Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Hello, my name is Lar Turner. I'm a postdoc in the Department of Neurology at the University of Ingham, Germany. Hi, my name is Lin Gunda.

I'm a medical student and I'm doing my thesis work in this lab. Today we are going to show you the clinical testing of the SODG 93, A Moss model of atrophic lateral sclerosis. Furthermore, we'll demonstrate the motor function test with a so-called rotor test and additionally, another motor function test with the hanging Wire grip in order to perform an immuno logic analysis.

After functional testing of the a s mice, a necessary procedure consists in the removal of the spinal cord, the STIC nerve, and the gastroc muscle. We want to show these surgical procedure in detail and one present some exemplary immuno logic findings at the end. Okay, but Now let's first start with the clinical testing.

This is a free moving wild type MOE that does not show any clinical symptoms like muscle tremor or Parsis in our clinical classification system. That would be score four. This is a transgenic A LS moe.

In an early disease stage, it exhibits a severe tremor of the right hin limb that classifies it as SCORE three E.The severe gait abnormalities Classify this animal as score two. Dragging of at least one hind limb are classified as clinical score one in the Hanging wire test, crude muscular rib strength is evaluated. Each mouse is placed on a CustomMade wire and cautiously turned upside down above the straw covered button.

After Training for three consecutive times of at least 180 seconds, the latency to falls measured. Each mouse is given up to three attempts to hold onto the inverted lid for a maximum of 180 seconds, and the longest period is recorded In comparison To the white wire type mouse on the right side. The plaque transgenic mouse on the left is very weak and falls off the lid before reaching the Maximum.

The Rotor rod apparatus is used to measure motor coordination, balance and motor learning ability. A good performance requires a high decree of sensory motor coordination. It consists of a computer controlled, motor driven rotating spindle, and five lanes for five m.

Faults of the mice are detected automatically by pressure on the bottom plastic plate. After training for three consecutive times of at least 180 seconds at a constant speed of 15 rounds per minute, the time for which an animal can remain on the rotating rot is measured. Each animal undergoes three trials and the longest latency without falling is recorded.

The healthy wild type mos on the left hand side is running without difficulty on the rotor rod. In contrast, the transgenic a s mo on the right side has severe problems to even remain on the rotor rod and slips off after a while observed from behind. You see the severe running difficulties of the left retre mouse.

In comparison with the healthy wire tap mouse, it is not able to keep up with the rotor rod speed and cannot push upwards with the HIN limbs. In order to perform an immunochemical analysis of the spinal cord, the animals have to be sacrificed. It is very important to always strictly follow the local guidelines for proper conduct of animal experiments.

In this context, In this Very simplified overview of the vertebral column and its surrounding tissues, the main anatomical structures are depicted. We will place the animal on its ventral side and start to prepare the muscles from the dorsal bag. After the muscular tissue is removed, we will access the vertebral column that encloses the spinal cord.

After the animal has been sacrificed and perfused with a paraform height containing solution, the spinal cord can be removed. The animal is placed onto an operation table and the full limbs are fixed on the sides and order to expose the backside of the mouse. The animal is wetted to flatten the hair coat and facilitate the following skin incision.

For this, a sharp scalpel is used. Be careful to stretch the skin to both sides. To facilitate cutting the leg muscle shall be prepared.

Their skin has also to be incised. After the skin incision is completed, it is gently pulled aside. With a pair of tweezers having Removed the leg muscles and the sciatic nerve, we continue to approach the spinal cord and placed the animal on a new operation table.

Now it is easy to identify the cranium neck, muscles, and spinal column. Having identified all these structures, it is easy to proceed to the next step. The muscles of the neck and the nu ligament have to be removed.

Be careful not to incise too deeply and lead the vertebral column. In the next Step, the paravertebral muscles are removed entirely to expose the vertebral column. In order To access the spinal column, several ectomies have to be done.

We start from the cervical part of the vertebral column, including the Atlantic occipital joint. To perform the laminectomy, we have to cut the vertebral arch to be able to remove all the vertebral processes from the vertebral Body. As explained before the laminectomy start with the cutting of the vertebral arches.

Then the laina can be pulled away. Again, take care not to lesion the spinal cord as many more ral have to be removed. It is quite helpful to use angled scissors and inci several vertebral arches of both sides at a time.

Then pull on the dorsal processes to entirely remove the vertebral lamine and the closeup. You see that the pair of scissors have to be carefully inserted most laterally at the side of the spinal canal. Remaining Lateral parts of the vertebral column have to be removed to facilitate the later complete removal of the spinal cord.

An Anatomical landmark of the spinal cord are the so-called cervical and lumbar esencia. Having finished the ectomies of the entire spinal cord, make sure that you also transect all ventral roots of the spinal cord and release it from the dur matter of the meninges. Next, The cervical spinal cord is cut.

Cranially and its removal is started again. Touch the spinal cord very cautiously in order not to harm this tissue, because this could cause severe limitations for later immuno immunochemical Analysis. The removal Of the spinal cord is further continued at the thoracic level and proceeded to the lumbar part.

It is Finally cut again this time at the side of the cord equina. Now we are able to completely release it. Here You see the spinal cord placed right next to its original location in the RAL column.

Ultimately, it is placed into a Paraform Hyde post fixating Solution. As a classical example for spinal cord immunohistochemical analysis, you can apply an antibody that recognizes choline, acetyl transferase to identify motor neurons in the spinal cord anterior horn. Here you see a large number of motor neurons in a healthy wild type Animal.

In Contrast, the transgenic a LS animal at end stage does exhibit a much smaller number of motor neurons in the Anterior horn. Watching This video article, you should have learned the basic clinical examination of the SODG 93. A mouse model of atrophic lateral sclerosis.

Two motor behavioral tests have been demonstrated the rotor test and the hanging wire test. They provide a good possibility to evaluate basic motor function. They are easy to perform, sensitive in detection of motor deficit and time efficient.

The main part of this video consists in a demonstration of the preparation technique of the spinal cord. This can of course be applied to any other mouse disease model and is an essential step for the later immunochemical analysis of disease pathology in the spinal cord. Thank you for watching.

We hope that this video article will be helpful for your future experiments.