The goal of our research is to study the pathogenesis of Multiple sclerosis. To achieve this goal we have established a rapid autopsy program to obtain and to study the brains from individuals with MS.There are two special aspects of our autopsy program, the first is that it's rapid. We collect the brain's under six to eight hours of death.
This allows for state of the art molecular studies. The second aspect is that we do an in situ MRI before we move the brain. This allows us to correlate pathological changes with brain imaging changes.
Visual demonstration allows viewers to understand how we organize our space and time to achieve optimal results. Tissue processing that happens rapidly and carefully allows for RNA analysis, immunostaining, immunoblotting and MRI correlations. New researchers may struggle with processing tissues with poor tissue integrity.
Avoiding long post mortem fixation times is important and MRI tissue corde striations can be challenging so consultation with MRI analysis experts can be very useful. Demonstrating the procedure will be Emilie Christie and Christina Volsko. Technicians from our lab.
After In situ magnetic resonance imaging, weigh and photograph the brain and place any attached dura in a container of 4%Paraformaldehyde or PFA. Separate the cerebellum and brainstem from the cerebrum and photograph the cerebrum. Use a probe to separate the optic nerves, chiasm and tracts and resect the structure with a scalpel.
Separate the cerebral hemispheres longitudinally and photograph each hemisphere individually. Ink the primary motor cortex or PMC for the left hemisphere, rephotograph the tissue and place the tissue in a 3.3 liter container for long fixation. To excise the PMC for the right hemisphere remove the covering meninges and rephotograph the tissue.
Next resect the PMC using a scalpel. Then photograph the resected PMC next to the hemisphere. Cut the PMC into six equally sized sections and ink the rostral aspect of each section.
Then place every other section into PFA filled containers for short fixation. And snap freeze the remaining sections for storage in sealed freezer bags, in cooler number one. Cut the right hemisphere anterior to posterior into one centimeter thick coronal sections.
Fix every other section in PFA and snap freeze the remaining sections for storage in sealed freezer bags, in cooler number one. Separate the brainstem from the cerebellum and place the brainstem in a PFA filled container for short fixation. Cut each cerebellar hemisphere into four equally fixed sagittal sections and photograph the medial and lateral views.
Then place the left cerebellar hemispheric slices in a container of PFA for short fixation. And snap freeze the right cerebellar hemispheric slices for storage in sealed freezer bags, in cooler number one. After obtaining the spinal cord with the nerve roots remove the spinal cord dura mater and store the dura in PFA.
Separate the left and right anterior and posterior nerve roots and cut the left anterior and posterior nerve roots from the spinal cord for short fixation in PFA. Cut the right anterior and posterior nerve roots from the spinal chord and snap freeze these tissues for storage in sealed freezer bags, in cooler number two. Then photograph the caudal-most 20 centimeter of the spinal cord and cut two centimeter transfer sections from the caudal to rostral direction of the cord for PFA short fixation and snap freezing.
Then photograph the remaining rostral portion of the spinal cord. And cut two centimeter transfer sections from the caudal to rostral direction of the cord for PFA short fixation and snap freezing. Cut sections of interest from the short or long fixed tissues.
Place the sections into individual wells of a six well plate and wash the tissues with three five minute washes in two milliliters of fresh PBS per wash, with shaking. For antigen retrieval, microwave the sections for two to three minutes in a glass beaker containing roughly 30 milliliters of 10 millimollar citrate buffer. When the sections have cooled to room temperature transfer the samples into a new six well plate for three five minute washes and two milliliters of PBS, plus 0.3%Triton X-100 per well, per wash.
Followed by endogenous peroxidase blocking in two milliliters of 3%hydrogen peroxide plus 0.3%Triton X-100 in PBS for 30 minutes, at room temperature. At the end of the incubation, wash the sections three times in two milliliters of PBS plus Triton X-100 per wash, as demonstrated. Followed by blocking in two milliliters of 3%normal goat serum and PBS plus Triton X-100 for one hour at room temperature.
Next, incubate the sections from overnight to five days in the primary antibodies of interest at four degrees celsius followed by three five minute washes in two milliliters of PBS per wash. Incubate the sections in avidin-biotin complex solution or ABC complex for one hour, at room temperature. Follow this up with three five minute washes in PBS.
Label the sections with two milliliter of filtered Diaminobenzidine supplemented with hydrogen peroxide per well for three to eight minutes. When the color has adequately developed, wash the sections three times in PBS and individually transfer each section to a PBS filled container. Position each section flatly, under individual tissue slides.
And gently lift each slide out of the PBS, keeping the sections as flat as possible. Use two paintbrushes to gently flatten and stretch out each tissue. And use a paper towel to dab away the excess water.
Then mount the sections with an appropriate mounting medium and seal each cover slip with clear nail polish. Tissue sections are examined for the presence of white matter lesions and the selected regions are stained for immune activity and demyelination. In acute Multiple Sceloris lesions many of these demyelinated axons act as axonal retraction bulbs which reflect the proximal ends of transected axons.
The density of transected axons in acute lesions exceeds 11000 per cubic millimeter in active lesions compared to just 15 in adjacent non-lesional white matter regions. Newly generated oligodendrocytes are also present in many chronic MS lesions. These processes associate with but do not myelinate demyelinated axons.
A representative, unbiased search for neuronal gene changes in rapidly frozen motor cortext obtained from chronic MS patients, identified significant reductions in 23 nuclear and coded mitochondrial messenger RNA's Credentialing studies using immunocytochemistry and in situ hybridization indicated that these genes were highly enriched in cortical projection neurons and that mitochondria isolated from projection axons display reduced glycolysis. Comparison of the neuronode gene expression in myelinated and demyelinated hippocampi reveals reductions in neuronal messenger RNA's including proteins involved in memory and learning. Further, compared to control cortices, cortical neuronal loss is significantly greater in a sub population of MS patients that have demyelination of the spinal cord and cerebral cortext but not of the cerebral white matter.
During tissue processing, make sure that when the coronal sections are cut they are cut consistently with respect to thickness and orientation. And that they lie flat when floating in a fixative or when frozen. In addition to the immunohistochemical analysis the frozen tissue collection provides us with the opportunity to conduct genetic and proteomic analysis.
These techniques have aided in the identification of a new cohort of MS patients with cerebral white matter lesions on MRI but without any significant white matter demyelination. Termed Myelocortical MS.
