Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

The overall goal of this protocol is to describe the procedures involved in establishing a symptomatic, pharmacological mouse model for DYT/PARK-ATP1A3 dystonia by using brain cannulas in osmotic pumps and by inducing stress via challenging motor tasks. This method details how to establish a pharmacological mouse model using chronic drug delivery via osmotic pumps to multiple brain cannulas and how to induce the phenotype via challenging motor tasks. The mild motor stress paradigm also provides insights into the role of gene-environmental interactions in movement disorders, and can be applied to other rodent models.

Animal experiments were performed in accordance with applicable national and institutional guidelines for care and use of animals. Begin by preparing the osmotic pumps in a sterile environment. First, fill a one milliliter syringe with a 27 gauge filling cannula with sterile filtered ouabain-solution or vehicle solution.

Hold the osmotic pump in upright position, insert the cannula all the way into the pump and slowly fill the reservoir until excess solution appears on top. Next, insert the flow moderator into the pump. Carefully take off the white flange of the flow moderator with scissors and connect a piece of vinyl tubing to the flow moderator.

Submerge each pump into Eppendorf tubes filled halfway with saline solution and put them into a thermocycler 37 degrees in order to ensure that the pumping rate reaches a steady state before beginning surgery. After the animal is deeply anesthetized according to approved protocols, place the animal into a stereotactic frame and fix the head using rubber tips or non-ruptured ear bars. After thorough disinfection of the surgical area, use a scalpel to place incision at the top of the head and use scissors to continue the incision down to the fore limbs.

Expose the skull with the help of bulldog clamps and wipe the periosteum with a sterile cotton-tipped applicator. Align either a pen or the tip of a cannular stained in black ink with bregma and use the appropriate coordinates to mark the three entry points for the brain cannulas on the skull. Next, carefully drill the holes for the double cannular designated for the basal ganglia and the single cannula designated for the cerebellum.

Drill a fourth hole for a small screw in-between the striatum and the cerebellum. This screw will eventually be embedded in dental cement and provide additional hold for the cannulas. In order to create a small pocket for the osmotic pump on each side of the animals back, use tissue forceps to separate the subcutaneous tissue layers.

Advance the forceps towards one hind leg and open the forceps slightly in order to widen the subcutaneous pocket. Repeat the same procedure for the other side, first removing the forceps from the incision and then pushing them gently towards the second hind leg. With the help of forceps, carefully insert the pumps and the connected catheter into each subcutaneous pocket.

The pocket should allow the pump to easily slide in. Using a mini pump holder, introduce a single osmotic cannula with a custom length of 3.0 millimeters into the hole drilled in the midline of the cerebellum. Detach the cannula head carefully and fix the cannula as well as the small screw with dental cement, taking care as not to cover the connecting piece for the tubing of the osmotic pump.

Ensure that the dentist cement surrounding the cannula has fully dried before continuing with surgery. Before inserting the double cannula into the bilaterally drilled holes above the basal ganglia, attach two 0.5 centimeter long pieces of vinyl tubing to the two connecting pieces of the double cannula. Connect the vinyl tubings with a bifurcation adaptor and carefully prefill the entire tubing system with sterile ouabain solution or vehicle.

Next, repeat the same steps as shown previously for the single cannula and end by fixing the double cannula with dental cement. Connect the catheters of the osmotic pumps to the bifurcation adaptor as well as a single cannula respectively. Both pumps have an equal flow rate so be careful to connect the osmotic pump with a double concentrated solution to the bifurcation adaptor to ensure that the same concentration reaches both the basal ganglia and the cerebellum.

Close the incision on the back of the animals with stitches as far as possible without overstretching the skin. Subcutaneously inject sterile saline, which should have body temperature to avoid dehydration. Remove the animal from the stereotactic frame and place it into recovery cage.

In order to induce dystonia-like movements, ouabain-perfused mice should be exposed to challenging motor tasks as a mild stress paradigm four hours after surgery and repetitively every 24 hours afterwards. This does not include a behavioral characterization of the animals. We demonstrate here the example of a vehicle-perfused animal, which is placed onto a 50 centimeter rough surface wooden pole, nose facing downwards.

Provide enough bedding at the base of the pole in case of falls. For ouabain-perfused mice, the animals should start to present first symptoms like bradykinesia four hours post surgery. However, 24 hours after surgery, you should see involuntary hyperextension of the front or hind limbs as a sign of dystonia-like movements during descent.

The mice should descend the pole three times, allow at least two minutes of recovery in between each descent. The time of descent does not need to be measured. The pole test should be followed by a second motor task, using a rotating rod.

As done for the Rotarod performance test, the ouabain and vehicle-perfused mice from the stress groups are placed on the rotating rod. The latency to fall does not need to be measured. As done for the previous test, mice should be placed on the rotating rod three times, allowing two minutes of recovery in-between runs.

The modified dystonia rating scale can be used to assess the frequency, severity, and body distribution of dystonia-like movements of mice. The animals should be placed in a plastic or wooden box and movements should be recorded for time period of four minutes. The raters should be blinded to the group assignment.

Movements or postures that are considered as dystonia-like are the involuntary hyperextension of front limbs, a wide stance, or hyperextension of hind limbs, as well as kyphosis. If only one body part is affected, dystonia should be considered as focal. If the trunk and at least two other body parts are affected, dystonia should be considered as generalized.

A second newly developed scoring system from zero to eight points, evaluates the presence and severity of dystonia-like movements during a tail suspension test. For the test, hold up the mouse by the tail near its base for two minutes. It is recommended to record the tail suspension test and assign a score to subsequent analysis of the footage.

Front limbs are scored from zero to four points. Repeated or sustained tonic retractions of one of both front limbs, as well as the hyperextension combined with crossing of the front limbs are scored as dystonia-like. Dystonia-like movements of the hind limbs, meaning retraction and clenching, as well as sustained hyperextension, are scored from zero to three points.

A truncal distortion present over 80%of the recorded time is scored with an additional point. Hind limb clasping is nonspecific sign for motor impairment and should not be scored. This graph shows the results of the dystonia rating scale.

The ouabain-perfused stressed mice present significantly more dystonia-like movements over an observational period of 72 hours than the non-stress ouabain-perfused group, as well as the vehicle animals. This image depicts the results from the tail suspension test and shows a similar result to the dystonia rating scale. Note that the results of the mean from the total score of each animal for each time point.

After watching this video, you should have a good understanding how to stereotaxically implant multiple brain cannulas and how to subcutaneously implant osmotic pumps. The main advantage of this method is that repetitive and stressful injections can be avoided and that a substrate like ouabain, which otherwise would have system-wide side effects can be directly delivered to a specific brain structures.