3D MR fingerprinting-derived myelin water fraction characterizing brain development and leukodystrophy.

BACKGROUND: Magnetic resonance fingerprinting (MRF) enables fast myelin quantification via the myelin water fraction (MWF), offering a noninvasive method to assess brain development and disease. However, MRF-derived MWF lacks histological evaluation and remains unexamined in relation to leukodystrophy. This study aimed to access MRF-derived MWF through histology in mice and establish links between myelin, development, and leukodystrophy in mice and children, demonstrating its potential applicability in animal and human studies. METHODS: 3D MRF was performed on normal C57BL/6 mice with different ages, megalencephalic leukoencephalopathy with subcortical cyst 1 wild type (MLC1 WT, control) mice, and MLC 1 knock-out (MLC1 KO, leukodystrophy) mice using a 3 T MRI. MWF values were analyzed from 3D MRF data, and histological myelin quantification was carried out using immunohistochemistry to anti-proteolipid protein (PLP) in the corpus callosum and cortex. The associations between 'MWF and PLP' and 'MWF and age' were evaluated in C57BL/6 mice. MWF values were compared between MLC1 WT and MLC1 KO mice. MWF of normal developing children were retrospectively collected and the association between MWF and age was assessed. RESULTS: In 35 C57BL/6 mice (age range; 3 weeks-48 weeks), MWF showed positive relations with PLP immunoreactivity in the corpus callosum (ß = 0.0006, P = 0.04) and cortex (ß = 0.0005, P = 0.006). In 12-week-old C57BL/6 mice MWF showed positive relations with PLP immunoreactivity (ß = 0.0009, P = 0.003, R2 = 0.54). MWF in the corpus callosum (ß = 0.0022, P < 0.001) and cortex (ß = 0.0010, P < 0.001) showed positive relations with age. Seven MLC1 WT and 9 MLC1 KO mice showed different MWF values in the corpus callous (P < 0.001) and cortex (P < 0.001). A total of 81 children (median age, 126 months; range, 0-199 months) were evaluated and their MWF values according to age showed the best fit for the third-order regression model (adjusted R2 range, 0.44-0.94, P < 0.001). CONCLUSION: MWF demonstrated associations with histologic myelin quantity, age, and the presence of leukodystrophy, underscoring the potential of 3D MRF-derived MWF as a rapid and noninvasive quantitative indicator of brain myelin content in both mice and humans.

Distribution of vascular endothelial growth factor receptor-3/Flt4 mRNA in adult rat central nervous system.

Vascular endothelial growth factor receptor (VEGFR)-3/Flt4 binds VEGF-C and VEGF-D with high affinity. It has been suggested to be involved in neurogenesis and adult neuronal function. However, little is known about the localization of VEGFR-3 in the adult central nervous system (CNS). The present study presents, to our knowledge, the first detailed mapping of VEGFR-3 mRNA expression in adult rat brain and spinal cord by using in situ hybridization and reverse transcription-polymerase chain reaction analysis (RT-PCR). Varying VEGFR-3 expression intensity was detected in functionally diverse nuclei, with the highest levels in the mitral cells of the olfactory bulb, piriform cortex, anterodorsal thalamic nucleus, several nuclei of the hypothalamus, and the brainstem cranial nerve nuclei. VEGFR-3 mRNA was abundantly expressed in the ventral motor neurons of the spinal cord and in some circumventricular organs such as the median eminence and the area postrema. Moreover, the locus coeruleus and some of the nuclei of the reticular formation showed moderate-to-high hybridization signals. VEGFR-3 expression appeared to be localized mostly within neurons, but weak labeling was also found in some astrocytes. In particular, VEGFR-3 was highly expressed in ependymal cells of the ventral third ventricle and the median eminence, which were co-labeled with vimentin but not with glial fibrillary acidic protein, suggesting that these cells are tanycytes. RT-PCR analysis revealed similar levels of VEGFR-3 expression in all regions of the adult rat CNS. The specific but widespread distribution of VEGFR-3 mRNA in the adult rat CNS suggests that VEGFR-3 functions more broadly than expected, regulating adult neuronal function playing important roles in tanycyte function.

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.

Expression of vascular endothelial growth factor receptor-3 mRNA in the developing rat cerebellum.

Vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, has recently been suggested to play an important role during neuronal development. To characterize its potential role in CNS ontogenesis, we investigated the spatiotemporal and cellular expression of VEGFR-3 in developing and mature rat cerebellum using in situ hybridization. VEGFR-3 expression appeared as early as E15, and was restricted to the ventricular zone of the cerebellar primordium, the germinative neuroepithelium, but was absent by E20. Instead, the expression area of VEGFR-3 in the cerebellum grew in parallel with cerebellar development. From E20 on, two populations of VEGFR-3-expressing cells can be clearly distinguished in the developing cerebellum: a population of differentiating and postmitotic neurons and the Bergmann glia. VEGFR-3 expression in neurons occurred during the period of neuronal differentiation, and increased with maturation. In particular, the expression of VEGFR-3 mRNA revealed different temporal patterns in different neuronal populations. Neurons generated early, Purkinje cells, and deep nuclear neurons expressed VEGFR-3 mRNA during late embryonic stages, whereas VEGFR-3 transcription in local interneurons appeared by P14 with weaker expression. In addition, Bergmann glia expressed VEGFR-3 throughout cerebellar maturation into adulthood. However, receptor expression was absent in the progenitors in the external granular layer and during further migration. The results of this study suggest that VEGFR-3 has even broader functions than previously thought, regulating both developmental processes and adult neuronal function in the cerebellum.

Induction of vascular endothelial growth factor receptor-3 mRNA in glial cells following focal cerebral ischemia in rats.

To identify whether vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, is involved in pathophysiology of stroke, we investigated the spatiotemporal regulation of VEGFR-3 mRNA after transient focal cerebral ischemia. Most of the increase in VEGFR-3 expression in the ischemic core could be attributed to brain macrophages, whereas VEGFR-3 in the peri-infarct penumbra region was predominantly expressed in reactive astrocytes. A subpopulation of VEGFR-3-expressing brain macrophages was positive for NG2 proteoglycan and showed proliferative activity. In addition, in vitro model of stroke revealed no significant induction of VEGFR-3 in activated microglial cells, indicating that infiltrating exogenous macrophages expressed VEGFR-3 after focal ischemia. These data suggest that VEGFR-3 may be involved in the glial reaction and possibly in the recruitment of monocytic macrophages during ischemic insults.

Enhanced expression of the sweet taste receptors and alpha-gustducin in reactive astrocytes of the rat hippocampus following ischemic injury.

The heterodimeric sweet taste receptors, T1R2 and T1R3, have recently been proposed to be associated with the brain glucose sensor. To identify whether sweet taste signaling is regulated in response to an ischemic injury inducing acute impairment of glucose metabolism, we investigated the spatiotemporal expression of the sweet taste receptors and their associated taste-specific G-protein α-gustducin in the rat hippocampus after ischemia. The expression profiles of both receptor subunits and α-gustducin shared overlapping expression patterns in sham-operated and ischemic hippocampi. Constitutive expression of both receptors and α-gustducin was localized in neurons of the pyramidal cell and granule cell layers, but their upregulation was detected in reactive astrocytes in ischemic hippocampi. Immunoblot analysis confirmed the immmunohistochemically determined temporal patterns of sweet-taste signaling proteins. These results suggest that the expression of sweet taste signaling proteins in astrocytes might be regulated in response to altered extracellular levels of glucose following an ischemic insult.

Spatial and temporal changes of osteopontin in oxygen-glucose-deprived hippocampal slice cultures.

We have examined the temporal changes and cellular localization of osteopontin (OPN) mRNA and protein in organotypic hippocampal slice cultures subjected to ischemia-like oxygen-glucose deprivation (OGD). The sequential induction pattern response consisted of neuronal and microglial OPN upregulation, followed by a later extended phase of expression in reactive astrocytes. OPN immunoreactivity after OGD matched the mRNA induction patterns. Activated microglia revealed OPN staining in focal deposits, whereas neurons and reactive astrocytes showed perinuclear staining with a punctate cytosolic pattern of OPN, typical of secreted proteins. These data demonstrated that the temporal and cellular patterns of OPN induction in reactive glial cells in this in vitro model closely correlated with that in the in vivo model, suggesting that OPN has a multifunctional role in the pathogenesis of ischemic injury.

Expression of vascular endothelial growth factor receptor-3 mRNA in the rat developing forebrain and retina.

Vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, is expressed in neural progenitor cells, but there has been no comprehensive study of its distribution in the developing brain. Here, the temporal and cell-specific expression of VEGFR-3 mRNA was studied in the developing rat forebrain and eye. Expression appeared along the ventricular and subventricular zones of the lateral and third ventricles showing ongoing neurogenesis as early as embryonic day 13 but was progressively down-regulated during development and remained in the subventricular zone and rostral migratory stream of the adult forebrain. VEGFR-3 expression was also detectable in some differentiating and postmitotic neurons in the developing cerebral cortex, including Cajal-Retzius cells, cortical plate neurons, and subplate neurons. Expression in the subplate increased significantly during the early postnatal period but was absent by postnatal day 14. It was also highly expressed in nonneural tissues of the eye during development, including the retinal pigment epithelium, the retinal ciliary margin, and the lens, but persisted in a subset of cells in the pigmented ciliary epithelium of the adult eye. In contrast, there was weak or undetectable expression in the early neural retina, but a subset of retinal neurons in the postnatal and mature retina showed intense signals. These unique spatiotemporal mRNA expression patterns suggest that VEGFR-3 might mediate the regulation of both neurogenesis and adult neuronal function in the rat forebrain and eye.

Enhanced expression of vascular endothelial growth factor receptor-3 in the subventricular zone of stroke-lesioned rats.

Vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, has recently been proposed to be involved in adult hippocampal neurogenesis in response to cerebral ischemia. To identify whether VEGFR-3 is involved in poststroke neurogenesis, we investigated the temporal regulation of VEGFR-3 mRNA expression in the subventricular zone (SVZ) of rats with transient focal cerebral ischemia by in situ hybridization analysis, and identified the phenotypes of cells expressing VEGFR-3 by double- and triple-labeling techniques. In sham-operated rats, hybridization signals for VEGFR-3 mRNA were evident at a weaker intensity in the SVZ of the lateral ventricle. VEGFR-3 was transiently increased in the dorsolateral SVZ of the infarcted hemisphere on days 3-7 after reperfusion. Almost all VEGFR-3-expressing cells in the ipsilateral SVZ were colabeled with glial fibrillary acidic protein and the neural progenitor marker nestin, and were highly proliferative. In addition, a subset of VEGFR-3-labeled cells in the ipsilateral SVZ expressed the immature neuronal marker, polysialic acid-neural cell adhesion molecule. These data indicate that VEGFR-3 is upregulated in SVZ astrocytes and immature neurons after focal ischemia, suggesting that VEGFR-3 might mediate the adult neurogenesis after ischemic stroke.

Enhanced expression of SOCS-2 in the rat hippocampus after transient forebrain ischemia.

Suppressor of cytokine signaling-2 (SOCS-2) has recently been identified as an important regulator involved in neuronal differentiation and maturation. However, the role of SOCS-2 in ischemia-induced hippocampal neurogenesis remains to be clarified. Here we investigated the spatiotemporal expression of SOCS-2 in the rat hippocampus following transient forebrain ischemia, and particular attention was paid to changes in the dentate gyrus. SOCS-2 mRNA was constitutively expressed in hippocampal neurons and astrocytes in control animals. However, its upregulation occurred specifically in reactive astrocytes in the hippocampus proper, in particular the CA1 and dentate hilar regions, at day 3 after reperfusion, and was sustained for more than 2 weeks. In addition to the CA1 and hilar regions, SOCS-2 was transiently increased in the subgranular zone (SGZ) of the dentate gyrus on days 3-7 after reperfusion. This correlated with the post-ischemic upregulation of SOCS-2 in the CA1 or dentate gyrus subfield, including the SGZ detected by semiquantitative reverse transcriptase-polymerase chain reaction analysis. The majority of the SOCS-2-expressing cells in the SGZ were co-labeled with glial fibrillary acidic protein (GFAP), and a subpopulation of GFAP/SOCS-2 double-labeled cells in the SGZ co-expressed the neural progenitor marker nestin, or the proliferation marker proliferating cellular nuclear antigen. In addition, a subset of SOCS-2-labeled cells in the SGZ expressed the immature neuronal marker polysialic acid-neural cell adhesion molecule. These data suggest that SOCS-2 may be involved in glial reactions, and possibly adult hippocampal neurogenesis during ischemic insults.

Transient expression of Bis protein in midline radial glia in developing rat brainstem and spinal cord.

Bis (Bcl-2 interacting death suppressor) has been reported to contribute to the differentiation and maturation of specific neuronal populations in the developing rat forebrain, in addition to its well-established functions as a stress or survival-related protein. In the present study, we have analyzed the expression of Bis in the rat brainstem and cervical spinal cord during development by using immunohistochemistry. Bis immunoreactivity was detected in radial glial cells flanking the midline from embryonic day 14. During embryonic and early postnatal development, Bis expression persisted in differentiating radial glia at the midline but disappeared first in the spinal cord by postnatal day 7 (P7) and later also in the brainstem by P14. Bis expression was restricted to a subpopulation of the midline radial glia, i.e., the dorsal midline of the midbrain and spinal cord and the ventral midline of the hindbrain, which were double- or triple-labeled with vimentin and nestin, markers for radial glia, and S100B. However, these markers also labeled all radial glia including the ventral midline glia in the midbrain and spinal cord, with Bis being absent from these structures. In addition, the dorsal midline glia in the midbrain and spinal cord expressed Bis prior to the timing of expression for radial glial markers. Therefore, our results demonstrate the early and transient expression of Bis in the subpopulation of midline glia in the developing brainstem and spinal cord, suggesting that Bis has a unique role in association with the radial glial cells in the developing central nervous system.

Differential regulation of vascular endothelial growth factor-C and its receptor in the rat hippocampus following transient forebrain ischemia.

We investigated the changes in the expression of vascular endothelial growth factor-C (VEGF-C) and its receptor, VEGFR-3, in the rat hippocampus following transient forebrain ischemia. The expression profiles of VEGF-C and VEGFR-3 were very similar in the control hippocampi, where both genes were constitutively expressed in neurons in the pyramidal cell and granule cell layers. The spatiotemporal expression pattern of VEGF-C was similar to that of VEGFR-3 in the ischemic hippocampus, and in the CA1 and dentate hilar regions both VEGF-C and VEGFR-3 were strongly expressed in activated glial cells rather than in neurons. Most of the activated glial cells expressing both genes were reactive astrocytes, although some were a subpopulation of brain macrophages. In the dentate gyrus, however, VEGFR-3 expression was transiently increased in the innermost layer of granule cells on days 7-10 after reperfusion, coinciding with an increase in polysialylated neural cell adhesion molecule staining--a marker for immature neurons. These data suggest that VEGF-C may be involved in glial reaction via paracrine or autocrine mechanisms in the ischemic brain and may carry out specific roles in adult hippocampal neurogenesis during ischemic insults.

Differential regulation of osteopontin receptors, CD44 and the alpha(v) and beta(3) integrin subunits, in the rat hippocampus following transient forebrain ischemia.

We have examined the spatiotemporal regulation of CD44 and the alpha(v)beta(3) integrin subunits, which have been identified as receptors for osteopontin (OPN), in the rat hippocampus following transient forebrain ischemia. Immunoreactivity for CD44 and the integrin subunits, alpha(v) and beta(3), showed characteristic time- and cell-dependent patterns in the ischemic hippocampus. CD44 immunoreactivity was induced at day 1 after reperfusion, reached a peak at day 3, and returned to basal levels by day 7. CD44 was induced in a subset of activated microglial cells within sites of intense neural damage, and the concomitant induction of OPN and CD44 was observed in the same cells in the ischemic hippocampus. In contrast, increased immunoreactivity for alpha(v) and beta(3), which shared overlapping expression patterns in the ischemic hippocampus, occurred in the majority of reactive astrocytes and only a few microglia at day 3 after reperfusion, and was sustained for more than 2 weeks. Immunoreactivity for both integrin subunits colocalized with OPN immunoreactivity in reactive astrocytes, and OPN immunoreactivity was also diffusely localized over the extracellular matrix around the reactive astrocytes. These data indicated that the rapid and transient induction of CD44 and OPN occurred in activated microglia/macrophages, whereas the long-lasting induction of alpha(v) and beta(3) integrin subunits and OPN occurred in reactive astrocytes, suggesting that the multifunctional role of OPN in the ischemic brain may be attributed, in part, to a time- and cell-dependent interaction with CD44 or integrin alpha(v)beta(3).

Induction of suppressor of cytokine signaling-3 in astrocytes of the rat hippocampus following transient forebrain ischemia.

We investigated the spatiotemporal expression of suppressor of cytokine signaling-3 (SOCS-3) in the rat hippocampus following transient forebrain ischemia using in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Messenger RNA for SOCS-3 was constitutively expressed in neurons of the pyramidal cell and granule cell layers in control animals; however, significant induction was detected in reactive astrocytes preferentially located in the CA1 and the dentate hilar regions of the ischemic hippocampus. SOCS-3 mRNA was induced within 3 days of ischemia and maintained for more than 2 weeks. The in situ hybridization data agreed with the semiquantitative RT-PCR analysis. These results demonstrate SOCS-3 induction occurs in reactive astrocytes of the post-ischemic hippocampus, suggesting that SOCS-3 is involved in regulating the astroglial reaction to an ischemic insult.

Expression of vascular endothelial growth factor receptors Flt-1 and Flk-1 in embryonic rat forebrain.

To define better the putative targets of vascular endothelial growth factor (VEGF) in the developing brain we have examined the ontogeny of the two VEGF tyrosine kinase receptors, Flt-1 and Flk-1, in embryonic rat forebrain. Semiquantitative reverse transcriptase-polymerase chain reaction and immunoblot analysis showed expression of both receptors in the forebrain at all embryonic ages studied. Messenger RNAs for Flt-1 and Flk-1 appeared along most of the ventricular zone of the lateral ventricle as early as embryonic day (E) 13. Messages gradually became restricted to a limited ventricular zone at E20. Expression of VEGF receptors was also observed in the cerebral cortex, hippocampus and thalamic nuclei. In the cortex, expression of mRNA for both receptors was detected in the cortical plate around E15, and became relatively weak and restricted to the deeper layers of the cortical plate at E20. These data suggest that VEGF may contribute to early developmental processes including the proliferation, differentiation and maturation of specific neuronal populations via specific VEGF receptors in the developing rat forebrain.

Auranofin blocks interleukin-6 signalling by inhibiting phosphorylation of JAK1 and STAT3.

Auranofin (AF) is a sulphur-containing gold compound. Because of its anti-inflammatory and immunosuppressive activities, AF has been widely used for the therapeutic treatment of rheumatoid arthritis. However, little is known about its mechanism of action. To elucidate the molecular mechanism underlying the anti-inflammatory effect of AF, we studied the effects of AF on cellular responses to interleukin-6 (IL-6). In HepG2 human hepatoma cells, AF markedly inhibited IL-6-induced phosphorylation of janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) and STAT3 translocation into the nucleus. Consistent with this, AF diminished IL-6-induced production of the acute-phase proteins, haptoglobin, fibrinogen, C3 complement and alpha(1)-acid glycoprotein, and gene expression of vascular endothelial growth factor, all of whose transcriptional activities are regulated by STAT3. The inhibitory activity of AF on STAT3 phosphorylation was also demonstrated in primary cells, i.e. fibroblast-like synoviocytes from rheumatoid arthritis patients, human umbilical vein endothelial cells and rat astrocytes. Auranofin-mediated inhibition of STAT3 phosphorylation was recovered by pretreatment with antioxidants containing thiol groups. These findings suggest that the anti-inflammatory action of AF is associated with a blockade of JAK1/STAT3 signalling. Thiol-group-reactive proteins may be involved in AF-induced suppression of JAK1/STAT3 phosphorylation.

Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 in rat hippocampus after transient forebrain ischemia.

This study characterizes the distribution of the two tyrosine kinase receptors for vascular endothelial growth factor (VEGF), Flt-1 and Flk-1, in the rat hippocampus following transient forebrain ischemia. The semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of Flt-1 and Flk-1 in hippocampal CA1 showed upregulation of these receptors following ischemic injury. Expression of Flt-1 and Flk-1 mRNA was restricted to neurons in the pyramidal cell and granule cell layers in control animals; however, upregulation was detected in activated glial cells and in the vascular endothelial cells rather than in neurons, in ischemic hippocampi. Most of the activated glial cells expressing Flt-1 and Flk-1 were reactive astrocytes, although some were microglial cells. The spatiotemporal expression of Flt-1 in the ischemic hippocampus mirrored that of Flk-1 expression. Expression of mRNA for both receptors was induced after 12 h, appeared to be increased progressively until 3 days when the highest expression was reached, and was sustained for more than 2 weeks. Flt-1 and Flk-1 immunoreactivity in the ischemic hippocampus matched the mRNA induction patterns except for a somewhat delayed onset. These data suggest that VEGF may be involved in the glial response via specific VEGF receptors in the rat hippocampus following transient forebrain ischemia.

Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 following acute spinal cord contusion in rats.

To investigate the possible role of vascular endothelial growth factor (VEGF) in the injured spinal cord, we analyzed the distribution and time course of the two tyrosine kinase receptors for VEGF, Flt-1 and Flk-1, in the rat spinal cord following contusion injury using a weight-drop impactor. The semi-quantitative RT-PCR analysis of Flt-1 and Flk-1 in the spinal cord showed slight upregulation of these receptors following spinal cord injury. Although mRNAs for Flt-1 and Flk-1 were constitutively expressed in neurons, vascular endothelial cells, and some astrocytes in laminectomy control rats, their upregulation was induced in association with microglia/macrophages and reactive astrocytes in the vicinity of the lesion within 1 day in rats with a contusion injury and persisted for at least 14 days. The spatiotemporal expression of Flt-1 in the contused spinal cord mirrored that of Flk-1 expression. In the early phase of spinal cord injury, upregulation of Flt-1 and Flk-1 mRNA occurred in microglia/macrophages that infiltrated the lesion. In addition, the expression of both receptors increased progressively in reactive astrocytes within the vicinity of the lesion, predominately in the white matter, and almost all reactive astrocytes coexpressed Flt-1 or Flk-1 and nestin. These results suggest that VEGF may be involved in the inflammatory response and the astroglial reaction to contusion injuries of the spinal cord via specific VEGF receptors.

Ischemic preconditioning upregulates expression of phospholipase D2 in the rat hippocampus.

To investigate the possible involvement of phospholipase D2 (PLD2) in the induction of ischemic tolerance, we analyzed the distribution and time course of PLD2 expression in the rat hippocampus after a sublethal period of ischemia. Forebrain ischemia was induced by four-vessel occlusion for 3 min. Increased PLD2 immunoreactivity after this sublethal ischemia was observed in CA1 pyramidal neurons of the rat hippocampus. In tolerance-acquired CA1 neurons, PLD2 immunoreactivity was upregulated as early as 12 h post-ischemia and was most prominent at 1-3 days, with expression sustained for at least 7 days, as shown by a time course of immunoblotting and measurement of the enzymatic activity of PLD. PLD2 expression was also increased in ischemia-resistant CA3 neurons and dentate granule cells, although weaker staining intensity was noted. Further, we showed that, in cultured SK-N-BE(2)C human neuroblastoma cells, overexpression of PLD2 inhibited cell death by chemical hypoxia induced with potassium cyanide and deoxyglucose. These data suggest that upregulation of PLD2 might be involved in the neuroprotective mechanism of ischemic tolerance in the rat hippocampus.

Transient microglial and prolonged astroglial upregulation of osteopontin following transient forebrain ischemia in rats.

Osteopontin (OPN) is an adhesive glycoprotein linked to a variety of pathophysiological processes, with neuroprotective properties in ischemic injury. We examined the postischemic expression and localization of OPN in the rat brain after transient forebrain ischemia. The semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that OPN expression in the hippocampal CA1 region was biphasic, peaking at day 3 after reperfusion and again between days 14 and 28. The two phases of OPN induction occurred in a time- and cell-dependent manner in the ischemic hippocampus. OPN mRNA expression in activated microglia was first induced 1 day after reperfusion, reached a peak at 3 days, and returned to basal levels by 7 days. In contrast, OPN expression in reactive astrocytes was first induced by 10 days after reperfusion in the hippocampal CA1. Astroglial OPN expression further increased, reaching a peak at day 14 and was maintained up to day 28, the latest time point we examined. OPN immunoreactivity in the ischemic hippocampus matched the mRNA induction patterns. OPN protein was first localized in the astroglial cytoplasm and later in the extracellular matrix of the hippocampal CA1. The temporal and cellular patterns of OPN induction in the ischemic hippocampus suggest a multifunctional capacity in the pathogenesis of ischemic injury, with the increased OPN production and secretion by reactive astrocytes being involved in subsequent tissue repair and reorganization.