the toe pinch reflex. After shaving the animal scalp, place it in the stereotaxic frame on a heating pad and position the head using the ear bars and bite bar.

Mount the syringe containing two microliters of lentiviral vector solution into the automated microinjection pump. Set the pump parameters, including a speed of 0.25 microliters per minute, a volume per hemisphere of two microliters, and the appropriate syringe type. Prepare the surgical site using alternating rounds of povidone iodine or chlorhexidine-based scrub and 70%alcohol repeated three times before making the incision.

Using a sterile scalpel blade, make a midline incision of approximately one to 1.5 centimeters from between the ears to the eyes. Expose the skull and clean the surface by removing the periosteum with cotton swabs. Locate bregma and record the stereotaxic zero point manually or digitally.

Determine the injection coordinates relative to bregma with anteroposterior at plus 0.6 millimeters and mediolateral at plus or minus 1.8 millimeters. Drill carefully at the marked anteroposterior and mediolateral coordinates slowly to avoid penetrating the brain. Lower the injection needle slowly to the dorsoventral coordinate, and then test for needle clogging by injecting a portion of the solution until a drop is clearly visible.

Test that the hole is in the correct position by lowering the injection needle. Inject two microliters of lentivirus encoding for the wild-type ataxin into one hemisphere at a rate of 0.25 microliters per minute. After five minutes, slowly retract the needle to avoid reflux.

Inject the mutant or expanded protein into the contralateral side similarly. Close the incision with three to five interrupted 5-O nylon sutures. Administer a mixture of antagonistic drugs subcutaneously to reverse the effects of the initial anesthetics before returning the animal to its recovery cage and isolate the brain after 12 weeks.

Fill the wells of a 48 well plate with PBS containing 0.05%sodium azide. Then, transport the brain on dry ice and allow it to equilibrate inside the precooled cryostat for five to 10 minutes to prevent tissue damage. Using a scalpel, remove the olfactory bulbs as they are soft and easy to cut.

Apply a pea size drop of fresh OCT compound onto the specimen disc and place the cerebellum in contact. Once frozen, add additional OCT to embed the entire brain. Mount the specimen disc in the holder and trim until the striatum visible as two white circles in each hemisphere is observed.

If hemispheres received different treatments, pierce one hemisphere with a needle to maintain orientation and record this for later analysis. Next, switch to sectioning mode on the instrument panel and collect free floating sections in a 48-well plate using a fine brush or a fire polished glass pasture pipette. Place the first section in well A1 and the second in A2, continuing sequentially.

When the plate is full, either continue in a new plate or proceed until the entire striatum is sectioned. Seal the plates with transparent film and store at four degrees Celsius until use. Perform immunohistochemistry on the sections using appropriate antibodies and develop the staining with diaminobenzadine.

Monitor the development of the brown color and allow the reaction to reach the expected intensity level. Capture images of coronal brain sections spanning the entire striatum. For manual counting, open the image in Fiji to load it and calibrate the pixel size if required when metadata is not available.

Select, analyze and choose set scale to enter the known distance, such as one pixel equals 0.5 micrometers. Navigate to plugins, then analyze cell counter and select cell counter. Click initialize and select the image.

Select a marker type such as type one for aggregates. Manually click on each visible aggregate to label it. Record the count displayed in the cell counter panel for each image or section, and export the counts manually or compile results using a macro.

For semi-automated quantification, open IHC Aggregount and click upload image to load the image. View the image in the original image display and navigate to the processing tab. Apply high-low to enhance aggregate and background contrast.

Apply a brightness threshold to filter aggregates from the background and use the min-size filter to remove small particles. Finally, navigate to the general tab for immunohistochemistry aggregate detection. Click Calculate Total Aggregates and Calculate Aggregate Area.

Observe detected aggregates highlighted and listed in the matrix table with identification, area, and mean size. Robust expression of both wild type and mutant ataxin-2 was detected 12 weeks after the stereotaxic injection of lentiviral vectors into the striatum of C57BL6J mice. Immunostaining for ataxin-2 revealed a diffuse cytoplasmic distribution of wild-type ataxin-2 without forming discrete inclusions.

In contrast, the mutant ataxin-2 exhibited cytoplasmic aggregates with a distinct spherical morphology. Immunolabeling with DARP-32, a marker for medium spiny neurons, demonstrated that both wild-type and mutant ataxin-2 were expressed within the striatal neuronal population. While wild-type ataxin-2 expression did not alter the gross morphology of DARP-32 positive neurons, the mutant protein produced neuronal marker loss.

This protocol allowed a segment of aggregation and neuronal loss within specific brain region, namely striatum, in response to therapeutic intervention. The main challenge of this protocol is to achieve precise lintiviral delivery via accurate stereotoxic surgery, controlled biodosage and minimizing tissue damage for consistent results. Future work can use this model to identify molecular mechanism underlying these pathogenesis and to accelerate the development of target therapy and biomarkers.