Poster
# 118
Poster |
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6th Internet World Congress for Biomedical Sciences |
Domizio Suvā(1), Isabelle Favre(2), Rudolf Kraftsik(3), Monica Esteban(4), Alexander Lobrinus(5), Judit Miklossy(6)
(1)(2)(4)(5)(6)CHUV, Institute of Pathology - Lausanne. Switzerland
(3)IBCM - Lausanne. Switzerland
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[Neuroscience] |
[Pathology] |
The brains of 29 autopsy cases were analyzed. The brains were fixed in 10% formalin for 4 weeks. From all the 29 brains about 3 x 3 x 0.5 cm large samples were taken from the following cortical regions: entorhinal cortex, hippocampus, frontal associative cortex (Brodmannīs area (Br) 8, 9), parietal associative cortex (Br. 39, 40), primary motor cortex (Br. 4) and primary sensory cortex (Br. 3,1,2). In 9 cases an additional sample from the primary visual cortex (Br. 17) was also taken. After embedding in paraffin, from all these blocks 5um thick paraffin sections were cut and stained with haematoxylin and eosin, Thioflavine S, Congo red, Gallyas silver technique (30) and immunostained with monoclonal antibody to beta-amyloid protein (DAKO, M872, dil.1:100). In some cases, sections were also stained with the cresyl-violet technique and immunostained with a rabbit polyclonal anti-myelin basic protein (MBP) (DAKO, A623, dil. 1:100) for the analysis of cyto- and myelo-architectonics, respectively.
For immunostaining the avidin-biotin-peroxidase technique was used. The sections were deparaffinized, rehydrated and incubated in 0.3% mathanolic peroxide for 30 minutes, in order to eliminate endogenous peroxidase activity. The sections were incubated overnight at 4°C with the primary antibody. The biotinylated secondary antibody and the avidin-biotin complex were used following recommendations of the manufacturer (DAKO, ABComplex/HRP-Kit, K0355). For the visualization of the immunoreaction diaminobenzidine was used as chromogen. After immunostaining, the sections were counterstained with haematoxylin. For the detection of beta-amyloid, before immunostaining, the sections were pre-treated with formic acid for 20 minutes.
For the neuropathological diagnosis of AD, in all cases, a semi-quantitative analysis of senile plaques and neurofibrillary tangles was performed on different cortical regions (entorhinal cortex, hippocampus, frontal and parietal associative areas), as described in a previous study (32). For the diagnosis of AD, criteria proposed by the Consortium to Establish Registery for Alzheimerīs Disease (CERAD) (9) and those proposed by Khachaturian were both considered (8). The severity of cortical involvement by neurofibrillary tangles was graded following Braak (20). Based on these staging procedures 17 cases with severe AD-type cortical changes fulfilled the criteria of the neuropathological diagnosis of AD (Table 1, cases 1-17). In these AD cases the severity of the cortical AD-type changes corresponded to Braak stages V-VI. The age of these patients varied between and 94 years. Dementia was clinically documented in all cases, therefore these 17 cases also fit the diagnosis of AD following the guidlines of CERAD (9). In addition to dementia signs of pyramydal involvement was clinically documented in three cases which included Babinski sign and/or spacticity. Seven patients, aged 76 to 89 years, had discrete to moderate AD-type cortical changes, insufficient for a diagnosis of AD (Table1, cases 18-24). These patients were not demented, except one, in which the neuropathological examination revealed hypertensive encephalopathy. In this group the severity of cortical lesions following Braak corresponded to stages I-II in 5 cases (Table1 cases 18-22) and to III-IV in two cases (Table1, cases 18-22). The remaining 5 non-demented cases, aged 37 to 61 years, without any AD-type cortical changes formed the third group and served as controls (Table1, cases 25-29).
In all 29 cases, a quantitative analysis of senile plaques and neurofibrillary tangles was performed on sections derived from the following cortical regions: entorhinal, frontal associative (Br. 8, 9), parietal associative (Br. 39, 40), primary motor (Br. 4) and primary sensory cortex (Br. 3, 1, 2). In order to verify the reproducibility and reliability of the results, in all cases and on all sections the quantitative analysis of plaques and tangles was repeated independently by another investigator.
Morphometric analysis of senile plaques was performed on beta-amyloid stained sections. Immunostained deposits of beta-amyloid of all type of senile plaque were considered (33). The analyzed cortical areas were always selected from regions free of vessels with beta-amyloid deposits. Using a computer assisted Leitz microscope microscopical images each corresponding to 0.16 mm2 of cortical surface visualized using X10 objective were captured with a video camera and Samba Immuno v.4.05 software (for Microsoft Windows). The morphometric analysis was made on these online images. In selecting the brown color of beta-amyloid deposits we were able to eliminate the blue color of haematoxylin-stained nuclei. After the subtraction of background noise and histology artifacts, the total surface occupied by beta-amyloid plaques was measured and expressed as a percentage of the totalcortical area studied. The analysis was performed in 5 fields selected from the most severely affected parts of each cortical areas. Of the 5 fields a mean was calculated.
The quantitative analysis of neurofibrillary tangles was performed on Gallyas-stained sections using a Leitz microscope. The number of tangles were counted in 0.4mm2 microscopic fields obtained using 10x power eye-piece and 25x power objective lens (250x magnification). Like for senile plaques, the quantitative analysis of neurofibrillary tangles was performed in 5 different fields of the histologically identified most severely affected regions of each cortical area. Tangles localized on the border of the upper half field were counted, those situated on the borderline of the lower half field were ignored. The results were expressed as the mean number of tangles per 0.4mm2 microscopic field (the total cortical area studied).
In order to determine the reliability of the quantitative study, the analysis of variance (ANOVA) was used to compare the results obtained by the two investigators. The same statistical method was used to compare the mean percentage of cortical surface occupied by senile plaques and the mean number of tangles between the 5 different cortical areas.
The Pearson correlation was calculated and tested for significance in order to analyze if an association exists between the severity of cortical changes among different cortical areas.
beta-amyloid and Gallyas-stained sections were used to analyze the laminar distribution of plaques and tangles with the aid of a Zeiss computer microscope system (34). In some cases on Nissl stained sections, the neuronal loss in layer Va and Vb of the primary motor cortex was documented using the same computer microscope and the Neurolucida mapping software from Microbrightfield Inc.
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[Neuroscience] |
[Pathology] |
