UNIVERSITY OF MARYLAND
School of Medicine
Brain and Tissue Bank
for Developmental Disorders
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UNIVERSITY OF MARYLAND |
Protocol Method 2 is the current method used for all brain tissue donations, where possible.
When possible, all brains should be chilled/cooled in wet ice for at least one half our prior to sectioning
to enhance the ease and quality of sectioning.
The preferred method of freezing the individual sections is in isopentane/dry ice at -30 to -40 degrees Centigrade.
The second method of choice is in liquid nitrogen. The third method of choice is freezing the samples on a tray
in a -80 degree freezer.
10 percent formalin is used for all fixed sections.
First, the medulla is removed from the brainstem by transecting at its juncture with the distal pons(Cut #1).
The medulla is sectioned in a coronal plane into five samples of 2-3mm thickness beginning at the pontine junction.
These five samples are assigned a sequential identifier from 1 to 5. Sections 1,3,5 are frozen; sections 2 and
4 are fixed in 10% formalin. After removal of the medulla, the entire brain is sectioned into its left and right
hemispheres (Cut #2). The right hemisphere is fixed in its entirety in 10% formalin.
A section is made just posterior to the cerebral peduncles and the midbrain/pons/cerebellum is removed as a
unit from the left hemisphere (Cut #3). The remaining cerebrum is sectioned coronally, at approximate 1 cm intervals
beginning from the frontal pole apex and proceeding caudally. As each section is isolated, it is gently rinsed
with water, blotted dry, assigned a sequential numeric identifier (odd numbers only!), and placed in the freezing
bath. The handling of sections is best aided by the use of a plastic spatula. Each frozen section is placed into
individual plastic bags appropriately labeled and sealed. All bags are then stored in a -80 degree Centigrade freezer
prior to shipping. Frozen sections of the cerebrum are identifed as sections 1,3,5,7,9...
The midbrain/pons (upper brainstem) is separated from the cerebellum (Cut #4). The midbrain/pons is placed on
a flat cutting board, mesial surface down, and sectioned into four or five sections at approximate 0.3 to 0.4 cm
intervals beginning at the midbrain and moving caudally. These sections are assigned a sequential identifier (odd
numbers only!). Frozen sections of the midbrain/pons are identified as sections 1,3,5...
The remaining cerebellum is placed in a verticle plane (its normal anatomic position) and sectioned at 0.5 to
0.6 cm intervals beginning from the medial surface (vermis) and moving laterally. Each resulting section is assigned
a sequential identifier (odd numbers only!). Frozen sections of the cerebellum are identified as sections 1,3,5,7,9...
The right hemisphere is fixed in its entirety in 10 percent formalin and is sectioned similarly to the left
hemisphere. Fixed sections of the cerebrum (right hemisphere) are identified as sections 2,4,6,8,10... Fixed sections
of the midbrain/pons (right hemisphere) are identified as sections 2,4,6... Fixed sections of the cerebellum (right
hemisphere) are identified as sections 2,4,6,8,10...
The above protocol may be modified in a certain number of cases due to the nature of the injury or diagnosis.
In the past, Protocol Method 1 was the first method used by the Brain and Tissue Bank for brain tissue donations. This method is no longer the current method used for tissue donations.
The brain is placed on a horizontal surface, dorsum down, with gyrus rectus parallel to the surface. A single
coronal section, perpendicular to the surface, is made at a point caudal to the mammillary bodies, but anterior
to the rostal origin of the crus cerebri. The brainstem/cerebellum portion is removed from the caudal half of the
cerebrum by transecting the midbrain at its junction with the rostral pons. A plexiglass cutting guide is used
to obtain one centimeter thick serial, coronal sections of the cerebrum. Each coronal section is then cut in the
sagittal plane through its midline, creating two hemisections. The cerebral hemisections are fixed and frozen in
an alternating sequence as shown.
Sectioning of the remaining brainstem/cerebellum is initiated by transecting the medulla from the pons in a
strict transverse plane, perpendicular to the long axis of the lower brainstem. The intact pons/cerebellum is sectioned
in a transverse plane at one centimeter intervals and hemisected. The pons/cerebellum is fixed and frozen in the
same alternating scheme as the cerebrum.
The medulla is cut transversely at the following four levels to create 5 sections: mid-olive, 4-5 mm distal,
caudal pyramidal decussation and 4-5 mm distal. Alternate sections of the medulla are fixed and frozen.
The above protocol may be modified in a certain number of cases due to the nature of the injury or diagnosis.