Astrocytes are involved in the formation of corpora amylacea-like structures from neuronal debris in the CA1 region of the rat hippocampus after ischemia.

Recently, we demonstrated that the corpora amylacea (CA), a glycoprotein-rich aggregate frequently found in aged brains, accumulates in the ischemic hippocampus and that osteopontin (OPN) mediates the entire process of CA formation. Therefore, this study aimed to elucidate the mechanisms by which astrocytes and microglia participate in CA formation during the late phase (4-12 weeks) of brain ischemia. Based on various morphological analyses, including immunohistochemistry, in situ hybridization, immunoelectron microscopy, and correlative light and electron microscopy, we propose that astrocytes are the primary cells responsible for CA formation after ischemia. During the subacute phase after ischemia, astrocytes, rather than microglia, express Opn messenger ribonucleic acid and OPN protein, a surrogate marker and key component of CA. Furthermore, the specific localization of OPN in the Golgi complex suggests that it is synthesized and secreted by astrocytes. Astrocytes were in close proximity to type I OPN deposits, which accumulated in the mitochondria of degenerating neurons before fully forming the CA (type III OPN deposits). Throughout CA formation, astrocytes remained closely attached to OPN deposits, with their processes exhibiting well-developed gap junctions. Astrocytic cytoplasmic protein S100ß, a calcium-binding protein, was detected within the fully formed CA. Additionally, ultrastructural analysis revealed direct contact between astroglial fibrils and the forming facets of the CA. Overall, we demonstrated that astrocytes play a central role in mediating CA formation from the initial stages of OPN deposit accumulation to the evolution of fully formed CA following transient ischemia in the hippocampus.

Osteopontin mediates the formation of corpora amylacea-like structures from degenerating neurons in the CA1 region of the rat hippocampus after ischemia.

We previously demonstrated that osteopontin (OPN) is closely associated with calcium precipitation in response to ischemic brain insults. The present study was designed to elucidate the possible association between deposition of OPN and progressive neurodegeneration in the ischemic hippocampus. To address this, we analyzed the OPN deposits in the rat hippocampus after global cerebral ischemia in the chronic phase (4 to 12 weeks) after reperfusion using immunoelectron microscopy and correlative light and electron microscopy. We identified three different types of OPN deposits based on their morphological characteristics, numbered according to the order in which they evolved. Dark degenerative cells that retained cellular morphology were frequently observed in the pyramidal cell layer, and type I OPN deposits were degenerative mitochondria that accumulated among these cells. Type II deposits evolved into more complex amorphous structures with prominent OPN deposits within their periphery and within degenerative mitochondria-like structures. Finally, type III had large concentric laminated structures with irregularly shaped bodies in the center of the deposits. In all types, OPN expression was closely correlated with calcification, as confirmed by calcium fixation and Alizarin Red staining. Notably, type II and III deposits were highly reminiscent of corpora amylacea, glycoprotein-rich aggregates found in aged brains, or neurodegenerative disease, which was further confirmed by ubiquitin expression and periodic acid-Schiff staining. Overall, our data provide a novel link between ongoing neurodegeneration and the formation of corpora amylacea-like structures and calcium deposits in the ischemic hippocampus, suggesting that OPN may play an important role in such processes.

Infiltration of meningeal macrophages into the Virchow-Robin space after ischemic stroke in rats: Correlation with activated PDGFR-ß-positive adventitial fibroblasts.

Macrophages play a crucial role in wound healing and fibrosis progression after brain injury. However, a detailed analysis of their initial infiltration and interaction with fibroblasts is yet to be conducted. This study aimed to investigate the possible route for migration of meningeal macrophages into the ischemic brain and whether these macrophages closely interact with neighboring platelet-derived growth factor beta receptor (PDGFR-ß)-positive adventitial fibroblasts during this process. A rat model of ischemic stroke induced by middle cerebral artery occlusion (MCAO) was developed. In sham-operated rats, CD206-positive meningeal macrophages were confined to the leptomeninges and the perivascular spaces, and they were not found in the cortical parenchyma. In MCAO rats, the number of CD206-positive meningeal macrophages increased both at the leptomeninges and along the vessels penetrating the cortex 1 day after reperfusion and increased progressively in the extravascular area of the cortical parenchyma by 3 days. Immunoelectron microscopy and correlative light and electron microscopy showed that in the ischemic brain, macrophages were frequently located in the Virchow-Robin space around the penetrating arterioles and ascending venules at the pial surface. This was identified by cells expressing PDGFR-ß, a novel biomarker of leptomeningeal cells. Macrophages within penetrating vessels were localized in the perivascular space between smooth muscle cells and PDGFR-ß-positive adventitial fibroblasts. In addition, these PDGFR-ß-positive fibroblasts showed morphological and molecular characteristics similar to those of leptomeningeal cells: they had large euchromatic nuclei with prominent nucleoli and well-developed rough endoplasmic reticulum; expressed nestin, vimentin, and type I collagen; and were frequently surrounded by collagen fibrils, indicating active collagen synthesis. In conclusion, the perivascular Virchow-Robin space surrounding the penetrating vessels could be an entry route of meningeal macrophages from the subarachnoid space into the ischemic cortical parenchyma, implying that activated PDGFR-ß-positive adventitial fibroblasts could be involved in this process.

Temporal dynamics of cells expressing NG2 and platelet-derived growth factor receptor-ß in the fibrotic scar formation after 3-nitropropionic acid-induced acute brain injury.

Neuron-glia antigen 2 (NG2) proteoglycan and platelet-derived growth factor receptor beta (PDGFR-ß) are widely used markers of pericytes, which are considered cells that form fibrotic scars in response to central nervous system insults. However, the exact phenotypes of NG2- and PDGFR-ß-expressing cells, as well as the origin of the fibrotic scar after central nervous system insults, are still elusive. In the present study, we directly examined the identities and distributions of NG2- and PDGFR-ß-positive cells in the control and lesioned striatum injured by the mitochondrial toxin 3-nitropropionic acid. Immunoelectron microscopy and correlative light and electron microscopy clearly distinguished NG2 and PDGFR-ß expression in the vasculature during the post-injury period. Vascular smooth muscle cells and pericytes expressed NG2, which was prominently increased after the injury. NG2 expression was restricted to these vascular mural cells until 14 days post-lesion. By contrast, PDGFR-ß-positive cells were perivascular fibroblasts located abluminal to smooth muscle cells or pericytes. These PDGFR-ß-expressing cells formed extravascular networks associated with collagen fibrils at 14 days post-lesion. We also found that in the injured striatal parenchyma, PDGFR-ß could be used as a complementary marker of resting and reactive NG2 glia because activated microglia/macrophages shared only the NG2 expression with NG2 glia in the lesioned striatum. These data indicate that NG2 and PDGFR-ß label different vascular mural and parenchymal cells in the healthy and injured brain, suggesting that fibrotic scar-forming cells most likely originate in PDGFR-ß-positive perivascular fibroblasts rather than in NG2-positive pericytes.

Cellular and subcellular localization of endogenous phospholipase D6 in seminiferous tubules of mouse testes.

Phospholipase D6 (PLD6) plays pivotal roles in mitochondrial dynamics and spermatogenesis, but the cellular and subcellular localization of endogenous PLD6 in testis germ cells is poorly defined. We examined the distribution and subcellular localization of PLD6 in mouse testes using validated specific anti-PLD6 antibodies. Ectopically expressed PLD6 protein was detected in the mitochondria of PLD6-transfected cells, but endogenous PLD6 expression in mouse testes was localized to the perinuclear region of pachytene spermatocytes, and more prominently, to the round (Golgi and cap phases) and elongating spermatids (acrosomal phase); these results suggest that PLD6 is localized to the Golgi apparatus. The distribution of PLD6 in the round spermatids partially overlapped with that of the cis-Golgi marker GM130, indicating that the PLD6 expression corresponded to the GM130-positive subdomains of the Golgi apparatus. Correlative light and electron microscopy revealed that PLD6 expression in developing spermatids was localized almost exclusively to several flattened cisternae, and these structures might correspond to the medial Golgi subcompartment; neither the trans-Golgi networks nor the developing acrosomal system expressed PLD6. Further, we observed that PLD6 interacted with tesmin, a testis-specific transcript necessary for successful spermatogenesis in mouse testes. To our knowledge, these results provide the first evidence of PLD6 as a Golgi-localized protein of pachytene spermatocytes and developing spermatids and suggest that its subcompartment-specific distribution within the Golgi apparatus may be related to the specific functions of this organelle during spermatogenesis.

Induction of BIS Protein During Astroglial and Fibrotic Scar Formation After Mitochondrial Toxin-Mediated Neuronal Injury in Rats.

B cell leukemia/lymphoma-2 (Bcl-2)-interacting death suppressor (BIS), also identified as Bcl-2-associated athanogene 3 (BAG3), has been reported to be upregulated in reactive astrocytes after brain insults. The present study was designed to further substantiate the involvement of BIS protein in the astroglial reaction in the striatum of rats treated with the mitochondrial toxin, 3-nitropropionic acid. Weak constitutive immunoreactivity for BIS was observed in astrocytes in the control striatum, whereas its expression was upregulated, along with that of nestin, in the lesioned striatum. In the lesion core, where astrocytes are virtually absent, BIS/nestin double-labeled cells were associated with the vasculature and were identified as perivascular adventitial fibroblasts. By contrast, BIS/nestin double-labeled cells in the perilesional area were reactive astrocytes, which were confined to the border zone contributing to the formation of the astroglial scar; this was evident 3 days post-lesion and increased thereafter progressively throughout the 28-day experimental period. At the ultrastructural level, BIS protein was diffusely localized throughout the cytoplasm within the stained cells. Collectively, our results demonstrate the phenotypic and functional heterogeneity of BIS-positive cells in the lesioned striatum, suggesting the involvement of BIS in the formation of astroglial scar and its potential role in the development of fibrotic scar after brain insults.

Spatiotemporal Profile and Morphological Changes of NG2 Glia in the CA1 Region of the Rat Hippocampus after Transient Forebrain Ischemia.

Neuron-glial antigen-2 (NG2) glia undergo proliferation and morphological changes following brain insults. Here, we show that NG2 glia is activated in a characteristic time- and layer-specific manner in the ischemia-vulnerable CA1 region of the rat hippocampus. Resting NG2 glia of the pyramidal cell layer (somatic region) shared morphological features with those of the neighboring dendritic stratum radiatum. During the postischemic period, reactive NG2 glia of the pyramidal cell layer exhibited shortened, scarcely branched processes, while those of the stratum radiatum had multiple branching processes with their arborization being almost indiscernible 7~14 days after reperfusion. Immunoelectron microscopy demonstrated that NG2 immunoreactivity was specifically associated with the plasma membrane and the adjacent extracellular matrix of NG2 glia in the stratum radiatum at 14 days. NG2 glia also exhibited differences in their numbers and proliferation profiles in the two examined hippocampal strata after ischemia. In addition, induced NG2 expression in activated microglia/macrophages exhibited a characteristic strata-dependent pattern in the ischemic CA1 hippocampus. NG2 induction was prominent in macrophage-like phenotypes which were predominantly localized in the pyramidal cell layer, compared with activated stellate microglial cells in the stratum radiatum. Thus, our data demonstrate that activation of NG2 glia and the induction of NG2 expression in activated microglia/macrophages occur in a distinct time- and layer-specific manner in the ischemic CA1 hippocampus. These characteristic profiles of reactive NG2 glia could be secondary to the degeneration processes occurring in the cell bodies or dendritic domains of hippocampal CA1 pyramidal neurons after ischemic insults.

Ultrastructural and Molecular Characterization of Platelet-derived growth factor Beta-Positive Leptomeningeal Cells in the Adult Rat Brain.

The leptomeninges, referring to the arachnoid and pia mater and their projections into the perivascular compartments in the central nervous system, actively participate in diverse biological processes including fluid homeostasis, immune cell infiltrations, and neurogenesis, yet their detailed cellular and molecular identities remain elusive. This study aimed to characterize platelet-derived growth factor beta (PDGFR-ß)-expressing cells in the leptomeninges in the adult rat brain using light and electron microscopy. PDGFR-ß+ cells were observed in the inner arachnoid, arachnoid trabeculae, pia mater, and leptomeningeal sheath of the subarachnoid vessels, thereby forming a cellular network throughout the leptomeninges. Leptomeningeal PDGFR-ß+ cells were commonly characterized by large euchromatic nuclei, thin branching processes forming web-like network, and the expression of the intermediate filaments nestin and vimentin. These cells were typical of active fibroblasts with a well-developed rough endoplasmic reticulum and close spatial correlation with collagen fibrils. Leptomeningeal PDGFR-ß+ cells ensheathing the vasculature in the subarachnoid space joined with pial PDGFR-ß+ cells upon entering the cortical parenchyma, yet perivascular PDGFR-ß+ cells in these penetrating vessels underwent abrupt changes in their morphological and molecular characteristics: they became more flattened with loss of immunoreactivity for nestin and vimentin and deficient collagen deposition, which was indicative of inactive fibroblasts termed fibrocytes. In the cortical parenchyma, PDGFR-ß immunoreactivity was almost exclusively localized to larger caliber vessels, and significantly decreased in capillary-like microvessels. Collectively, our data identify PDGFR-ß as a novel cellular marker for leptomeningeal fibroblasts comprising the leptomeninges and perivascular adventitial cells of the subarachnoid and penetrating large-sized cortical vasculatures.

14-3-3ζ targeting induced senescence in Hep-2 laryngeal cancer cell through deneddylation of Cullin1 in the Skp1-Cullin-F-box protein complex.

OBJECTIVES: Despite of the aberrant expression of 14-3-3ζ in head and neck squamous cell carcinoma (HNSCC), little is known about the role of 14-3-3ζ in the regulation of senescence in HNSCC. This study was performed to investigate whether 14-3-3ζ is implicated in senescence evasion of Hep-2 laryngeal cancer cells. METHODS: The expression of 14-3-3ζ was suppressed using RNA interference strategy. Senescence induction was determined by senescence-associated ß-galactosidase staining and the numbers of promyelocytic leukaemia nuclear body. Real-time PCR, western blotting and immunohistochemistry were applied for the expression of corresponding proteins. Xenograft experiment was performed to show in vivo effect of 14-3-3ζ silencing on tumour growth. RESULTS: 14-3-3ζ silencing significantly induced senescence phenotypes via 27 accumulations. Subsequently, we demonstrated that p27 accumulation is linked to inactivation of SCFSkp2 complex activity, probably due to the deneddylation of cullin-1 (Cul-1) as follows. (a) Neddylated Cul-1 is decreased by 14-3-3ζ silencing. (b) Blocking neddylation using MLN4924 reproduces senescence phenotypes. (c) Knockdown of CSN5, which functions as a deneddylase, was shown to restore the senescence phenotypes induced by 14-3-3ζ depletion. Finally, we demonstrated that 14-3-3ζ depletion effectively hindered the proliferation of Hep-2 cells implanted into nude mice. CONCLUSION: 14-3-3ζ negatively regulates senescence in Hep-2 cells, suggesting that 14-3-3ζ targeting may serve to suppress the expansion of laryngeal cancer via induction of senescence through the Cul-1/SCFSkp2 /p27 axis.

Osteopontin and its spatiotemporal relationship with glial cells in the striatum of rats treated with mitochondrial toxin 3-nitropropionic acid: possible involvement in phagocytosis.

BACKGROUND: Osteopontin (OPN, SPP1) is upregulated in response to acute brain injury, and based on its immunoreactivity, two distinct forms have been identified: intracellular OPN within brain macrophages and small granular OPN, identified as OPN-coated degenerated neurites. This study investigates the spatiotemporal relationship between punctate OPN deposition and astroglial and microglial reactions elicited by 3-nitropropionic acid (3-NP). METHODS: Male Sprague-Dawley rats were intraperitoneally injected with mitochondrial toxin 3-NP and euthanized at 3, 7, 14, and 28 days. Quantitative and qualitative light and electron microscopic techniques were used to assess the relationship between OPN and glial cells. Statistical significance was determined by Student's t test or a one-way analysis of variance followed by Tukey's multiple comparisons test. RESULTS: Punctate OPN-immunoreactive profiles were synthesized and secreted by amoeboid-like brain macrophages in the lesion core, but not by reactive astrocytes and activated microglia with a stellate shape in the peri-lesional area. Punctate OPN accumulation was detected only in the lesion core away from reactive astrocytes in the peri-lesional area at day 3, but had direct contact with, and even overlapped with astroglial processes at day 7. The distance between the OPN-positive area and the astrocytic scar significantly decreased from days 3 to 7. By days 14 and 28 post-lesion, when the glial scar was fully formed, punctate OPN distribution mostly overlapped with the astrocytic scar. Three-dimensional reconstructions and quantitative image analysis revealed numerous granular OPN puncta inside the cytoplasm of reactive astrocytes and brain macrophages. Reactive astrocytes showed prominent expression of the lysosomal marker lysosomal-associated membrane protein 1, and ultrastructural analysis confirmed OPN-coated degenerating neurites inside astrocytes, suggesting the phagocytosis of OPN puncta by reactive astrocytes after injury. CONCLUSIONS: Punctate OPN-immunoreactive profiles corresponded to OPN-coated degenerated neurites, which were closely associated with, or completely engulfed by, the reactive astrocytes forming the astroglial scar in 3-NP lesioned striatum, suggesting that OPN may cause astrocytes to migrate towards these degenerated neurites in the lesion core to establish physical contact with, and possibly, to phagocytose them. Our results provide novel insights essential to understanding the recovery and repair of the central nervous system tissue.

Spatiotemporal Expression of GRP78 in the Blood Vessels of Rats Treated With 3-Nitropropionic Acid Correlates With Blood-Brain Barrier Disruption.

Glucose-regulated protein (GRP78) or BiP, a 78-kDa chaperone protein located in the endoplasmic reticulum (ER), has recently been reported to be involved in the neuroglial response to ischemia-induced ER stress. The present study was designed to study the expression patterns of this protein and the cell types involved in the induction of GRP78 expression in rats treated with the mitochondrial toxin 3-nitropropionic acid (3-NP). GRP78 immunoreactivity was almost exclusively localized to striatal neurons in saline-treated controls, but GRP78 expression was induced in activated glial cells, including reactive astrocytes and activated microglia/macrophages, in the striata of rats treated with 3-NP. In the lesion core, increased GRP78 immunoreactivity was observed in the vasculature; this was evident in the lesion periphery of the core at 3 days after lesion induction, and was evenly distributed throughout the lesion core by 7 days after lesion induction. Vascular GRP78 expression was correlated, both temporally and spatially, with infiltration of activated microglia into the lesion core. In addition, this was coincident with the time and pattern of blood-brain barrier (BBB) leakage, detected by the extravasation of fluorescein isothiocyanate-albumin, an established BBB permeability marker. Vascular GRP78-positive cells in the lesion core were identified as endothelial cells, smooth muscle cells, and adventitial fibroblast-like cells, in which GRP78 protein was specifically localized to the cisternae of the rough ER and perinuclear cisternae, but not to other organelles such as mitochondria or nuclei. Thus, our data provide novel insights into the phenotypic and functional heterogeneity of GRP78-positive cells within the lesion core, suggesting the involvement of GRP78 in the activation/recruitment of activated microglia/macrophages and its potential role in BBB impairment in response to a 3-NP-mediated neurotoxic insult.

PDGFR-ß-Positive Perivascular Adventitial Cells Expressing Nestin Contribute to Fibrotic Scar Formation in the Striatum of 3-NP Intoxicated Rats.

Perivascular cells expressing platelet-derived growth factor receptor beta (PDGFR-ß) have recently been implicated in fibrotic scar formation after acute brain injury, but their precise identity and detailed morphological characteristics remain elusive. This study sought to characterize and define the cellular phenotype of vascular-associated cells expressing PDGFR-ß in the striatum of rats treated with the mitochondrial toxin 3-nitropropionic acid (3-NP). In the control striatum, PDGFR-ß-positive cells were invariably localized on the abluminal side of smooth muscle cells of larger caliber vessels, and demonstrated morphological features typical of perivascular fibroblasts. PDGFR-ß expression increased and expanded to almost all vessels, including microvessels in the lesion core, at 7 days after 3-NP injection. The cells expressing PDGFR-ß had ultrastructural features of fibroblasts undergoing active collagen synthesis: large euchromatic nuclei with a prominent nucleolus, well-developed rough endoplasmic reticulum (rER) with dilated cisterns and extracellular collagen fibrils. By 14 days, PDGFR-ß-positive cells had somata located at a distance from the vasculature, and their highly ramified, slender processes overlapped with those from other cells, thus forming a plexus of processes in the extravascular space of the lesion core. In addition, their ultrastructural morphology and spatial correlation with activated microglia/macrophages were elaborated by three-dimensional reconstruction. Using a correlative light- and electron-microscopy technique, we found that the intermediate filament proteins nestin and vimentin were induced in PDGFRß-positive fibroblasts in the lesion core. Collectively, our data suggest that perivascular PDGFR-ß-positive fibroblasts are distinct from other vascular cell types, including pericytes and contribute to fibrotic scar formation in the lesion core after acute brain injury. Nestin and vimentin play critical roles in the structural dynamics of these reactive fibroblasts.

Cellular and Subcellular Localization of Endoplasmic Reticulum Chaperone GRP78 Following Transient Focal Cerebral Ischemia in Rats.

The 78-kDa glucose-regulated protein (GRP78), a chaperone protein located in the endoplasmic reticulum (ER), has been reported to have neuroprotective effects in the injured central nervous system. Our aim was to examine the expression profiles and subcellular distributions of GRP78 and its association with the neuroglial reaction in the rat striatum after transient, focal cerebral ischemia. In sham-operated rats, constitutive, specific immunoreactivity for GRP78 was almost exclusively localized to the rough ER of striatal neurons, with none in the resting, ramified microglia or astrocytes. At 1 day post reperfusion, increased expression was observed in ischemia-resistant cholinergic interneurons, when most striatal neurons had lost GRP78 expression (this occurred earlier than the loss of other neuronal markers). By 3 days post reperfusion, GRP78 expression had re-emerged in association with the activation of glial cells in both infarct and peri-infarct areas but showed different patterns in the two regions. Most of the expression induced in the infarct area could be attributed to brain macrophages, while expression in the peri-infarct area predominantly occurred in neurons and reactive astrocytes. A gradual, sustained induction of GRP78 immunoreactivity occurred in reactive astrocytes localized to the astroglial scar, lasting for at least 28 days post reperfusion. Using correlative light- and electron-microscopy, we found conspicuous GRP78 protein localized to abnormally prominent, dilated rough ER in both glial cell types. Thus, our data indicate a link between GRP78 expression and the activated functional status of neuroglial cells, predominantly microglia/macrophages and astrocytes, occurring in response to ischemia-induced ER stress.

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.

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.

Progressive accumulation of autofluorescent granules in macrophages in rat striatum after systemic 3-nitropropionic acid: a correlative light- and electron-microscopic study.

A variety of tissue biomolecules and intracellular structures are known to be autofluorescent. However, autofluorescent signals in brain tissues often confound analysis of the fluorescent markers used for immunohistochemistry. While investigating tissue and cellular pathologies induced by 3-nitropropionic acid, a mitochondrial toxin selective for striatal neurons, we encountered many autofluorescent signals confined to the lesion core. These structures were excited by blue (wavelength = 488 nm) and yellow-orange (555 nm), but not by red (639 nm) or violet (405 nm) lasers, indicating that this autofluorescence overlaps with the emission spectra of commonly used fluorophores. Almost all of the autofluorescence was localized in activated microglia/macrophages, while reactive astrocytes emitted no detectable autofluorescence. Amoeboid brain macrophages filled with autofluorescent granules revealed very weak expression of the microglial marker, ionized calcium-binding adaptor molecule 1 (Iba1), while activated microglia with evident processes and intense Iba1 immunoreactivity contained scant autofluorescent granules. In addition, immunolabeling with two lysosomal markers, ED1/CD68 and lysosomal-associated membrane protein 1, showed a pattern complementary with autofluorescent signals in activated microglia/macrophages, implying that the autofluorescent structures reside within cytoplasm free of intact lysosomes. A correlative light- and electron-microscopic approach finally revealed the ultrastructural identity of the fluorescent granules, most of which matched to clusters of lipofuscin-like inclusions with varying morphology. Thus, autofluorescence in the damaged brain may reflect the presence of lipofuscin-laden brain macrophages, which should be taken into account when verifying any fluorescent signals that are likely to be correlated with activated microglia/macrophages after brain insults.

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.

Increased Expression of Slit2 and its Robo Receptors During Astroglial Scar Formation After Transient Focal Cerebral Ischemia in Rats.

Slit2, a secreted glycoprotein, has recently been implicated in the post-ischemic astroglial reaction. The objective of this study was to investigate the temporal changes and cellular localization of Slit2 and its receptors, Robo1, Robo2, and Robo4, in a rat transient focal ischemia model induced by middle cerebral artery occlusion. We used double- and triple-immunolabeling to determine the cell-specific changes in Slit2 and its receptors during a 10-week post-ischemia period. The expression profiles of Slit2 and the Robo receptors shared overlapping expression patterns in sham-operated and ischemic striatum. Constitutive expression of Slit2 and Robo receptors was observed in striatal neurons with weak intensity, whereas in rats reperfused after ischemic insults, these immunoreactivities were increased in reactive astrocytes. Astroglial induction of Slit2 and Robo in the peri-infarct region was distinct on days 7-14 after reperfusion and thereafter increased progressively throughout the 10-week experimental period. Slit2 and Robo were prominently expressed in the perinuclear cytoplasm and main processes of reactive astrocytes forming the astroglial scar. This observation was confirmed by quantification of the mean fluorescence intensity of Slit2 and Robo receptors over reactive astrocytes localized at the edge of the infarct area. However, activated microglia/macrophages in the peri-infarct area were devoid of any specific labeling for Slit2 and Robo. Thus, our data revealed a selective and sustained induction of Slit2 and Robo in astrocytes localized throughout the astroglial scar after ischemic stroke, suggesting that Slit2/Robo signaling participates in glial scar formation and brain remodeling following ischemic injury.

Increased expression of suppressor of cytokine signaling 2 in the subventricular zone after transient focal cerebral ischemia in adult rats.

Suppressor of cytokine signaling 2 (SOCS2) is a well-established negative regulator of growth hormone signaling that acts on adult hippocampal neurogenesis during ischemic insults. To explore whether SCOS2 is involved in poststroke neurogenesis, we studied the temporal expression of SOCS2 mRNA in the subventricular zone (SVZ) of rats after transient focal cerebral ischemia. We found that SOCS2 expression was upregulated in the SVZ of the infarcted hemisphere. The number of SOCS2-expressing cells was significantly increased in the ipsilateral SVZ compared with that on the contralateral side on days 7-10 after reperfusion, and SOCS2-expressing cells were highly proliferative, coinciding both spatially and temporally with stroke-induced neurogenesis. Almost all SOCS2-expressing cells in the SVZ were colabeled with the neural stem cell markers nestin and musashi1 and the neural/glial progenitor transcription factor Sox-2. In addition, SOCS2 was highly expressed in newly generated neurons that were immunoreactive for polysialic acid-neural cell adhesion molecule, indicating that SOCS2 expression may be persistent during neuronal differentiation. Thus, our data demonstrated that SOCS2 mRNA was highly expressed in proliferating neural stem/precursor cells and postmitotic migratory neuroblasts in the SVZ niche after focal cerebral ischemia, suggesting that SOCS2 may be actively involved in regulating adult neurogenesis induced by ischemic stroke.

Enhanced expression of the calcium-sensing receptor in reactive astrocytes following ischemic injury in vivo and in vitro.

We recently demonstrated that the G protein-coupled calcium-sensing receptor (CaSR) is associated with the pathogenesis of ischemic stroke and may be involved in vascular remodeling and astrogliosis. To further substantiate the involvement of CaSR in the astroglial reaction common to ischemic insults, we investigated the temporal and cell type-specific expression patterns of CaSR in the hippocampus after transient forebrain ischemia. CaSR was constitutively expressed in neurons of the pyramidal and granule cell layers, whereas increased CaSR immunoreactivity was observed in reactive astrocytes, but not in activated microglia or macrophages, in the CA1 region of the post-ischemic hippocampus. Astroglial induction of CaSR expression was evident on days 3-7 after reperfusion and appeared to increase progressively through day 28, at which time CaSR expression was prominent in astrocytes with a highly reactive hypertrophic phenotype and elevated levels of glial fibrillary acidic protein. This expression pattern was supported by results of immunoblot analyses. Furthermore, CaSR expression was upregulated in rat primary cortical astrocytes exposed to oxygen-glucose deprivation, which undergo reactive gliosis-like changes. Thus, our results demonstrate that selective and long-lasting astroglial induction of CaSR expression is a common characteristic of ischemic injury and suggest its involvement in the ischemia-induced astroglial reaction.