Lisa A. Collier

The spleen contributes to stroke-induced neurodegeneration

Stroke, a cerebrovascular injury, is the leading cause of disability and third leading cause of death in the world. Recent reports indicate that inhibiting the inflammatory response to stroke enhances neurosurvival and limits expansion of the infarction. The immune response that is initiated in the spleen has been linked to the systemic inflammatory response to stroke, contributing to neurodegeneration. Here we show that removal of the spleen significantly reduces neurodegeneration after ischemic insult. Rats splenectomized 2 weeks before permanent middle cerebral artery occlusion had a >80% decrease in infarction volume in the brain compared with those rats that were subjected to the stroke surgery alone. Splenectomy also resulted in decreased numbers of activated microglia, macrophages, and neutrophils present in the brain tissue. Our results demonstrate that the peripheral immune response as mediated by the spleen is a major contributor to the inflammation that enhances neurodegeneration after stroke.

Sigma Receptor Activation Reduces Infarct Size at 24 Hours After Permanent Middle Cerebral Artery Occlusion in Rats

The only available treatment for embolic stroke is recombinant tissue plasminogen activator, which must be administered within three hours of stroke onset. We examined the effects of 1,3-di-o-tolyguanidine (DTG), a high affinity sigma receptor agonist, as a potential treatment for decreasing infarct area at delayed time points. Rats were subjected to permanent embolic middle cerebral artery occlusion (MCAO) and allowed to recover before receiving subcutaneous injections of 15 mg/kg of DTG at 24, 48, and 72 hours. At 96 hours the rats were euthanized, and brains harvested and sectioned. Infarct areas were quantified at the level of the cortical/striatal and cortical/hippocampal regions in control (MCAO-only) and DTG treated animals using a marker for neurodegeneration, Fluoro-Jade. DTG treatment significantly reduced infarct area in both cortical/striatal and cortical/hippocampal regions by >80%, relative to control rats. These findings were confirmed by immunohistochemical experiments using the neuronal marker, mouse anti-neuronal nuclei monoclonal antibody (NeuN), which showed that application of DTG significantly increased the number of viable neurons in these regions. Furthermore, DTG blocked the inflammatory response evoked by MCAO, as indicated by decreases in the number of reactive astrocytes and activated microglia/macrophages detected by immunostaining for glial fibrillary acidic protein (GFAP) and binding of isolectin IB4, respectively. Thus, our results demonstrate that the sigma receptor-selective agonist, DTG, can enhance neuronal survival when administered 24 hr after an ischemic stroke. In addition, the efficacy of sigma receptors for stroke treatment at delayed time points is likely the result of combined neuroprotective and anti-inflammatory properties of these receptors.

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.

NF-κB protects neurons from ischemic injury after middle cerebral artery occlusion in mice

Knowledge about the molecular mechanisms of neuronal survival following ischemia is crucial to the development of therapeutic interventions for victims of stroke. Previous research in our laboratory has implicated nuclear factor-kappaB (NF-kappaB) as contributing to neuronal survival in response to toxic or ischemic brain insult, with in vivo models having focused on the rat. To take advantage of genetic alterations available in the mouse, we utilized a murine transient endovascular middle cerebral artery occlusion (MCAO) model to examine the influence of NF-kappaB on neuronal survival. When brains were immunostained for the nuclear localization sequence (NLS) of the p50 subunit of NF-kappaB, a unilateral increase in immunoreactivity was seen, especially in pyramidal cell layers of the ipsilateral (stroked) hippocampus. When transgenic mice lacking p50 were compared with non-transgenic counterparts using Fluoro-Jade, a marker for neurodegeneration, both the hippocampus and striatum showed enhanced neurodegeneration at various survival times after 1 h of MCAO. In the hippocampus specifically, there was an eightfold increase in Fluoro-jade staining in the p50 knockout group vs. the non-transgenic group. Sections double stained for Fluoro-Jade and NF-kappaB activity (using a mouse engineered with a NF-kappaB responsive promoter driving a LacZ gene to produce beta galactosidase) demonstrated neuronal degeneration only in regions sparsely showing NF-kappaB activity, and those demonstrating NF-kappaB activity failed to degenerate. These data provide evidence that NF-kappaB participates in survival signaling following temporary focal ischemia, and thus may represent an attractive target for pharmacologic activation in the treatment of stroke.

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.

Temporary focal ischemia in the mouse: Technical aspects and patterns of Fluoro-Jade evident neurodegeneration

Animal models of cerebral infarction are crucial to understanding the mechanisms of neuronal survival following ischemic brain injury and to the development of therapeutic interventions for victims of all types of stroke. Rodents have been used extensively in such research. One rodent model of stroke utilizes either permanent or temporary occlusion of the middle cerebral artery (MCAO) to produce ischemia. Since the development of an endovascular method for this was published in 1989, MCAO has been applied commonly to the rat, and often paired with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining for stroke volume measurement. Meanwhile, advances in the ability to genetically alter mice have allowed exciting lines of research into ischemia. Because of technical demands and issues with survival, relatively few laboratories have investigated the MCAO method in the mouse. Our present work utilizes a mouse middle cerebral occlusion (MCAO) model of embolic stroke to study neuronal degeneration following temporary focal cerebral ischemia. C57Bl/6J mice were used to examine the exact effects of MCAO using Fluoro-Jade, a marker of neurodegeneration that allows observation of specific brain regions and cells destined to die. A time course of escalating neuronal degeneration from 10 min to 7 days following MCAO was established. Technical aspects of this popular method for transient focal ischemia as it applies to the mouse are discussed.

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.

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.

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.