Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis

We're interested in understanding how autoimmunity and multiple sclerosis, or MS, gets started. This research uses a variety of approaches to study how risk factors associated with MS, lead to the development of autoimmunity. Experimental Autoimmune Encephalomyelitis, or EAE, is a mouse model of MS that can be used to examine the effect of various risk factors on autoimmunity.

We have recently discovered the biological pathway of how one MS risk factor, obesity, leads to heightened autoimmune inflammation in mice. Studies in the EAE model were critical to unraveling these mechanisms. EAE disease severity is often scored on a five-point clinical scale, however, this scale has limited sensitivity and cannot distinguish whether clinical deficits are driven by inflammation or tissue damage.

Histology on the spinal cord and brain is the gold standard method to assess inflammation and tissue damage in EAE. This protocol provides a simple method for measuring inflammation and tissue damage in the spinal cord and brain during EAE, our protocol can be used alongside the clinical scale to better characterize the severity of EAE, Our protocol measures the key histological features of EAE, including immune infiltration, demyelination, axonal damage in the spinal cord, and brain inflammation. It also includes measuring neurofilament light in mouse serum, which indicates the extent of CNS injury.

This comprehensive protocol provides a more detailed characterization of EAE, than using only one marker. To begin, use forceps to grasp the skin on top of the head of a euthanized mouse, then make a 2.5-centimeter incision using surgical scissors. Use fingers to push the skin on the head laterally to visualize the underlying skull.

Then, with the standard Adson forceps, grasp the eye sockets to stabilize the head. Using fine scissors, make small snips in the skull along the midline from the cervical spine to the olfactory bulbs. Use Adson forceps with teeth to reflect the open skull to reveal the underlying brain.

Hold the brain using the non-dominant hand. With the closed scissors in the dominant hand, scoop the spinal cord out from the cervical spine, and gently push the brain out from the skull, snipping the cranial nerves. Place the brain in a conical tube containing 10 milliliters of 10%neutral buffered formalin.

Next, incise the fur along the midline along the midline of the mouse's torso from the neck to the tail. Use fingers to push the skin laterally to visualize the spine. Using surgical scissors cut downwards through the spine at the point of femur attachment to the hip, then cut the body wall on each side of the spine, from the hip to the neck.

Place the spine containing the spinal cord in the same tube containing the brain and formalin. To begin, use scissors to cut a small piece of lens paper and place it in the Petri dish. Next, pour the formalin containing the fixed brain and spine isolated from a mouse into a funnel lined with filter paper.

Transfer the brain and spine to an empty Petri dish. Use a scalpel to divide the brain into six coronal pieces. Using forceps, transfer the brain specimens onto one half of the lens paper in the Petri dish.

Cut the spinal cord into three pieces using the scalpel, then cut the sacral spine piece at the caudal end until the spinal cord becomes visible. Pick up the thoracic spine with the non-dominant hand. Take the Adson forceps with teeth tightly closed in the dominant hand, and gently push the end into the smaller opening of the spinal column with a gentle twisting motion.

Next, using forceps, gently pull the emerging spinal cord out of the column. Place the cord piece in the Petri dish containing the lens paper. Using a scalpel, divide three spinal cord pieces into smaller cross-sectional pieces.

Arrange these pieces on the same half of the lens paper containing the brain pieces. Fold the lens paper to sandwich the tissue pieces, and place it in a labeled cassette. Transfer the cassette into a specimen jar containing formalin.

After incubation, transfer the cassette from the specimen container into the first formalin bath in the automatic tissue processor, and run the processor overnight. The next day, remove the cassettes from the tissue processor and transfer them to the warm holding chamber of the paraffin embedding station. Pour the paraffin wax to cover the bottom of the mold.

Using fine forceps, place brain coronal and spinal cord cross-sectional pieces into the paraffin at the bottom of the mold. Transfer the mold to the cooling surface for several seconds, to fix the brain and specimens in place. Move the mold back to the heated surface, and fill it to the top with hot paraffin.

Place the cassette lid labeled with specimen ID onto the mold. Pour paraffin on top of the cassette lid. Transfer the mold to the cooling station to allow the wax to set.

Once the blocks are completely cool, secure the block on a rotary microtome. Trim the blocks and cut five-micrometer sections of each paraffin block. Move the paraffin ribbon to a 42-degree Celsius water bath.

Collect the section from the water bath onto a labeled slide. Place the slides in a plastic slide rack, and bake the sections overnight at 37 degrees Celsius in a dry oven. After scanning, open the Image J, then drag and drop the TIFF image of the section for analysis.

Observe the spinal cord section in four quadrants and score for the presence of demyelination lesions in each quadrant. This section has a score of four because there are lesions in all four quadrants. This section scores one because lesions are only in one quadrant.

CD45 staining indicates the presence of leukocytes in lesions. New lesions contain many CD45 cells compared to older ones. In contrast, new lesions may contain fewer SMI-32 positive axons compared to older lesions.

The mice in the wild-type group developed severe EAE with complete paralysis, while the OGR1 Knockout group developed mild disease. This difference in clinical score corresponded to variations in submeningeal demyelination lesions, in percent myelin area stained in the spinal cord. Furthermore, the percent myelin fraction also differed significantly between OGR1 and wild-type mice, and it correlated with a cumulative EAE score.

In EAE, inflammation in the brain is primarily localized in the cerebellum and brainstem. Additional areas of inflammation may be evident in the brain, including in the meninges, near the ventricles, and in other white matter tracts, such as the optic nerve and corpus callosum. Serum neurofilament light, measured with a small molecule array assay, detected higher serum neurofilament light levels in mice with EAE, compared to healthy mice, and these elevated levels correlated with the density of SMI-32 positive in the spinal cord.