Hong Lim Kim

Osteopontin: correlation with phagocytosis by brain macrophages in a rat model of stroke.

Osteopontin (OPN) is an adhesive glycoprotein linked to a variety of pathophysiological processes. We investigated whether OPN might act as an opsonin in the diseased brain by studying the postischemic expression and localization of OPN mRNA and protein in a rat model of ischemic stroke. In addition, we characterized the subcellular localization of OPN protein in the ischemic brain core. Induction of OPN mRNA occurred in activated microglia/macrophages in the ischemic core on days 3–7 after reperfusion and this was sustained up to day 28, at least. OPN protein was synthesized and secreted by brain macrophages, which first surrounded damaged striatal white matter tracts and then infiltrated into them. Punctate OPN‐immunoreactive profiles were scattered throughout the infarction core except in white matter bundles. Electron microscopy showed the localization of OPN protein along the membranes lining what appeared to be the debris of dead neurons. These were located in the extracellular space and within the cytoplasm of brain macrophages, indicating that the OPN protein accumulated selectively on the surface of dead cells, most of which were phagocytosed subsequently by brain macrophages. However, no significant induction of OPN occurred in degenerating striatal white matter tracts or in brain macrophage‐engulfed axonic or myelin debris. These data suggest that OPN secreted by brain macrophages in this rat model of stroke might be involved in the phagocytosis of fragmented cell debris and possibly not in the phagocytosis of axonic or myelin debris.

Overlapping Distribution of Osteopontin and Calcium in the Ischemic Core of Rat Brain after Transient Focal Ischemia

Osteopontin (OPN), an adhesive glycoprotein, has recently been proposed to act as an opsonin that facilitates phagocytosis of neuronal debris by macrophages in the ischemic brain. The present study was designed to elucidate the process whereby OPN binds to neuronal cell debris in a rat model of ischemic stroke. Significant co-localization of the OPN protein and calcium deposits in the ischemic core were observed by combining alizarin red staining and OPN immunohistochemistry. In addition, electron microscopy (EM) using the osmium/potassium dichromate method revealed that electron-dense precipitates, typical of calcium deposits, were localized mainly along the periphery of putative degenerating neurites. This topical pattern of calcium precipitates resembled the distribution of OPN as detected by immunogold-silver EM. Combining immunogold-silver EM and electron probe microanalysis further demonstrated that the OPN protein was localized at the periphery of cell debris or degenerating neurites, corresponding with locally higher concentrations of calcium and phosphorus, and that the relative magnitude of OPN accumulation was comparable to that of calcium and phosphorus. These data suggest that calcium precipitation provides a matrix for the binding of the OPN protein within the debris or degenerating neurites induced by ischemic injury. Therefore, OPN binding to calcium deposits may be involved in phagocytosis of such debris, and may participate in the regulation of ectopic calcification in the ischemic brain.

Sustained Expression of Osteopontin Is Closely Associated with Calcium Deposits in the Rat Hippocampus After Transient Forebrain Ischemia

The present study was designed to evaluate the extent and topography of osteopontin (OPN) protein expression in the rat hippocampus 4 to 12 weeks following transient forebrain ischemia, and to compare OPN expression patterns with those of calcium deposits and astroglial and microglial reactions. Two patterns of OPN staining were recognized by light microscopy: 1) a diffuse pattern of tiny granular deposits throughout the CA1 region at 4 weeks after ischemia and 2) non-diffuse ovoid to round deposits, which formed conglomerates in the CA1 pyramidal cell layer over the chronic interval of 8 to 12 weeks. Immunogold-silver electron microscopy and electron probe microanalysis demonstrated that OPN deposits were indeed diverse types of calcium deposits, which were clearly delineated by profuse silver grains indicative of OPN expression. Intracellular OPN deposits were frequently observed within reactive astrocytes and neurons 4 weeks after ischemia but rarely at later times. By contrast, extracellular OPN deposits progressively increased in size and appeared to be gradually phagocytized by microglia or brain macrophages and some astrocytes over 8 to 12 weeks. These data indicate an interaction between OPN and calcium in the hippocampus in the chronic period after ischemia, suggesting that OPN binding to calcium deposits may be involved in scavenging mechanisms.

Desmin expression profile in reactive astrocytes in the 3-nitropropionic acid–lesioned striatum of rat: Characterization and comparison with glial fibrillary acidic protein and nestin

Desmin, a muscle-specific, type-III intermediate-filament protein, is reportedly expressed in astrocytes in the central nervous system. These cells become reactive astrocytes in response to brain injuries. To elucidate whether desmin is involved in this process, we examined the spatiotemporal expression profiles of desmin and their relationship with two astroglial intermediate filaments, glial fibrillary acidic protein (GFAP) and nestin, in the striatum of rats treated with the mitochondrial toxin 3-nitropropionic acid (3-NP). Weak, constitutive immunoreactivity for desmin was observed in astrocytes generally, and in reactive astrocytes in the peri-lesional area, its expression increased in parallel with that of GFAP over 3 d post-lesion and was maintained until at least day 28. Desmin, GFAP, and nestin showed characteristic time-dependent expression patterns in reactive astrocytes forming the astroglial scar; delayed and long-lasting induction of desmin and GFAP, and rapid but transient induction of nestin. In the lesion core, desmin was expressed in two categories of perivascular cells: nestin-negative and nestin-positive. These findings show that desmin, together with GFAP and nestin, is a dynamic component of intermediate filaments in activated astroglia, which may account for the dynamic structural changes seen in these cells in response to brain injuries.

Ultrastructural investigation of microcalcification and the role of oxygen–glucose deprivation in cultured rat hippocampal slices

Intracellular calcium accumulation is associated with cell death in several neuropathological disorders including brain ischemia, but the exact mechanisms of calcification need to be clarified. We used organotypic hippocampal slice culture - cultures subjected to oxygen-glucose deprivation (OGD) mimicking the in vivo situation to investigate the events underlying ectopic calcification. Alizarin red staining indicating calcium deposition was observed in the cornu ammonis (CA)1 and dentate gyrus regions in control hippocampal slices despite no specific labeling for cell death markers. Electron microscopy using the osmium/potassium dichromate method revealed scattered degenerated cells throughout the normally appearing CA1 region. They contained electron-dense precipitates within mitochondria, and electron probe microanalysis confirmed that they were calcifying mitochondria. Selective calcium deposition was noted within, but not beyond, mitochondria in these mineralized cells. They showed ultrastructural features of non-necrotic, non-apoptotic cell death and retained their compact ultrastructure, even after the majority of mitochondria were calcified. Unexpectedly, no intracellular calcification was noted in necrotic CA1 pyramidal cells after OGD, and there was no progression of calcification in OGD-lesioned slices. In addition, mineralized cells in both control and OGD-lesioned slices were closely associated with or completely engulfed by astrocytes but not microglia. These astrocytes were laden with heterogeneous cytoplasmic inclusions that appeared to be related with their phagocytic activity. These data demonstrate that microcalcification specifically associated with mitochondria might lead to a novel type of cell death and suggest that astrocytes may be involved in the phagocytosis of these mineralized cells and possibly in the regulation of ectopic calcification.

Spatiotemporal expression of osteopontin in the striatum of rats subjected to the mitochondrial toxin 3-nitropropionic acid correlates with microcalcification

Our aim was to elucidate whether osteopontin (OPN) is involved in the onset of mineralisation and progression of extracellular calcification in striatal lesions due to mitochondrial toxin 3-nitropropionic acid exposure. OPN expression had two different patterns when observed using light microscopy. It was either localised to the Golgi complex in brain macrophages or had a small granular pattern scattered in the affected striatum. OPN labelling tended to increase in number and size over a 2-week period following the lesion. Ultrastructural investigations revealed that OPN is initially localised to degenerating mitochondria within distal dendrites, which were then progressively surrounded by profuse OPN on days 7–14. Electron probe microanalysis of OPN-positive and calcium-fixated neurites indicated that OPN accumulates selectively on the surfaces of degenerating calcifying dendrites, possibly via interactions between OPN and calcium. In addition, 3-dimensional reconstruction of OPN-positive neurites revealed that they are in direct contact with larger OPN-negative degenerating dendrites rather than with fragmented cell debris. Our overall results indicate that OPN expression is likely to correlate with the spatiotemporal progression of calcification in the affected striatum, and raise the possibility that OPN may play an important role in the initiation and progression of microcalcification in response to brain insults.

Morphological characterization of NG2 glia and their association with neuroglial cells in the 3-nitropropionic acid–lesioned striatum of rat

Our aim was to examine the spatiotemporal profiles and phenotypic characteristics of neuron-glia antigen 2 (NG2) glia and their associations with neuroglial cells in striatal lesions due to the mitochondrial toxin 3-nitropropionic acid (3-NP). In control striatum, weak NG2 immunoreactivity was restricted to resting NG2 glia with thin processes, but prominent NG2 expression was noted on activated microglia/macrophages, and reactive NG2 glia in the lesion core after 3-NP injection. Activation of NG2 glia, including enhanced proliferation and morphological changes, had a close spatiotemporal relationship with infiltration of activated microglia into the lesion core. Thick and highly branched processes of reactive NG2 glia formed a cellular network in the astrocyte-free lesion core and primarily surrounded developing cavities 2–4 weeks post-lesion. NG2 glia became associated with astrocytes in the lesion core and the border of cavities over the chronic interval of 4–8 weeks. Immunoelectron microscopy indicated that reactive NG2 glia had large euchromatic nuclei with prominent nucleoli and thick and branched processes that ramified distally. Thus, our data provide detailed information regarding the morphologies of NG2 glia in the lesion core, and support the link between transformation of NG2 glia to the reactive form and microglial activation/recruitment in response to brain insults.

Spatiotemporal Progression of Microcalcification in the Hippocampal CA1 Region following Transient Forebrain Ischemia in Rats: An Ultrastructural Study.

Calcification in areas of neuronal degeneration is a common finding in several neuropathological disorders including ischemic insults. Here, we performed a detailed examination of the onset and spatiotemporal profile of calcification in the CA1 region of the hippocampus, where neuronal death has been observed after transient forebrain ischemia. Histopathological examinations showed very little alizarin red staining in the CA1 pyramidal cell layer until day 28 after reperfusion, while prominent alizarin red staining was detected in CA1 dendritic subfields, particularly in the stratum radiatum, by 14 days after reperfusion. Electron microscopy using the osmium/potassium dichromate method and electron probe microanalysis revealed selective calcium deposits within the mitochondria of degenerating dendrites at as early as 7 days after reperfusion, with subsequent complete mineralization occurring throughout the dendrites, which then coalesced to form larger mineral conglomerates with the adjacent calcifying neurites by 14 days after reperfusion. Large calcifying deposits were frequently observed at 28 days after reperfusion, when they were closely associated with or completely engulfed by astrocytes. In contrast, no prominent calcification was observed in the somata of CA1 pyramidal neurons showing the characteristic features of necrotic cell death after ischemia, although what appeared to be calcified mitochondria were noted in some degenerated neurons that became dark and condensed. Thus, our data indicate that intrahippocampal calcification after ischemic insults initially occurs within the mitochondria of degenerating dendrites, which leads to the extensive calcification that is associated with ischemic injuries. These findings suggest that in degenerating neurons, the calcified mitochondria in the dendrites, rather than in the somata, may serve as the nidus for further calcium precipitation in the ischemic hippocampus.