Julie A. Ellison

IL-10 reduces rat brain injury following focal stroke

The effects of the anti-inflammatory cytokine, IL-10, on brain injury following permanent focal ischemia were determined. Rats subjected to occlusion of the right middle cerebral artery (MCAO) were administered IL-10 (1 microg) centrally into the lateral ventricle 30 min and 3 h post MCAO or systemically into the tail vein (5 or 15 microg/h) starting 30 min post MCAO for 3 h. Brains were removed 24 h later and infarct size was measured. IL-10 administered centrally significantly (P < 0.01) reduced infarct size by 20.7% +/- 6.0 compared to vehicle. Systemic IL-10 administration at 5 and 15 microg/h significantly (P < 0.05) decreased infarct size (40.3% +/- 14.0 and 30.7% +/- 13.7, respectively). These studies indicate that an anti-inflammatory therapeutic approach using IL-10 can provide neuroprotection in ischemic stroke.

Caspase-8 and Caspase-3 Are Expressed by Different Populations of Cortical Neurons Undergoing Delayed Cell Death after Focal Stroke in the Rat

A number of studies have provided evidence that neuronal cell loss after stroke involves programmed cell death or apoptosis. In particular, recent biochemical and immunohistochemical studies have demonstrated the expression and activation of intracellular proteases, notably caspase-3, which act as both initiators and executors of the apoptotic process. To further elucidate the involvement of caspases in neuronal cell death induced by focal stroke we developed a panel of antibodies and investigated the spatial and temporal pattern of both caspase-8 and caspase-3 expression. Our efforts focused on caspase-8 because its “apical” position within the enzymatic cascade of caspases makes it a potentially important therapeutic target. Constitutive expression of procaspase-8 was detectable in most cortical neurons, and proteolytic processing yielding the active form of caspase-8 was found as early as 6 hr after focal stroke induced in rats by permanent middle cerebral artery occlusion. This active form of caspase-8 was predominantly seen in the large pyramidal neurons of lamina V. Active caspase-3 was evident only in neurons located within lamina II/III starting at 24 hr after injury and in microglia throughout the core infarct at all times examined. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, gel electrophoresis of DNA, and neuronal cell quantitation indicated that there was an early nonapoptotic loss of cortical neurons followed by a progressive elimination of neurons with features of apoptosis. These data indicate that the pattern of caspase expression occurring during delayed neuronal cell death after focal stroke will vary depending on the neuronal phenotype.

Brain-Specific Knock-Out of Hypoxia-Inducible Factor-1α Reduces Rather Than Increases Hypoxic-Ischemic Damage

Hypoxia-inducible factor-1α (HIF-1α) plays an essential role in cellular and systemic O2 homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1α in the brain, we exposed adult mice with late-stage brain deletion of HIF-1α to hypoxic injuries. Contrary to expectations, the brains from the HIF-1α-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreasing the level of HIF-1α can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1α-deficient mice. Although the role of HIF-1α has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1α in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1α-deficient mice is mechanistically consistent with a predominant role of HIF-1α as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1α, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes.

Astrocytic demise precedes delayed neuronal death in focal ischemic rat brain

Active neuronal-glial interaction is important in the maintenance of brain homeostasis and is vital for neuronal survival following brain injury. The time course of post-ischemic astroglial dysfunction and neuronal death was studied in the spontaneously hypertensive rat (SHR) brain following permanent middle cerebral artery occlusion (MCAO). In situ hybridization with 35S-labeled riboprobes for GFAP and GLUT3 was used to monitor mRNA expression in glia and neurons. Astrocytic proteins GFAP, vimentin, S100, Glutathione-S-Transferase Yb (GST Yb) and neuronal protein TG2 were detected by immunofluorescence. Cells were co-stained with in situ end labeling (ISEL) to detect DNA fragmentation, a hallmark of cell death. GFAP mRNA expression declined rapidly in the ischemic region of the cortex and was almost absent by 12 h. Immunohistochemical studies revealed a parallel decline in the corresponding protein: a reduction in GFAP staining was apparent in the infarct after 3 h and by 24 h, there was essentially no remaining GFAP. Three other glial proteins (vimentin, S100 and GST Yb) disappeared from infarct over a similar time course. A few ISEL positive cells were observed in the infarct at 6 h, but maximal detection was not seen until 24-48 h. Most of the ISEL-positive cells were neurons, identified by co-staining with the neuronal marker TG2. Few cells expressing GFAP or other glial markers were positive at any time point. Neuronal GLUT3 mRNA declined more slowly than GFAP mRNA in the ischemic core and disappeared during the period of neuronal death. Concurrent with the loss of GFAP mRNA and protein expression in the infarct, there was a rapid rise in GFAP mRNA in the peri-infarct region of ipsilateral hemisphere and proximal region of the contralateral hemisphere. This was followed by the enhanced GFAP protein expression characteristic of reactive astrocytes, but over a significantly slower time course. These studies show that MCAO leads to a rapid decline of GFAP mRNA and glial proteins, which appears to precede the decline in neuronal mRNA and neuronal death within the infarct. Early astroglial dysfunction may play a critical role in determining the outcome of acute hypoxic-ischemic injury by compromising neuronal-glial interactions.

Benign focal ischemic preconditioning induces neuronal Hsp70 and prolonged astrogliosis with expression of Hsp27

We have established a focal preconditioning (PC) paradigm that produces significant and prolonged ischemic tolerance (IT) of the brain to subsequent permanent middle cerebral artery occlusion (MCAO). PC using 10 min of MCAO induces brain tolerance at 1-7 days of reperfusion that requires active protein synthesis. The protective protein(s) involved are unknown. In these studies the increased transcription and translation of the inducible 70-kDa heat shock protein (Hsp70) and the 27-kDa heat shock protein (Hsp27), and astrogliosis/glial fibrillary acidic protein (GFAP) were determined by Northern analysis and immunohistochemistry following PC. Cellular localization of proteins was determined by double labeling. PC produced no brain injury but did increase Hsp70 mRNA transiently at 6 h and increased Hsp27 mRNA later at 24 h for at least 5 days. Protein expression induced by PC exhibited a similar profile. Hsp70 protein was primarily expressed in neurons from 1 to 5 days post-PC throughout the PC cortex. Hsp27 protein expression was initiated later for a much longer period of time. A remarkable astroglyosis was verified with increased astrocytic Hsp27 from 1 to 7 days after PC. Gliosis with increased Hsp27 in the PC cortex was still present but reduced 4 weeks after PC. Therefore, PC that results in brain tolerance/neuroprotection increases neuronal Hsp70 in the PC cortex and activated astrocytic Hsp27 in the PC cortex in a temporal fashion associated with developing IT. The short duration of benign ischemia (PC) that produces IT produces a robust, long-lived cellular and protein synthetic response that extends throughout the entire cortex (i.e. well beyond the MCA perfusion territory). The resulting IT is associated with changes in astrocyte-activation that might provide increased support and protection from injury. Although both Hsp70 and Hsp27 may participate in the neuroprotection/brain tolerance induced by PC, the temporal expression patterns of these proteins indicate that they are not solely responsible for the tolerance to brain injury.

Delayed Expression of Osteopontin after Focal Stroke in the Rat

Focal brain ischemia induces inflammation, extracellular matrix remodeling, gliosis, and neovascularization. Osteopontin (OPN) is a secreted glycoprotein that has been implicated in vascular injury by promoting cell adhesion, migration, and chemotaxis. To investigate the possible involvement of OPN in brain matrix remodeling after focal stroke, we examined the expression of OPN in ischemic cortex after permanent or temporary occlusion of the middle cerebral artery (MCAO) of the rat. OPN mRNA and protein levels in nonischemic cortex were not detected consistently, although significant induction of OPN was observed in the ischemic cortex. OPN mRNA increased 3.5-fold at 12 hr and reached peak levels 5 d (49.5-fold; p < 0.001) after permanent MCAO. The profile of OPN mRNA induction after transient MCAO (160 min) with reperfusion was essentially the same as that of permanent MCAO. In situ hybridization and immunohistochemical studies demonstrated strong induction of OPN in the ischemic cortex, which was localized primarily in a subset of ED-1-positive macrophages that accumulated in the ischemic zone. Moreover, OPN immunoreactivity was detected in the matrix of ischemic brain, suggesting a functional role of the newly deposited matrix protein in cell–matrix interactions and remodeling. Indeed, using a modified Boyden chamber, we demonstrated a dose-dependent chemotactic activity of OPN in C6 astroglia cells and normal human astrocytes. Taken together, these data suggest that the upregulation of OPN after focal brain ischemia may play a role in cellular (glia, macrophage) migration/activation and matrix remodeling that provides for new matrix–cell interaction.

Osteopontin and its Integrin Receptor αvβ3 Are Upregulated During Formation of the Glial Scar After Focal Stroke

Background and Purpose —Microglia and astrocytes in the peri-infarct region are activated in response to focal stroke. A critical function of activated glia is formation of a protective barrier that ultimately forms a new glial-limiting membrane. Osteopontin, a provisional matrix protein expressed during wound healing, is induced after focal stroke. The present study was performed to determine the spatial and temporal expression of osteopontin and its integrin receptor αvβ3 during formation of the peri-infarct gliotic barrier and subsequent formation of a new glial-limiting membrane. Methods —Spontaneously hypertensive rats (n=19) were subjected to permanent occlusion of the middle cerebral artery and killed 3, 6, and 24 hours and 2, 5, and 15 days after occlusion. The spatial and temporal expression of osteopontin mRNA was determined by in situ hybridization, and that of osteopontin ligand and its integrin receptor αvβ3 was determined by immunohistochemistry. Results —Osteopontin mRNA was expressed de novo in the peri-infarct region from 3 to 48 hours; by 5 days osteopontin mRNA expression was restricted to the infarct. Osteopontin protein was expressed by peri-infarct microglia beginning at 24 hours and by microglia/macrophages at 48 hours in the infarct. Integrin receptor αvβ3 was expressed in peri-infarct astrocytes at 5 and 15 days. Conclusions —Early microglial/macrophage expression of osteopontin mRNA defines the borders and final infarct area at 24 hours. At 5 days osteopontin ligand is at a distance from the peri-infarct astrocytes expressing integrin receptor αvβ3. By 15 days astrocytes expressing integrin receptor αvβ3 are localized in an osteopontin-rich region concomitant with formation of the new glial-limiting membrane. The de novo expression and interaction of osteopontin ligand with its receptor integrin αvβ3 suggest a role in wound healing after focal stroke.

α-Synuclein maps to a quantitative trait locus for alcohol preference and is differentially expressed in alcohol-preferring and -nonpreferring rats

Total gene expression analysis (TOGA) was used to identify genes that are differentially expressed in brain regions between the alcohol-naïve, inbred alcohol-preferring (iP), and -nonpreferring (iNP) rats. α-Synuclein, expressed at >2-fold higher levels in the hippocampus of the iP than the iNP rat, was prioritized for further study. In situ hybridization was used to determine specific brain regions and cells expressing α-synuclein in the iP and iNP rats. Similar to α-synuclein mRNA levels, protein levels in the hippocampus were higher in iP rats than iNP rats. Higher protein levels were also observed in the caudate putamen of iP rats compared with iNP rats. Sequence analysis identified two single nucleotide polymorphisms in the 3′ UTR of the cDNA. The polymorphism was used to map the gene, by using recombination-based methods, to chromosome 4, within a quantitative trait locus for alcohol consumption that was identified in the iP and iNP rats. A nucleotide exchange in the iNP 3′ UTR reduced expression of the luciferase reporter gene in SK-N-SH neuroblastoma cells. These results suggest that differential expression of the α-synuclein gene may contribute to alcohol preference in the iP rats.

Prolonged expression of interferon-inducible protein-10 in ischemic cortex after permanent occlusion of the middle cerebral artery in rat

Abstract: Focal cerebral ischemia elicits local inflammatory reaction as demonstrated by the accumulation of inflammatory cells and mediators in the ischemic brain. Interferon‐inducible protein‐10 (IP‐10) is a member of the C‐X‐C chemokine family that possesses potent chemoattractant actions for monocytes, T cells, and smooth muscle cells. To investigate a potential role of IP‐10 in focal stroke, we studied the temporal expression of IP‐10 mRNA after occlusion of the middle cerebral artery in rat by means of northern analysis. IP‐10 mRNA expression after focal stroke demonstrated a unique biphasic profile, with a marked increase early at 3 h (4.9‐fold over control; p < 0.01), a peak level at 6 h (14.5‐fold; p < 0.001) after occlusion of the middle cerebral artery, and a second wave induction 10–15 days after ischemic injury (7.2‐ and 9.3‐fold increase for 10 and 15 days, respectively; p < 0.001). In situ hybridization confirmed the induced expression of IP‐10 mRNA and revealed its spatial distribution after focal stroke. Immunohistochemical studies demonstrated the expression of IP‐10 peptide in neurons (3–12 h) and astroglial cells (6 h to 15 days) of the ischemic zone. To explore further the potential role of IP‐10 in focal stroke, we demonstrated a dose‐dependent chemotactic action of IP‐10 on C6 glial cells and enhanced attachment of rat cerebellar granule neurons. Taken together, the data suggest that ischemia induces IP‐10, which may play a pleiotropic role in prolonged leukocyte recruitment, astrocyte migration/activation, and neuron attachment/sprouting after focal stroke.

CNTF regulation of astrogliosis and the activation of microglia in the developing rat central nervous system

In response to physical or chemical brain injury, the mammalian central nervous system (CNS) often reacts by evoking astrogliosis. The most prominent feature describing this state is an upregulation of glial fibrillary acidic protein (GFAP). The agent(s) responsible for inducing astrogliosis remains unclear; however, recent observations have shown cytokines may play a pivotal role. During CNS trauma, macrophages and lymphocytes infiltrate the CNS where they are thought to synthesize and secrete cytokines; moreover, activated microglia and reactive astrocytes are known to be capable of cytokine production. We are the first to report that an intracerebral injection of the pleiotropic cytokine, ciliary neurotrophic factor (CNTF), increases astrogliosis and the appearance of activated microglia in the neonatal rat. This response to CNTF was comparable to the response observed in animals receiving a well known pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). Only a moderate increase was observed in the proliferative index of cytokine-injected animals; therefore, we conclude that GFAP is largely upregulated in a pre-existing GFAP negative cell population. Interestingly, coinjections of CNTF and TNF-alpha appeared to act synergistically. Coinjected animals displayed a wave of hypertrophied astrocytes reaching far into the contralateral hemisphere. No contralateral spreading of microglia was observed. This article clearly provides interesting information regarding the regulatory mechanisms that govern astrogliosis and discusses the probable relationship of reactive astrocytes to microglia.