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.

Oligodendrocytes (OLs), the predominant cell type found in cerebral white matter, are essential for structural integrity and proper neural signaling. Very little is known concerning stroke-induced OL dysfunction. Our laboratory has shown that infusion of human umbilical cord blood (HUCB) cells protects striatal white matter tracts in vivo and directly protects mature primary OL cultures from oxygen glucose deprivation (OGD). Microarray studies of RNA prepared from OL cultures subjected to OGD and treated with HUCB cells showed an increase in the expression of 33 genes associated with OL proliferation, survival, and repair functions, such as myelination. The microarray results were verified using quantitative RT-PCR for the following eight genes: U2AF homology motif kinase 1 (Uhmk1), insulin induce gene 1 (Insig1), metallothionein ( Mt3), tetraspanin 2 (Tspan2), peroxiredoxin 4 (Prdx4), stathmin-like 2 (Stmn2), myelin oligodendrocyte glycoprotein (MOG), and versican (Vcan). Immunohistochemistry showed that MOG, Prdx4, Uhmk1, Insig1 and Mt3 protein expression were upregluated in the ipsilateral white matter tracts of rats infused with HUCB cells 48 hrs after middle cerebral artery occlusion (MCAO). Furthermore, promoter region analysis of these genes revealed common transcription factor binding sites, providing insight into the shared signal transduction pathways activated by HUCB cells to enhance transcription of these genes. These results show expression of genes induced by HUCB cell therapy that could confer oligoprotection from ischemia.

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 have shown efficacy in rodent models of focal ischemia and in vitro systems that recapitulate stroke conditions. One potential mechanism of protection is through secretion of soluble factors that protect neurons and oligodendrocytes (OLs) from oxidative stress. To overcome practical issues with cellular therapies, identification of soluble factors released by HUCB and other stem cells may pave the way for treatment modalities that are safer for a larger percentage of stroke patients. Among these soluble factors is leukemia inhibitory factor (LIF), a cytokine that exerts pleiotropic effects on cell survival. Here, data show that LIF effectively reduced infarct volume, reduced white matter injury and improved functional outcomes when administered to rats following permanent middle cerebral artery occlusion. To further explore downstream signaling, primary oligodendrocyte cultures were exposed to oxygen glucose deprivation (OGD) to mimic stroke conditions. LIF significantly reduced lactate dehydrogenase release from OLs, reduced superoxide dismutase activity, and induced peroxiredoxin 4 (Prdx4) transcript. Additionally, the protective and antioxidant capacity of LIF was negated by both Akt inhibition and co-incubation with Prdx4 neutralizing antibodies, establishing a role for the Akt signaling pathway and Prdx4-mediated antioxidation in LIF protection.

A major limitation of current stroke therapies is the need to treat candidate patients within three hours of stroke onset. Human umbilical cord blood cell (HUCBC) and the sigma receptor agonist 1,3, dio-tolylguanidine (DTG) administration both caused significant reductions in brain damage in the rat middle cerebral artery occlusion model of stroke when administered at delayed timepoints. In vivo, these treatments suppress the infiltration of peripheral lymphocytes into the brain in addition to decreasing neurodegeneration. An ex vivo organotypic slice culture (OTC) model was utilized to characterize the efficacy of these treatments in mitigating neurodegeneration in ischemic brain tissue in the absence of the peripheral immune system. Slice cultures subjected to oxygen glucose deprivation (OGD) had significantly elevated levels of degenerating neurons and microglial nitric oxide production when compared to their normoxic counterparts. In cultures subjected to OGD, HUCBC but not DTG treatment reduced the number of degenerating neurons and the production of microglial derived nitric oxide back to levels detected in normoxic controls. These data show that HUCBC treatment can mediate direct neuroprotection and suppress innate inflammation in ischemic brain tissue in the absence of the peripheral immune system, whereas DTG requires peripheral effects to mediate neuroprotection. These experiments yield insight into the mechanisms by which these neuroprotective treatments function at delayed timepoints following stroke.

Traumatic brain injury (TBI) is complex and involves multiple processes that contribute to functional decline. Progressive neuropathies result from delayed cellular death following the initial impact. Although the precise mechanisms responsible for delayed injury are unknown, numerous data implicate a role for the peripheral immune system in perpetuating neuroinflammation after TBI. A previous report demonstrated that splenic CCL20 chemokine expression was upregulated 24 h after lateral fluid percussive impact (LFPI), prior to neuronal expression but consistent with neurodegeneration. Here, we expand on those data to report increased CCL20 protein expression in white matter 48 h after LFPI and demonstrate that CCL20 is directly toxic to primary neurons and oligodendrocytes subjected to oxygen glucose deprivation. The temporal expression profile of CCL20, coupled with in vitro toxicity to primary cells, suggests that this chemokine exerts deleterious effects on cell viability following TBI. These findings warrant further investigations into the use of CCL20 as a potential biomarker and/or therapeutic target.

Human umbilical cord blood (HUCB) cell therapies have shown promising results in reducing brain infarct volume and most importantly in improving neurobehavioral function in rat permanent middle cerebral artery occlusion, a model of stroke. In this study, we examined the gene expression profile in neurons subjected to oxygen-glucose deprivation (OGD) with or without HUCB treatment and identified signaling pathways (Akt/MAPK) important in eliciting HUCB-mediated neuroprotective responses. Gene chip microarray analysis was performed using RNA samples extracted from the neuronal cell cultures from four experimental groups: normoxia, normoxia + HUCB, OGD, and OGD + HUCB. Both quantitative RT-PCR and immunohistochemistry were carried out to verify the microarray results. Using the Genomatix software program, promoter regions of selected genes were compared to reveal common transcription factor-binding sites and, subsequently, signal transduction pathways. Under OGD condition, HUCB cells significantly reduced neuronal loss from 68% to 44% [one-way ANOVA, F(3, 16) = 11, p = 0.0003]. Microarray analysis identified mRNA expression of Prdx5, Vcam1, CCL20, Alcam, and Pax6 as being significantly altered by HUCB cell treatment. Inhibition of the Akt pathway significantly abolished the neuroprotective effect of HUCB cells [one-way ANOVA, F(3, 11) = 8.663, p = 0.0031]. Our observations show that HUCB neuroprotection is dependent on the activation of the Akt signaling pathway that increases transcription of the Prdx5 gene. We concluded that HUCB cell therapy would be a promising treatment for stroke and other forms of brain injury by modifying acute gene expression to promote neural cell protection.

Cerebral edema after stroke is associated with poor neurological outcomes. Current therapies are limited to osmotic agents, such as hypertonic saline (HS), which reduce intracranial pressure. Although studies have demonstrated edema reductions following HS, tissue survival has not been thoroughly examined. Additionally, the efficacy of promising pharmacological agents has not been evaluated for synergy with osmotic agents. Conivaptan is an FDA-approved vasopressin receptor antagonist that may exert both osmotic and anti-inflammatory effects. In this study, rats were subjected to middle cerebral artery occlusion prior to treatment with 5 % HS bolus +5 % HS maintenance (HS), conivaptan alone (Con), conivaptan +5 % HS maintenance (Con + HS), or conivaptan +5 % HS bolus +5 % maintenance (Con + HSb). Treatments were initiated at six (Early) or 24 h (Late) following stroke and rats were euthanized at 48 h to evaluate infarct volume, brain edema, and microglia/macrophage activation. Infarct volume and brain edema in the Early HS, Early Con, and Late HS groups were significantly reduced compared with controls. Interestingly, only the Early Con group demonstrated reduced microglia/macrophage activation. These data suggest an anti-inflammatory mechanism for conivaptan and provide support for a multipronged approach combining osmotic agents with compounds that inhibit the neuroinflammatory response to stroke.