Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

This protocol describes a method to track individual brain-aging trajectories through a brain donation program and proper characterization of brains. Brain donors are involved in a long-term longitudinal study including serial multi-dimensional assessments. The protocol contains a detailed description of brain processing and an accurate diagnostic methodology.

Transcript

As a neurocognitive disorder prevalence is continuously increasing, our aim is to obtain, characterize and store quality brain tissue samples to achieve accurate diagnosis and shed light on the neurodegenerative mechanisms. Considering brain assymetry, our sampling methodology allows all meta studies to be performed on histologically well-defined tissues from both hemispheres and the many data can be collected and correlated through deep learning. Additive effects on brain damage by multiple pathologies are frequently observed.

The definite diagnosis requires combining clinical syndrome with the neuropathological findings to discover disease pathogenesis, and possible therapies. Fresh cutting procedures as well as macro section management are tricky but are also essential to achieve good results. A trained and tight-knit team is crucial to overcome this difficulties.

Our protocol includes a series of steps that are required mammal dexterity, therefore visual demonstration can be more useful than descriptive texts for researcher wishing to master these techniques. After confirming thanatography, use a sharp scalpel to make a scalp incision through the hair skin and subcutaneous tissue on the coronal plate from the tip of the mastoid process on one side to the other, passing over the vertex. Separate the two folds of the scalp carefully from the skull and continue the incision until the yellow super orbital fat becomes visible.

Reflect the scalp anteriorly and pull the other section of tissue posteriorly. Using a scalpel and forceps, harvest a small sample of the temporal muscle at each side of the skull, placing one specimen in 4%formaldehyde and the other sample at four degrees Celsius. Using an electrical saw cut the skull in a V cut on the frontal side and remove the skullcap.

After cutting the meninges, harvest one piece of dura for fixation and another for freezing. Insert a 20 gauge 3.5 inch needle attached to a 10 milliliter syringe through the corpus callosum to reach the third ventricle and retract the plunger to obtain about 10 milliliters of cerebral spinal fluid. Assess the appearance color and turbidity of fluid and measure the pH.

Gently pull on both frontal lobes to lift the brain and cut both optic nerves infundibulum, the internal carotid arteries and the third, fourth, fifth and sixth cranial nerves. Cut the tentorium to reach the posterior fossa and cut the vertebral arteries and the lower cranial nerves. Then insert the scalpel as deep as possible through the foramen magnum to cut the most caudal portion of the medula and carefully remove the whole brain.

Use the scalpel to appears to the bone over Meckel's cave and collect the Gasserian ganglion from each side for fixation and freezing. Use a surgical mallet and chisel to fracture the bony sella turcica and remove the pituitary gland for fixation in 4%formaldehyde. Inspect the skull and the whole brain from macroscopic alterations and vascular changes.

Use a measuring tape to measure the transverse and anterior posture diameters of the brain and weigh the brain on a scale. Carefully retrieve the circle of Willis for macroscopic assessment for the presence of anatomic variants, lesions, or vessel stenosis. Separate and inspect the cerebrum, cerebellum, and brainstem before weighing the tissues individually.

Remove one to two, two to four centimeter squared pieces of leptomeninges from the convexity and preserve the samples in complete high glucose DMEM culture medium at four degrees Celsius. Harvest the pineal gland for fixation in 4%formaldehyde and remove the olfactory bulbs and optic nerves for the fixation and freezing of one of each pair. Place the cerebrum, cerebellum, and brainstem on ice at four degrees Celsius.

When all of the tissues of interest have been harvested use a super adhesive glue to reattach the bone and use a surgical needle, and non-absorbable sutures to stitch back the scalp. For brain bank dissection use a dissecting knife to cut the brainstem axially and make the first cut at the level of the rostral midbrain through the superior colliculus to obtain two slices to expose the substantia nigra. Cut the brainstem into 10, eight millimeter slices taking care to include cuts passing through the rostral pons near the superior margins of the fourth ventricle to observe the locus caeruleus and through the medulla oblongata inferior to the acoustic stria, just above the inferior apex of the fourth ventricle to obtain slices including the dorsal motor nucleus of the vagus.

Label all slices in a rostral caudal manner as BS for brainstem followed by Arabic numerals. After the last brainstem section, use the designation SC for spinal cord followed by Arabic numerals. Cut the cerebellum on the sagittal plane to separate the two cerebellar hemispheres at the level of the vermis and perform sagittal sectioning to obtain five slices from each hemisphere.

Name all of the slices from the vermis as CBR or CBL for the right and left cerebellum respectively, and use Arabic numerals to identify each section. Next, separate the two hemispheres of the cerebrum through the corpus callosum on the sagittal plane before slicing the hemisphere as singularly along the coronal plane passing between the optic chiasm and the mammillary bodies through the frontal lobe temporal pole, anterior cingulate, anterior commissure, nucleus of minored, and basal ganglia. Make a second cut about one centimeter posteriorly passing through the mammillary bodies and exposing the basal ganglia, anterior thalamus sub thalamus, and amygdala.

Continue slicing to dissect the anterior and posterior regions until 15 to 21 centimeter slices have been obtained for each hemisphere. Place the slice is on a flat surface as they're attained following their original position in the anterior posterior direction from the frontal to the occipital pole, with the portion continuous with the previous slides facing upwards. When all of the slices have been placed select the main sections to be fixed for histopathology including the previously acquired sections and the hippocampal, parietal and occipital sections, and label the slices as L or R followed by Arabic numerals including a label indicating whether the slice is to be fixed or frozen.

When all of the tissues have been sectioned and labeled, obtain an image for each series of sections before their fixation or freezing. To freeze the samples place the reserved alternate tissue sections on a pre frozen aluminum tray and cover the sections with an interlocking aluminum plate to keep them flat. Then freeze the slices in liquid nitrogen for three minutes before placing the frozen samples inside a plastic bag labeled with their corresponding identification codes and slice numbers for a storage in the appropriate cryogenic box at 80 degrees Celsius.

To fix the samples place the reserved alternate tissue sections in individual pieces of gauze and place the slices in 10%phosphate buffered formalin solution for five days in a four degrees Celsius cold room. 24 out of 27 brains harvested received the complete neuropathological characterization with a definite clinical pathological diagnosis. Preliminary quantitative electroencephalogram analysis of a series of 36 brain donors indicates that the main alpha rhythm percentage is significantly lower in major neurocognitive disorders than in normal elderly mild neurocognitive disorder.

Here an example of an asymmetric pathology is shown with a right side atrophy evident macroscopically with the severe ventricular dilations observed in the right coronal section compared to the left. Another case displays an infarct of the right hemisphere, while another specimen shows a clear atrophy of the right mammillary body. Macro sections can be stained to identify myelin loss or the distribution of immunoreactivity within hemispheres.

These samples are from twins who had a clinical onset of Alzheimer's disease in different timeframes due to epigenetic and environmental factors. Nevertheless, the microscopic analysis revealed a very similar neuropathological picture indicating a high Alzheimer's disease pathology and amyloid angiopathy. In these two patients however, the same clinical diagnosis of Alzheimer's disease did not exactly correspond to the neuropathological features, which revealed a dementia duo in multiple etiologies.

Indeed, the first case presented Lewy bodies in the gyrus single lie and substantia nigra. And the second case showed Lewy bodies associated with Lewy neurites in the amygdala and in minors nucleus. Photograph of the slices is carefully for an accurate identification of the anatomical areas during the microscopic analysis.

The fixation and freezing of singles slices are also important for preserving the tissue microstructure Omics the topography of gene activation and protein distribution and correlate these data with the histopathology. Cell cultures from well-characterized tissues, also a investigation into disease pathogenesis. Always wear adequate personal protective equipment as a vested brain tissue is considered potentially infectious and because the use of dangerous tools such as sharp instruments and liquid nitrogen is required.

Read Article

Journal (Neuroscience)

Journal (Neuroscience)

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

Chapters

Transcript

Related Videos

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Your free access has ended.