jeanflop/post-ocr-correction huggingface dataset:
immunostaining of Alzheimer-amyloid deposits (anti-amyloid) and microglial cells (anti-human L-ferritin) on autopsy samples. Material and methods Materials We used material from five patients (age ranging from 43 to 75 years), who fulfilled established neuropathological criteria for Alzheimer's disease. Biopsy specimens, taken from the second frontal gyrus of the right hemisphere, fixed in 1 % glutaraldehyde and 2 % paraformaldehyde, post-fixed in osmium tetroxide and embedded in Araldite, were processed for conventional electron microscopy or immuno-electron microscopy. Most patients were at an early stage of evolution: biopsies were performed for diagnostic purposes before CT-scan made easier, by elimination of focal lesion,
purposes before CT-scan made easier, by elimination of focal lesion, a non-invasive diagnosis of degenerative dementia. Brains were fixed at autopsy in formalin 1/7 v/v in tap water with 0.2 M sucrose added, and samples were embedded in paraffin and cut into 7 μm thick serial sections for routine staining or immunohistochemistry. We examined the frontal and temporal cortex, hippocampus and cerebellum. Light immunohistochemistry Serial 7 μm thick sections from paraffin-embedded blocks were alternately stained with routine methods (hematoxylin-eosine, Masson trichrome or thioflavine), impregnated with silver according to Bodian, or single or double immunostained with anti-amyloid (Dako or antiSP28) (1/400)
single or double immunostained with anti-amyloid (Dako or antiSP28) (1/400) and/or with anti-human L-ferritin antibody (1/500) (Dako). Anti-SP28, a gift of Dr Frangione, is a rabbit serum directed against the 28 residue synthetic peptide homologous to NH2-terminal region of β protein [13]. For single immunocytochemistry, the antibody was evidenced by the Avidin-Biotin Complex (1/100) (ABC Vectastain) and 3, 3' diaminobenzidine as chromogen (DAB). For double immunostaining, the anti-amyloid was revealed by DAB as chromogen; anti-human L-ferritin was evidenced by ABC with biotinylated anti-rabbit serum, alkaline phosphatase-conjugated streptavidin (1/100) (Vectastain) and 5-Bromo-4-Chloro-3-Indolyl-Phosphate and 4-Nitro Blue Tetrazolium chloride as chromogens. The second
5-Bromo-4-Chloro-3-Indolyl-Phosphate and 4-Nitro Blue Tetrazolium chloride as chromogens. The second antibodies were used in condition of maximal saturation. Electron microscopy immunohistochemistry We used a post-embedding procedure [14]. Ultrathin sections from Araldite-embedded blocks were mounted on gold grids, incubated with normal goat serum (1/40), monoclonal anti-β amyloid (Dako) or anti-SP28 (1/200), overnight at 4°C and reincubated respectively with goat anti-mouse IgG or goat anti-rabbit serum conjugated to colloidal gold particles (1/50) (Auroprobe GAMG or GARG, 15 nm, Jansen) during 2 h at room temperature. The sections were counterstained with lead citrate and uranyl acetate or with the latter only and finally
and uranyl acetate or with the latter only and finally observed with a Siemens 102 electron microscope. As was done with the light microscope immunohistochemistry, the specificity of immunostaining was controlled by incubating control sections with Tris buffer or preimmunized serum as primary antibody. Results Reactive microglial cells were identified through morphological characteristics. The nucleus was generally kidney-shaped with peripherally clumped chromatin. The ramified cytoplasm was filled with prominent endoplasmic reticulum, vacuoles, lipids droplets and lipofuscin (Fig. TA). Microglial cells emitted processes around degenerating neurons, the cytoplasm of which was filled with paired helical filaments (PHF) (Fig. 1B); around "ghost
filled with paired helical filaments (PHF) (Fig. 1B); around "ghost tangles", that is extracellular PHF (not shown); or around degenerating neurites (Fig. 1D). The plasma membrane of microglial cells associated with plaque cores was directly apposed to amyloid fibrils; their processes are often interdigitated with the periphery of the plaque core. In some instances, the cytoplasm of the microglial cells associated with plaque core contained parallel bundles of membrane-bound fibrils (Fig. IC, 1E and IF). No immunodecorated amorphous material was seen in these cells (Fig. 2E and 2F). Electron microscopy immunocytochemistry (anti-β amyloid) showed gold particles decoration of these fibrils
immunocytochemistry (anti-β amyloid) showed gold particles decoration of these fibrils as well as plaque core amyloid fibrils (Fig. 2E and 2F). Immunostaining was observed neither in secretory organelles nor in hyaloplasm. Microglia and β/A4 amyloid in Alzheimer's disease Figure 1. A (X: 8500) Reactive microglial cell in the vicinity of amyloid fibrils (arrow heads) and paired helical filaments (arrows). The cytoplasm of this microglial cell shows morphological characteristics of phagocytic function. B (X: 7000) Close association between microglial cell and degenerating neuron, the cytoplasm of which is filled with paired helical filaments (arrows). C (X: 8200) A microglial cell, with
helical filaments (arrows). C (X: 8200) A microglial cell, with part of its characteristic nucleus, engulfs a star-shaped mass of fibrillary amyloid (arrow heads), strongly reminiscent of the core of a mature plaque. Note that there is no evidence of protein secretion (very scanty ergastoplasm) and, conversely, that lysosomes (L) are abundant, indicative of exogenous material catabolism. D (X: 11 200) Microglial cell emitting processes around degenerating neurite (N) which is filled with paired helical filaments (arrow heads), degenerated mitochondria and dense bodies (arrows). E (X: 10200) Classical plaque with amyloid "star" (arrow heads) partially surrounded by microglial cell. This
amyloid "star" (arrow heads) partially surrounded by microglial cell. This cell also emits processes around degenerated neurites (N). F (X: 17260) High magnification of Fig. 1E shows intracytoplasmic bundles of amyloid fibrils, membrane bounded (arrows). Microglial/phagocytes and β/A4 amyloid in Alzheimer's disease Figure 2. A (X: 270) Anti-human L-ferritin stains intensively cells which are more abundant in the deep cerebral cortical layers and usually arranged in nodules (frontal cortex). B (X: 300) Double immunostaining, where microglial cells (blue) are mainly associated with β amyloid (brown) (temporal cortex). C (X: 460) Ferritin is often colocalized with β amyloid (temporal cortex). D
Ferritin is often colocalized with β amyloid (temporal cortex). D (X: 460) Double immunostaining: amyloid deposits of the superficial cortical layers are not associated with microglial cells (frontal cortex). E (X: 13 000) EM immunostaining of a microglial cell-amyloid star complex on serial section of the region shown on Figure 1E; intracytoplasmic and plaque core amyloid fibrils (arrow heads) are simultaneously immunodecorated; no immunostaining of secretory organelles (arrows) nor of hyaloplasm is seen. F (X: 21 000) Another associated-amyloid plaque core microglial cell; decoration is seen on amyloid fibrils only (arrow heads). The microglial cells that were not associated with
(arrow heads). The microglial cells that were not associated with plaque cores did not react to anti-p amyloid. Light microscopy immunohistochemistry coupled to acid formic pretreatment revealed immunoreactivity of the various types of deposits as previously reported by many authors (for review see [6, 7]). Single immunostaining with anti-ferritin showed, at the light microscope level, increased density of immunostained cells either dispersed or grouped in nodules (Fig. 2A). The cells were more numerous in deep cortical layers. They were small, bipolar or multipolar, with a scanty cytoplasm and delicate processes, presumably corresponding to resting microglia. Anti-human L-ferritin also stained intensively
presumably corresponding to resting microglia. Anti-human L-ferritin also stained intensively microglial cells with swollen cytoplasm and few branching processes, presumably corresponding to reactive microglia. As shown with double immunostaining, the majority of microglial cells were associated with senile plaques in frontal and temporal cortex. Ferritin and p amyloid were often colocalized (Fig. 2B and 2C). Some other microglial cells did not show any topographical relationship with amyloid deposits. Qualitative study showed that diffuse amyloid deposits, which were particularly abundant in the superficial cortical layers, were usually not associated with microglial cells (Fig. 2D). Discussion Several forms of amyloidosis have been
cells (Fig. 2D). Discussion Several forms of amyloidosis have been linked to the mononuclear phagocytic system (MPS), a link that could be constructed as a prima facie case for amyloid secretion by that system. Amyloid fibrils were found associated with MPS in experimental amyloidosis [15], human myeloma stem-cell cultures [16], Scrapie [17] and Gerstmann Straussler Scheinker syndrome [18]. Morphological evidence alone, however, would not allow a clear distinction between secreting or phagocytosing MPS cells. Experimental amyloidogenesis studies in animals could not establish unequivocally the origin of Aβ4 amyloid or its precursor. It has been suggested that astrocytes might be a
precursor. It has been suggested that astrocytes might be a source of APP [19], although it has been pointed out that increased biosynthesis of APP following damage is an event distinct from reactive gliosis [20]. A recent report [21] showed that after motor neuron axotomy the APP gene expression is increased in the rat brain; on the other hand, reactive microglial cells phagocytose APP produced by neurons following neuronal damage [22]. The two immunoreagents we used to detect Aβ4 amyloid (anti-SP28 and Dako antibody), coupled with formic acid pretreatment, show various forms of deposits as already documented in the literature:
various forms of deposits as already documented in the literature: classical and diffuse plaques, vascular, perivascular and subpial deposits. Anti-human L-ferritin is a marker of microglia, applicable to formalin-fixed and paraffin-embedded samples [12, 23]. It reacts with microglia in normal and Alzheimer's disease brain. Microglial cells exhibit various shapes, ranging from rod-like to oval-shaped appearance while varying in size. They are highly ramified with extending branches. Amyloid plaques were systematically associated with these cells; conversely, reactive microglial cells were not always associated with amyloid deposits. Conventional electron microscopy showed several morphological changes in microglial cells, with characteristics of phagocytic function
morphological changes in microglial cells, with characteristics of phagocytic function that were found in the vicinity of the neuropathological features of Alzheimer's disease, suggesting that microglial cells take part to the neurodegenerative phenomena operative in Alzheimer's disease. Electron microscopy and light microscopy immunostaining studies showed a consistent association of microglial cells with senile plaque cores, but not with diffuse plaques. Parallel bundles of fibrils were observed in microglial cells cytoplasm. They were immunodecorated simultaneously with the fibrils of the plaque core. On the basis of the morphological data alone, it is not possible to tell whether amyloid is streaming out
is not possible to tell whether amyloid is streaming out of or into microglia. The morphological characteristics of plaque-associated microglial cells, the fibrillar nature of the intracytoplasmic amyloid (inasmuch the amorphous form of Aβ4 peptide is believed to be precursor of the fibrillar form [13,24]) and the absence of immunoreactivity in secretion-related organelles lead us to tentatively conclude that, in Alzheimer's disease, microglial cells with phagocytic characteristics may participate to amyloid catabolism rather than to amyloid synthesis. This opinion has been indirectly confirmed by a recent report [25], according to which in situ hybridization failed to detect messenger RNA of
which in situ hybridization failed to detect messenger RNA of APP in these cells. The apparently contradictory results of experimental studies in primary microglial cells culture from rat brain [26] or in reactive microglia following rat brain damage [27] that showed expression of APP isoforms has no direct bearing on our problem relative to the production of Aβ4 amyloid, a process that appears to be far from specific than is the nearly ubiquitous APP production. However, although we do not confirm the hypothesis of de novo production of Aβ4 amyloid by microglia, we cannot rule out the possibility that microglia
by microglia, we cannot rule out the possibility that microglia may participate in the polymerization process of Aβ4 amyloid in Alzheimer's disease. Acknowledgments: we thank Pr O. Bugiani and Dr F. Tagliavini for helpful discussions and D. Lecren for iconography. This research was supported by Naturalia and Biologia, EPHE and INSERM. VERSION ABRÉGÉE La plaque sénile dans la maladie d'Alzheimer est constituée essentiellement de fibres amyloïdes (coeur) et de neurites dégénérés, de cellules astrocytaires et microgliales à sa périphérie. Le constituant majeur de l'amyloïde est un peptide de 4 kD appelé peptide A4 ou protéine P-amyloïde (BAP), qui dérive d'une
appelé peptide A4 ou protéine P-amyloïde (BAP), qui dérive d'une grande glycoprotéine dénommée « amyloid protein precursor » (APP). En plus de sa large répartition parmi les tissus et taxons, l'APP a été détectée dans le système nerveux au niveau de différentes cellules y compris les neurons et les astrocytes, et dans le liquide céphalorachidien. Les mécanismes de transformation de l'APP en BAP sont mal connus et une des questions fondamentales dans la compréhension de la physiopathologie de la maladie d'Alzheimer est la relation entre la dégénérescence neuronale et les dépôts d'amyloïde. Parmi les cellules associées à la plaque sénile,
dépôts d'amyloïde. Parmi les cellules associées à la plaque sénile, les cellules microgliales pourraient jouer un rôle primordial dans ce processus. Ces cellules, qui sont généralement activées dans le système nerveux lors de processus inflammatoires, neurodégénératifs ou traumatiques, migrent vers le site lésionnel et se transforment en macrophages chargés de vacuoles et de gouttelettes lipidiques. Elles sont aussi connues pour sécréter diverses protéines, par exemple des facteurs de croissance et l'interleukine 1. Notre travail se propose d'étudier la relation topographique et fonctionnelle entre les cellules microgliales et les plaques séniles afin de préciser si ces cellules sécrètent ou phagocytent la
afin de préciser si ces cellules sécrètent ou phagocytent la protéine βA4 amyloïde. Cette étude a été effectuée par une approche ultrastructurale, immuno-ultrastructurale (anti-β amyloïde) et par un double marquage immunohistochimique à l'échelle optique (anti-β amyloïde et anti-L ferritine humaine), sur des prélèvements autopsiques (5 patients) et des prélèvements biopsiques (5 patients). L'anti-L ferritine humaine est utilisé comme marqueur des cellules microgliales. L'étude immuno-ultrastructurale a été effectuée sur coupes ultrafines à partir de blocs provenant de notre collection, fixés dans le mélange de Karnovsky et inclus dans l'Araldite. Les coupes sont incubées dans l'anti-β amyloïde pendant une nuit à 4
sont incubées dans l'anti-β amyloïde pendant une nuit à 4 °C, réincubées en présence d'un anticorps de mouton anti IgG de lapin couplé à des particules d'or colloïdal de 15 nm de diamètre pendant 2 h à température ambiante. Les coupes sont contrastées à l'acétate d'uranyle et au citrate de plomb. L'immunohistochimie photonique est effectuée sur coupes de paraffine à partir de prélèvements fixés au formol, préalablement traitées à l'acide formique à 95 % pendant 9 min. Elle a été réalisée en simple et double marquage. Lors du double marquage immunohistochimique (anti-β amyloïde et anti-L ferritine humaine), nous avons utilisé
immunohistochimique (anti-β amyloïde et anti-L ferritine humaine), nous avons utilisé le complexe avidine-biotine (Vectastain) conjugué à la peroxydase et la 3,3' diaminobenzidine comme chromogène pour révéler l'anti-β amyloïde. L'anti-L ferritine humaine a été révélée par le complexe avidine-biotine conjugué à la phosphatase alcaline-strepavidine et le 5-Bromo-4-Chloro-3-Indolyl-Phosphate et le 4-Nitro Blue Tetrazolium chloride comme chromogènes. Les cellules microgliales ont été identifiées grâce à l'anti-L ferritine humaine, marqueur sélectif de ces cellules. L'étude ultrastructurale montre une mobilisation des cellules microgliales non seulement autour du cœur amyloïde, mais aussi autour des neurites et des neurons dégénérés et de paires de filaments en hélice
des neurons dégénérés et de paires de filaments en hélice en situation extracellulaire. Les cellules microgliales, avec lysosomes, corps densés et vacuoles lipidiques témoins d'activité phagocytique, contiennent des amas de fibrilles semblables à celles du cœur de la plaque. En périphérie du cœur de la plaque, le cytoplasme de ces cellules montre des interdigitations. L'étude immuno-ultrastructurale montre une décoration sélective des fibrilles intracytoplasmiques ainsi que de celles du cœur amyloïde. Au contraire, il n'a jamais été observé de structure amorphe immunoréactive vis-à-vis de l'anti-β amyloïde dans le cytoplasme des cellules microgliales. Les organites cellulaires sécrétoires et l'hyaloplasme, des cellules microgliales
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