Christopher C. Leonardo

Blockade of adrenoreceptors inhibits the splenic response to stroke

Recent studies have highlighted the involvement of the peripheral immune system in delayed cellular degeneration after stroke. In the permanent middle cerebral artery occlusion (MCAO) model of stroke, the spleen decreases in size. This reduction occurs through the release of splenic immune cells. Systemic treatment with human umbilical cord blood cells (HUCBC) 24 h post-stroke blocks the reduction in spleen size while significantly reducing infarct volume. Splenectomy 2 weeks prior to MCAO also reduces infarct volume, further demonstrating the detrimental role of this organ in stroke-induced neurodegeneration. Activation of the sympathetic nervous system after MCAO results in elevated catecholamine levels both at the level of the spleen, through direct splenic innervation, and throughout the systemic circulation upon release from the adrenal medulla. These catecholamines bind to splenic alpha and beta adrenoreceptors. This study examines whether catecholamines regulate the splenic response to stroke. Male Sprague-Dawley rats either underwent splenic denervation 2 weeks prior to MCAO or received injections of carvedilol, a pan adrenergic receptor blocker, prazosin, an alpha1 receptor blocker, or propranolol, a beta receptor blocker. Denervation was confirmed by reduced splenic expression of tyrosine hydroxylase. Denervation prior to MCAO did not alter infarct volume or spleen size. Propranolol treatment also had no effects on these outcomes. Treatment with either prazosin or carvedilol prevented the reduction in spleen size, yet only carvedilol significantly reduced infarct volume (p < 0.05). These results demonstrate that circulating blood borne catecholamines regulate the splenic response to stroke through the activation of both alpha and beta adrenergic receptors.

The spleen contributes to stroke induced neurodegeneration through interferon gamma signaling

Delayed neuronal death associated with stroke has been increasingly linked to the immune response to the injury. Splenectomy prior to middle cerebral artery occlusion (MCAO) is neuroprotective and significantly reduces neuroinflammation. The present study investigated whether splenic signaling occurs through interferon gamma (IFNγ). IFNγ was elevated early in spleens but later in the brains of rats following MCAO. Splenectomy decreased the amount of IFNγ in the infarct post-MCAO. Systemic administration of recombinant IFNγ abolished the protective effects of splenectomy with a concurrent increase in INFγ expression in the brain. These results suggest a role for spleen-derived IFNγ in stroke pathology.

Neuroinflammation and MMPs: potential therapeutic targets in neonatal hypoxic-ischemic injury.

Exposure to hypoxic-ischemic insults during the neonatal or perinatal developmental periods produces various forms of pathology. Injuries that occur in response to these events often manifest as severe cognitive and/or motor disturbances over time. Due to difficulties regarding the early diagnosis and treatment of hypoxic-ischemic injury, there is a growing need for effective therapies that can be delivered at delayed time points. Much of the research into mechanisms of neural injury has focused on molecular targets associated with excitotoxicity and free oxygen radicals. Despite repeated success in animal models, these compounds have failed to show efficacy in clinical trials. Increasing evidence indicates that hypoxic-ischemic injury in the neonate is progressive, and the resulting neuropathies are linked to the activation of neuroinflammatory processes that occur in response to the initial wave of cell death. Understanding this latter response, therefore, will be critical in the development of novel therapies to block the progression of the injury. In this review, we summarize emerging concepts from rodent models concerning the regulation of various cytokines, chemokines, and matrix metalloproteinases in response to ischemia, and the various ways in which the delayed neuroinflammatory response may contribute to the progressive nature of neonatal hypoxic-ischemic injury in rat. Finally, we discuss data that supports the potential to target these neuroinflammatory signals at clinically relevant time points.

Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat

BackgroundHypoxia-ischemia (H-I) can produce widespread neurodegeneration and deep cerebral white matter injury in the neonate. Resident microglia and invading leukocytes promote lesion progression by releasing reactive oxygen species, proteases and other pro-inflammatory mediators. After injury, expression of the gelatin-degrading matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are thought to result in the proteolysis of extracellular matrix (ECM), activation of cytokines/chemokines, and the loss of vascular integrity. Thus, therapies targeting ECM degradation and progressive neuroinflammation may be beneficial in reducing H-I – induced neuropathy. Minocycline has MMP-inhibitory properties and is both anti-inflammatory and neuroprotective. AG3340 (prinomastat) is an MMP inhibitor with high selectivity for the gelatinases. The purpose of this study was to determine whether these compounds could limit H-I – induced injury when administered at a delayed time point.MethodsSprague-Dawley rats were exposed to H-I at postnatal day 7 (P7), consisting of unilateral carotid artery ligation followed by 90 min exposure to 8% O2. Minocycline, AG3340, or vehicle were administered once daily for 6 days, beginning 24 hours after insult. Animals were sacrificed at P14 for neurohistological assessments. Immunohistochemistry was performed to determine the degree of reactive astrogliosis and immune cell activation/recruitment. Neural injury was detected using the Fluoro-Jade stain, a marker that identifies degenerating cells.ResultsCD11b and glial fibrillary acidic protein (GFAP) immunopositive cells increased in ipsilateral cortex after treatment with vehicle alone, demonstrating microglia/macrophage recruitment and reactive astrogliosis, respectively. Fluoro-Jade staining was markedly increased throughout the fronto-parietal cortex, striatum and hippocampus. Treatment with minocycline or AG3340 inhibited microglia/macrophage recruitment, attenuated astrogliosis and reduced Fluoro-Jade staining when compared to vehicle alone.ConclusionThe selective gelatinase inhibitor AG3340 showed equal efficacy in reducing neural injury and dampening neuroinflammation when compared to the anti-inflammatory compound minocycline. Thus, MMP-2 and MMP-9 may be viable therapeutic targets to treat neonatal brain injury.

Multimodal signaling by the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) promotes neurite extension

Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains associate with hyaluronan and various secreted proteins to form a complex of extracellular matrix (ECM) that inhibits neural plasticity in the central nervous system (CNS). Chondroitinase treatment depletes PGs of their CS side chains and enhances neurite extension. Increasing evidence from in vivo models indicates that proteolytic cleavage of the PG core protein by members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of glutamyl-endopeptidases also promotes neural plasticity. The purpose of this study was to determine whether proteolytic action of the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of primary rat neurons with ADAMTS4 cDNA induced longer neurites, whether the neurons were grown on a monolayer of astrocytes that secrete inhibitory PGs or on laminin/poly-L-lysine substrate alone. Similar results were found when neurons were transfected with a construct encoding a proteolytically inactive, point mutant of ADAMTS4. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures also enhanced neurite extension in a dose-dependent manner, an effect demonstrated to be dependent on the activation of MAP ERK1/2 kinase. These results suggest that ADAMTS4 enhances neurite outgrowth via a mechanism that does not require proteolysis but is dependent on activation of the MAP kinase cascade. Thus a model to illustrate multimodal ADAMTS activity would entail proteolysis of CS-bearing PGs to create a loosened matrix environment more favorable for neurite outgrowth, and enhanced neurite outgrowth directly stimulated by ADAMTS signaling at the cell surface.

Lateral fluid percussion injury of the brain induces CCL20 inflammatory chemokine expression in rats

BackgroundTraumatic brain injury (TBI) evokes a systemic immune response including leukocyte migration into the brain and release of pro-inflammatory cytokines; however, the mechanisms underlying TBI pathogenesis and protection are poorly understood. Due to the high incidence of head trauma in the sports field, battlefield and automobile accidents identification of the molecular signals involved in TBI progression is critical for the development of novel therapeutics.MethodsIn this report, we used a rat lateral fluid percussion impact (LFPI) model of TBI to characterize neurodegeneration, apoptosis and alterations in pro-inflammatory mediators at two time points within the secondary injury phase. Brain histopathology was evaluated by fluoro-jade (FJ) staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, polymerase chain reaction (qRT PCR), enzyme linked immunosorbent assay (ELISA) and immunohistochemistry were employed to evaluate the CCL20 gene expression in different tissues.ResultsHistological analysis of neurodegeneration by FJ staining showed mild injury in the cerebral cortex, hippocampus and thalamus. TUNEL staining confirmed the presence of apoptotic cells and CD11b+ microglia indicated initiation of an inflammatory reaction leading to secondary damage in these areas. Analysis of spleen mRNA by PCR microarray of an inflammation panel led to the identification of CCL20 as an important pro-inflammatory signal upregulated 24 h after TBI. Although, CCL20 expression was observed in spleen and thymus after 24h of TBI, it was not expressed in degenerating cortex or hippocampal neurons until 48 h after insult. Splenectomy partially but significantly decreased the CCL20 expression in brain tissues.ConclusionThese results demonstrate that the systemic inflammatory reaction to TBI starts earlier than the local brain response and suggest that spleen- and/ or thymus-derived CCL20 might play a role in promoting neuronal injury and central nervous system inflammation in response to mild TBI.

Human umbilical cord blood cell therapy blocks the morphological change and recruitment of CD11b-expressing, isolectin-binding proinflammatory cells after middle cerebral artery occlusion.

Secondary neurodegeneration resulting from stroke is mediated by delayed proinflammatory signaling and immune cell activation. Although it remains unknown which cell surface markers signify a proinflammatory phenotype, increased isolectin binding occurs on CD11b‐expressing immune cells within injured brain tissue. Several reports have confirmed the efficacy of human umbilical cord blood (HUCB) cell therapy in reducing ischemic injury in rat after middle cerebral artery occlusion (MCAO), and these effects were attributed in part to dampened neuroinflammation. The present study examined the time course of lectin binding to cells of microglia/macrophage lineage within 96 hr after MCAO and whether delayed HUCB cell treatment alters the migration and/or morphological characteristics of these cells throughout the period of infarct expansion. Isolectin binding was up‐regulated in response to injury, was maximal at 96 hr, and colocalized with cells that expressed the putative proinflammatory markers MMP‐9 and nitric oxide. Isolectin‐tagged fluorescence was also significantly increased at 72 hr and localized to greater numbers of amoeboid, CD11b‐expressing cells relative to 51 hr. Treatment with 1 × 106 HUCB cells significantly reduced total lectin binding at 72 hr, as well as the total area occupied by lectin‐tagged fluorescence at both 51 and 72 hr, relative to vehicle‐treated controls. This effect was accompanied by a shift in the morphology of CD11b‐positive cells from amoeboid to ramified shape. These data indicate that HUCB cell therapy suppressed the recruitment of proinflammatory, isolectin‐binding cells during the period of infarct expansion, thus offering a potential mechanism for the protective effects of HUCB cell therapy.

Dietary flavonoids are neuroprotective through Nrf2-coordinated induction of endogenous cytoprotective proteins

Abstract Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk for cardiovascular disease and stroke. Detailed investigations into the specific dietary components of these foods have revealed that many polyphenolic constituents exert anti-oxidant effects on key substrates involved in the pathogenesis and progression of ischemic injury. These data have perpetuated the belief that the protective effects of flavonoids result from direct anti-oxidant actions at the levels of the cerebral vasculature and brain parenchyma. While many in vitro studies using purified extracts support this contention, first-pass metabolism alters the bioavailability of flavonoids such that the achievable concentrations after oral consumption are not consistent with this mechanism. Importantly, oral consumption of flavonoids may promote neural protection by facilitating the expression of gene products responsible for detoxifying the ischemic microenvironment through both anti-oxidative and anti-inflammatory actions. In particular, the transcriptional factor nuclear factor erythroid 2-related factor 2 has emerged as a critical regulator of flavonoid-mediated protection through the induction of various cytoprotective genes. The pleiotropic effects associated with potent transcriptional regulation likely represent the primary mechanisms of neural protection, as the flavonoid concentrations reaching ischemic tissues in vivo are sufficient to alter intracellular signal transduction but likely preclude the one-to-one stoichiometry necessary to confer protection by direct anti-oxidation. These data reflect an exciting new direction in the study of complementary and alternative medicine that may lead to the development of novel therapies for ischemic/hemorrhagic stroke, traumatic brain injury, and other neurological disorders.

Human umbilical cord blood cells directly suppress ischemic oligodendrocyte cell death

Previous reports have shown that human umbilical cord blood cells (HUCBCs) administered intravenously 48 hr following middle cerebral artery occlusion reduce infarct area and behavioral deficits of rodents. This cellular therapy is potently neuroprotective and antiinflammatory. This study investigates the effect of HUCBC treatment on white matter injury and oligodendrocyte survival in a rat model of ischemia. Intravenous infusion of 106 HUCBCs 48 hr poststroke reduced the amount of white matter damage in vivo as seen by quantification of myelin basic protein staining in tissue sections. To determine whether HUCBC treatment was protective via direct effects on oligodendrocytes, cultured oligodendrocytes were studied in an in vitro model of oxygen glucose deprivation. Active caspase 3 immunohistochemistry and the lactate dehydrogenase assay for cytotoxicity were used to determine that HUCBCs provide protection to oligodendrocytes in vitro. Based on these results, it is likely that HUCBC administration directly protects oligodendrocytes and white matter. This effect is likely to contribute to the increased behavioral recovery observed with HUCBC therapy.

Human umbilical cord blood cells protect oligodendrocytes from brain ischemia through Akt signal transduction.

Background: Human umbilical cord blood cells are an effective experimental treatment for stroke. Results: These cells activate Akt to increase peroxiredoxin 4 and are essential for oligodendrocyte survival during ischemia. Conclusion: Akt and peroxiredoxin 4 are key molecules in transducing the cellular protection elicited by cord blood cells. Significance: Identifying this signaling pathway provides new pharmaceutical targets for stroke treatment. Human umbilical cord blood (HUCB) cells protect the brain against ischemic injury, yet the mechanism of protection remains unclear. Using both in vitro and in vivo paradigms, this study examined the role of Akt signaling and peroxiredoxin 4 expression in human umbilical cord blood cell-mediated protection of oligodendrocytes from ischemic conditions. As previously reported, the addition of HUCB cells to oligodendrocyte cultures prior to oxygen glucose deprivation significantly enhanced oligodendrocyte survival. The presence of human umbilical cord blood cells also increased Akt phosphorylation and elevated peroxiredoxin 4 expression in oligodendrocytes. Blocking either Akt or peroxiredoxin 4 activity with Akt Inhibitor IV or a peroxiredoxin 4-neutralizing antibody, respectively, negated the protective effects of human umbilical cord blood cells. In vivo, systemic administration of human umbilical cord blood cells 48 h after middle cerebral artery occlusion increased Akt phosphorylation and peroxiredoxin 4 protein expression while reducing proteolytic cleavage of caspase 3 in oligodendrocytes residing in the ipsilateral external capsule. Moreover, human umbilical cord blood cells protected striatal white matter bundles from degeneration following middle cerebral artery occlusion. These results suggest that the soluble factors released from human umbilical cord blood cells converge on Akt to elevate peroxiredoxin 4 levels, and these effects contribute to oligodendrocyte survival.