jeanflop/post_ocr_correction2 huggingface dataset:
(T-C) in codon 29 of the α2 globin gene produces a structural haemoglobin variant and α-thalassemia. Br. J. Haematol. 84 (suppl. 1): 63 (abstr. P153). C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317 : 437-44 C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317 : 445-51 Sciences médicales/Médical sciences Do microglial cells phagocyte the β/A4-amyloid sénile plaque core of Alzheimer disease? Les cellules microgliales phagocytent-elles la substance β/A4 amyloïde du cœur de la plaque sénile de la maladie d'Alzheimer? KHALID HAMID EL HACHIMI, JEAN-FRANÇOIS FONCIN EPHE Neurohistologie and INSERM U. 106, Hôpital de la Salpêtrière, 47, boulevard de l'Hôpital 75651 Paris Cedex 13, France. Tirés à part : K. H. El Hachimi RÉSUMÉ Nous avons étudié à l'échelle ultrastructurale, immuno-ultrastructurale et immuno-photonique la relation topographique et fonctionnelle entre les cellules microgliales et les plaques séniles de la maladie d'Alzheimer. Nos résultats montrent que ces cellules, présentant les caractéristiques cytologiques de la phagocytose, sont associées aux dépôts amyloïdes classiques ainsi qu'aux autres lésions neurodégénératives. En périphérie du cœur amyloïde, ces cellules contiennent des amas fibrillaires immunoréactifs à anti-β/A4 amyloïde et entourés d'une membrane. Les dépôts préamyloïdes des couches superficielles, essentiellement de la couche II, ne sont pas associés aux cellules microgliales. Les caractéristiques cytologiques de ces cellules, le caractère uniquement fibrillaire du matériel immunoréactif et l'absence d'immunoréactivité sur matériel amorphe ou dans les organites de sécrétion, dans ces cellules aussi bien que dans les cellules non associées aux plaques, sont en faveur d'une activité phagocytaire de la microglie vis-à-vis de l'amyloïde dans la maladie d'Alzheimer. ▲ Mots clés : cellules microgliales, maladie d'Alzheimer. ABSTRACT We performed EM, immuno-EM and light microscope immunohistochemistry studies on the topography and functional relationships between microglial cells and amyloid sénile plaque core in Alzheimer's disease. Microglial cells with cytological characteristics of phagocytic function were associated to amyloid fibrils and to other neuropathological degenerative processes. On the periphery of the amyloid plaque core, microglial cells contain intracytoplasmic bundles of membrane bound fibrils. These fibrils, like plaque core fibrils, are immunodecorated. Immunostaining was observed neither in secretory organelles nor in hyaloplasm.
ne sont pas associés aux cellules microgliales. Les caractéristiques cytologiques de ces cellules, le caractère uniquement fibrillaire du matériel immunoréactif et l'absence d'immunoréactivité sur matériel amorphe ou dans les organites de sécrétion, dans ces cellules aussi bien que dans les cellules non associées aux plaques, sont en faveur d'une activité phagocytaire de la microglie vis-à-vis de l'amyloïde dans la maladie d'Alzheimer. ▲ Mots clés : cellules microgliales, maladie d'Alzheimer. ABSTRACT We performed EM, immuno-EM and light microscope immunohistochemistry studies on the topography and functional relationships between microglial cells and amyloid sénile plaque core in Alzheimer's disease. Microglial cells with cytological characteristics of phagocytic function were associated to amyloid fibrils and to other neuropathological degenerative processes. On the periphery of the amyloid plaque core, microglial cells contain intracytoplasmic bundles of membrane bound fibrils. These fibrils, like plaque core fibrils, are immunodecorated. Immunostaining was observed neither in secretory organelles nor in hyaloplasm. Preamyloid deposits in superficial layers were not associated to microglial cells. These data lead us to conclude that the microglial cells participate to phagocytosis of β/A4 amyloid and do not secrete this substance in Alzheimer's disease. ▲ Key words : microglial cells, Alzheimer disease. Voir Version abrégée (p. 450) The generic term of amyloid covers a group of diverse proteins with spécifique conformational (β-pleated sheet) and staining (Congo red and thioflavine) characteristics. Human cerebral amyloidoses include Alzheimer's disease, cerebral amyloid angiopathies and transmissible spongiform encephalopathies (Kuru, Creutzfeldt-Jakob and Gerstmann-Straußler-Scheinker diseases). In Alzheimer's disease, a major constituent of the amyloid is a 4 kD peptide, β/A4 amyloid protein, BAP [1, 2] which derives from a larger glycoprotein called amyloid precursor protein, APP [3]. APP has been shown to be présent in various cells, including neurons, astrocytes and in extracellular fluid [4]. How BAP derives from APP is still unknown; one of the unanswered questions in Alzheimer's disease research is the relationship between neuronal degeneration and amyloid deposits. Several studies reported that degenerated neurons and dystrophic neurites associated with plaques accumulate various fragments of APP and suggested that neuronal APP is the most likely initial source of extracellular BAP [5]. Neuritic plaques
generic term of amyloid covers a group of diverse proteins with spécifique conformational (β-pleated sheet) and staining (Congo red and thioflavine) characteristics. Human cerebral amyloidoses include Alzheimer's disease, cerebral amyloid angiopathies and transmissible spongiform encephalopathies (Kuru, Creutzfeldt-Jakob and Gerstmann-Straußler-Scheinker diseases). In Alzheimer's disease, a major constituent of the amyloid is a 4 kD peptide, β/A4 amyloid protein, BAP [1, 2] which derives from a larger glycoprotein called amyloid precursor protein, APP [3]. APP has been shown to be présent in various cells, including neurons, astrocytes and in extracellular fluid [4]. How BAP derives from APP is still unknown; one of the unanswered questions in Alzheimer's disease research is the relationship between neuronal degeneration and amyloid deposits. Several studies reported that degenerated neurons and dystrophic neurites associated with plaques accumulate various fragments of APP and suggested that neuronal APP is the most likely initial source of extracellular BAP [5]. Neuritic plaques are composed mainly of amyloid fibrils, degenerating neurites, and reactive cells such as astrocytes and microglial cells. More recently, antibodies directed against various fragments of the β/A4 peptide have enabled to recognize, besides the amyloid core of classical plaques and the vascular amyloid deposits, another type of deposit that displays neither the ultrastructural fibrillary morphology nor the tinctorial characteristics of amyloid. These deposits have been called diffuse plaques [6] or preamyloid deposits [7]. The relationship with amyloid sensu stricto remains obscure. We correlated previously the amorphous amyloid of diffuse deposits with the dégénérescence of dendritic spines, whereas degenerating neurites were not immunostained [8, 9]; these studies however, had no bearing on the origin of the fibrillar amyloid of the classical plaque core. Among the cells associated with sénile plaques, microglial cells have been proposed as playing an important rôle in the formation of sénile plaques [10]. Microglial cells are activated by inflammatory, degenerative or traumatic events in the nervous System. They migrate to the site of injury and are transformed into activated macrophages filled with vacuoles and lipid droplets. Microglial cells are also known to secrete various proteins including glial growth factors, and interleukin 1 (for review see [11]). Ferritin
neither the ultrastructural fibrillary morphology nor the tinctorial characteristics of amyloid. These deposits have been called diffuse plaques [6] or preamyloid deposits [7]. The relationship with amyloid sensu stricto remains obscure. We correlated previously the amorphous amyloid of diffuse deposits with the dégénérescence of dendritic spines, whereas degenerating neurites were not immunostained [8, 9]; these studies however, had no bearing on the origin of the fibrillar amyloid of the classical plaque core. Among the cells associated with sénile plaques, microglial cells have been proposed as playing an important rôle in the formation of sénile plaques [10]. Microglial cells are activated by inflammatory, degenerative or traumatic events in the nervous System. They migrate to the site of injury and are transformed into activated macrophages filled with vacuoles and lipid droplets. Microglial cells are also known to secrete various proteins including glial growth factors, and interleukin 1 (for review see [11]). Ferritin is considered an sélective marker of these cells [12]. The présent study was designed to examine the topography and functional relationships between microglial cells and sénile plaques in order to specify whether they phagocyte or secrete β/A4 amyloid. This problem was addressed by means of conventional electron microscopy, immunoelectron microscopy study of anti-amyloid in biopsy specimens, and light microscopy double 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, 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
study of anti-amyloid in biopsy specimens, and light microscopy double 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, 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) 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 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)
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 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 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 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
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 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 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 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 cell also emits processes around degenerated neurites (N). F (X:
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 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 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 (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 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
(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 (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 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 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 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