PMC:2493521 / 27820-37471 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2493521","sourcedb":"PMC","sourceid":"2493521","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2493521","text":"Blood and CSF draw, brain removal, gross dissection and initial tissue processing\nPrior to removing the brain, cerebrospinal fluid (CSF) is drawn from the lateral ventricles and heart blood is drawn from the left ventricle by transthoracic puncture, using 30 ml disposable syringes fitted with 8 cm long, 18 guage needles. The CSF is ejected into 15 ml disposable Falcon tubes for centrifugation while the blood is converted to serum by introduction into standard serum separator vacuum tubes (7 ml) prior to centrifugation. Although a small fraction of blood is also taken into EDTA vacuum tubes (4 ml each) for preparation of plasma, this is not directly comparable to plasma from living subjects as extensive postmortem coagulation would have removed many clotting factors and other proteins. About 20–30 ml of CSF, 5–10 ml of serum and 4 ml of plasma are generally obtained. A 2–3 ml sample of blood serum is sent to a commercial clinical pathology laboratory for syphilis, HIV and hepatitis A, B and C serology. Remaining blood serum as well as CSF is centrifuged and supernatants from both blood serum and CSF are aliquotted into 1 ml microcentrifuge tubes and stored frozen at −80°C. Postmortem CSF has been said to be unsuitable for research purposes due to postmortem changes or due to its ventricular origin, but these opinions appear to be purely anecdotal as we have conducted an extensive literature search and found no published evidence supporting these claims. We have conducted extensive studies on postmortem CSF (Castano et al. 2006) using Western blot, ELISA and proteomic methodology and have obtained results similar to those published by many other groups, who have all used CSF from living subjects. In our opinion, the benefits obtained by having precise neuropathological diagnoses for subjects greatly outweighs the disadvantages conferred by the presence of any minor postmortem changes. We are presently conducting a detailed proteomic comparison of CSF from living and deceased subjects. Similar considerations apply to the usage of postmortem blood fractions, which we have also investigated extensively (Kuo et al. 1998, 1999, 2000a; Rogers et al. 2006).\nAlso prior to brain removal, and prior to CSF draw, samples are taken of temporalis muscle and scalp. Many neurological diseases can be diagnosed or studied in muscle or skin and therefore we fix and/or freeze these samples along with the brain tissue. We have published results for Aβ concentrations in these temporalis muscle samples (Kuo et al. 2000b) and have done unpublished work on scalp samples from AD and control subjects (finding no immunohistochemically-detectable Aβ).\nBrain removal is accomplished in the standard fashion with an oscillating electric saw. To minimize bacterial contamination of brain tissue, the scalp is washed with an antibacterial solution. After the skull cap is removed, the brain team leader changes to new sterile gloves before moving the brain to the cutting board, which has been prepared by wiping down with 70% ethanol. These precautions are in the interests of our postmortem cell culture teams, which regularly culture glial and vascular cells from the postmortem brain tissue (Lue et al. 1996; Lue and Walker 2002; Rogers et al. 2002; Walker and Lue 2005). The brain is removed with as much cervical spinal cord as possible. Following brain removal, the pituitary gland is dissected from the sella turcica. Gross neuropathologic examinations on brain external surfaces, coronal cerebral slices and parasagittal cerebellar slices are performed by the neuropathologist during working hours. After-hours brains are photographed at the time of removal, whole and after slicing with a digital camera (Nikon D50) and dedicated software that transmits the images directly to a computer hard-drive (Nikon Capture 4). By reviewing the digital images, the neuropathologist is then able to perform a gross description on brains removed and processed after-hours. The images may also be used for morphometric analysis without the usual complication of correcting for volume changes due to fixation.\nThe details of the brain dissection are as follows. The olfactory bulbs and tracts as well as the pineal gland are removed and the cerebellum and brainstem are severed from the cerebrum by a transverse cut across the rostral pons. The leptomeninges are stripped from both cerebrum and cerebellum/brainstem for storage as research material (Roher et al. 2003). The cerebellum is separated from the brainstem by severing the cerebellar peduncles. Each hemicerebellum is sliced into 4–5 segments in the parasagittal plane. The brainstem is sliced into left and right halves. The cerebrum is sliced into approximately 0.8–1 cm segments in the coronal plane. The left side slices are used for immersion fixation in freshly-prepared phosphate-buffered 4% paraformaldehyde while the right side slices are rapidly frozen between sheets of dry ice (20 × 20 × 3 cm).\nThis method of brain processing differs from the conventional neuropathological examination, in which the whole or half of the brain is immersed in formaldehyde for 7–14 days before slicing. Although it has been argued that fresh-slicing detracts from the neuropathologic gross exam, we feel that it is essential to slice the brain fresh, both to enable rapid freezing and optimum fixation. Freezing an intact hemisphere slowly on dry ice or by placing it in an ultralow-temperature freezer results in severe ice-crystal artifact (Vonsattel et al. 1995), rendering the tissue unsuitable for cryostat-section histological examination, which has become increasingly important as a method for molecular studies, especially those utilizing confocal laser-capture microscopic analysis. Also, the long-entrenched idea that rapid freezing in liquid nitrogen or similar fluids is essential for molecular biological studies is simplistic and incorrect. In fact, rapid freezing may cause membrane damage while slow freezing in iso-osmotic cyroprotectant is optimal for synaptosomal preparations (Hynd et al. 2003). Our experience has shown that freezing rapidly between sheets of dry ice gives morphologically intact tissue suitable for a wide range of molecular biological procedures, including Western blot, ELISA, Northern blot, in-situ hybridization, gene microarray, proteomics and RT-PCR.\nSimilarly, fixing the brain whole or as an intact hemisphere is not an optimal use of the resource as this results in extreme fixation gradients across the tissue, with brain surfaces becoming over-fixed while interior regions are under-fixed. These fixation gradients make even semi-quantitative immunohistohchemical evaluation very difficult, although a minority of epitopes are tolerant to this. While perfusion-fixation of the postmortem brain through the circle of Willis (the entire cerebrum or one hemisphere can be fixed this way) is the optimal fixation method, this is time-consuming and we have found that fixation of 1 cm-thick slices for 24–48 h at 4°C is a reasonable compromise (Beach et al. 1987).\nTo aid in the production of uniformly-thick fresh brain slices for freezing and fixation, we have developed a brain slicing apparatus (jig) that holds the brain together while being sliced. With the assistance of a retired engineer living in Sun City and a contracted fabrication company (AvTek, Inc, Phoenix, AZ), we have enabled its semi-automated duplication by entering the 3-dimensional plans for the apparatus onto a computer. We have made this available and known to other brain banks.\nFixation is continued for 2 days at 4°C. Following fixation, diagnostic tissue blocks are taken from 28 brain regions (Table 2) for embedding in paraffin wax; additionally, 8 large (about 3 × 4 cm) tissue blocks, representing all cerebral lobes as well as cerebellum and brainstem (Table 2), following cryoprotection in 2% DMSO/20% glycerol, are sectioned at 40 μm on a sledge-type freezing microtome. These thick sections are superior to standard autopsy brain paraffin blocks for both silver stains and immunohistochemistry, due to their relatively light fixation (Beach et al. 1987; Leong and Gilham 1989), thickness (allowing full visualization of 3-D structure), structural integrity (allowing processing as free-floating sections, which improves antibody access and washing) and lack of exposure to hot paraffin wax (Pollard et al. 1987). These large-format sections also offer the opportunity for grading of periventricular white matter rarefaction (Fig. 2a) and an extensive survey for microscopic infarctions. Pre-cut free-floating 40 μm sections, as well as unsectioned fixed wet brain blocks, are stored at −20°C in a solution composed of 30% ethylene glycol, 30% glycerol in 0.1 M phosphate buffer. Sections prepared in this manner have been used successfully for immunohistochemical studies after two decades of storage.\nTable 2 Brain regions sampled for diagnostic histopathology and inventory\nFig. 2 Photomicrographs of 40 μm frozen sections stained according to our standard protocols. (a) Frontal lobe stained with H \u0026 E, showing periventricular white matter rarefaction; (b) hippocampus stained with the Campbell-Switzer silver method, showing senile plaques and neurofibrillary tangles; (c) neurofibrillary tangles stained with the Gallyas silver method; (d) neuron in the dentate gyrus granular cell layer stained immunohistochemically for phosphorylated tau protein with the AT8 antibody; (e) tufted astrocyte stained with the Gallyas method; (f) thorned astrocyte stained with the Gallyas method; (g) oligodendroglial coiled body stained with the Gallyas method","divisions":[{"label":"Title","span":{"begin":0,"end":81}},{"label":"Table caption","span":{"begin":8902,"end":8975}}],"tracks":[{"project":"2_test","denotations":[{"id":"18347928-16551433-20694307","span":{"begin":1547,"end":1551},"obj":"16551433"},{"id":"18347928-9837771-20694308","span":{"begin":2147,"end":2151},"obj":"9837771"},{"id":"18347928-10208861-20694309","span":{"begin":2153,"end":2157},"obj":"10208861"},{"id":"18347928-16290270-20694311","span":{"begin":2180,"end":2184},"obj":"16290270"},{"id":"18347928-8725904-20694313","span":{"begin":3220,"end":3224},"obj":"8725904"},{"id":"18347928-12404514-20694314","span":{"begin":3241,"end":3245},"obj":"12404514"},{"id":"18347928-15957156-20694315","span":{"begin":3282,"end":3286},"obj":"15957156"},{"id":"18347928-12865947-20694316","span":{"begin":4472,"end":4476},"obj":"12865947"},{"id":"18347928-7815079-20694317","span":{"begin":5524,"end":5528},"obj":"7815079"},{"id":"18347928-12694381-20694318","span":{"begin":6074,"end":6078},"obj":"12694381"},{"id":"18347928-2437408-20694319","span":{"begin":7068,"end":7072},"obj":"2437408"},{"id":"18347928-2437408-20694320","span":{"begin":8148,"end":8152},"obj":"2437408"},{"id":"18347928-2483748-20694321","span":{"begin":8171,"end":8175},"obj":"2483748"},{"id":"18347928-3309048-20694322","span":{"begin":8406,"end":8410},"obj":"3309048"}],"attributes":[{"subj":"18347928-16551433-20694307","pred":"source","obj":"2_test"},{"subj":"18347928-9837771-20694308","pred":"source","obj":"2_test"},{"subj":"18347928-10208861-20694309","pred":"source","obj":"2_test"},{"subj":"18347928-16290270-20694311","pred":"source","obj":"2_test"},{"subj":"18347928-8725904-20694313","pred":"source","obj":"2_test"},{"subj":"18347928-12404514-20694314","pred":"source","obj":"2_test"},{"subj":"18347928-15957156-20694315","pred":"source","obj":"2_test"},{"subj":"18347928-12865947-20694316","pred":"source","obj":"2_test"},{"subj":"18347928-7815079-20694317","pred":"source","obj":"2_test"},{"subj":"18347928-12694381-20694318","pred":"source","obj":"2_test"},{"subj":"18347928-2437408-20694319","pred":"source","obj":"2_test"},{"subj":"18347928-2437408-20694320","pred":"source","obj":"2_test"},{"subj":"18347928-2483748-20694321","pred":"source","obj":"2_test"},{"subj":"18347928-3309048-20694322","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93d9ec","default":true}]}]}}