Neuroprotective effects of neuregulin-1 on B35 neuronal cells following ischemia.

We previously showed that neuregulin-1 (NRG-1) protected neurons from death in vivo following focal ischemia. The goal of this study was to develop an in vitro rat ischemia model to examine the cellular and molecular mechanisms involved in the neuroprotective effects of NRG-1 on ischemia-induced neuronal death. Rat B-35 neuroblastoma cells differentiated by serum withdrawal, developed enhanced neuronal characteristics including, neurite extension and upregulation of neuronal markers of differentiation. When B35 neurons were subjected to oxygen glucose deprivation (OGD)/reoxygenation or glutamate, widespread neuronal death was seen after both treatments. Treatment with NRG-1 immediately after OGD significantly increased neuronal survival. NRG-1 administration also resulted in a significant decrease in annexin V, an early marker of apoptosis. However, the neurotoxic actions of glutamate were unaffected by NRG-1. The neuroprotective effects of NRG-1 were prevented by an inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. These results provide a new model to gain insight into the mechanisms employed by NRG-1 to protect neurons from ischemic brain injury.

Neuroprotection by neuregulin-1 in a rat model of permanent focal cerebral ischemia.

Neuregulin-1 (NRG-1) is a growth factor with potent neuroprotective capacity in ischemic stroke. We recently showed that NRG-1 reduced neuronal death following transient middle cerebral artery occlusion (tMCAO) by up to 90% with an extended therapeutic window. Here, we examined the neuroprotective potential of NRG-1 using a permanent MCAO ischemia (pMCAO) rat model. NRG-1 reduced infarction in pMCAO by 50% when administered prior to ischemia. We previously demonstrated using gene expression profiling that pMCAO was associated with an exaggerated excitotoxicity response compared to tMCAO. Therefore, we examined whether co-treatment with an inhibitor of excitotoxicity would augment the effect of NRG-1 following pMCAO. Both NRG-1 and the N-methyl-D-aspartate (NMDA) antagonist MK-801 similarly reduced infarct size following pMCAO. However, combination treatment with both NRG-1 and MK-801 resulted in greater neuroprotection than either compound alone, including a 75% reduction in cortical infarction compared to control. Consistent with these findings, NRG-1 reduced neuronal death using an in vitro ischemia model and this effect was augmented by MK-801. These results demonstrate the efficacy of NRG-1 in pMCAO rat focal ischemia model. Our findings further indicate the potential clinically relevance of NRG-1 alone or as a combination strategy for treating ischemic stroke.

Extended therapeutic window and functional recovery after intraarterial administration of neuregulin-1 after focal ischemic stroke.

We have previously shown that neuregulin-1 (NRG-1) protects neurons from ischemic brain injury if administered before focal stroke. Here, we examined the therapeutic window and functional recovery after NRG-1 treatment in rats subjected to 90 mins of middle cerebral artery occlusion (MCAO) and 24 h of reperfusion. Neuregulin-1 (2.5 microg/kg [corrected] bolus, 1.25 microg/kg/min [corrected] infusion) reduced infarct volume by 89.2%+/-41.9% (mean+/-s.d.; n=8; P<0.01) if administered immediately after the onset of reperfusion. Neuroprotection was also evident if NRG-1 was administered 4 h (66.4%+/-52.6%; n=7; P<0.01) and 12 h (57.0%+/-20.8%; n=8; P<0.01) after reperfusion. Neuregulin-1 administration also resulted in a significant improvement of functional neurologic outcome compared with vehicle-treated animals (32.1%+/-5.7%; n=9; P<0.01). The neuroprotective effect of the single administration of NRG-1 was seen as long as 2 weeks after treatment. Neurons labeled with the neurodegeneration marker dye Fluoro-JadeB were observed after MCAO in the cortex, but the numbers were significantly reduced after NRG-1 treatment. These results indicate that NRG-1 is a potent neuroprotective compound with an extended therapeutic window that has practical therapeutic potential in treating individuals after ischemic brain injury.

Neuregulin-1 attenuates neointimal formation following vascular injury and inhibits the proliferation of vascular smooth muscle cells.

Neuregulin-1 (NRG-1) is expressed in vascular endothelial cells, and its receptors are localized to the underlying smooth muscle cells. However, the role of NRG-1 in vascular function and injury is largely unknown. First, the expression of NRG-1 and its receptors (erbB receptors) was analyzed after balloon injury to the rat carotid artery. NRG-1 and erbB expression levels were low in uninjured vessels; however, NRG-1 and erbB4 were upregulated following injury. We then examined the effect of NRG-1 on neointimal formation following balloon injury. NRG-1 was administered by tail-vein injection prior to injury and every 2 days following injury. Two weeks after injury, NRG-1-treated animals demonstrated a 50% reduction in lesion size compared with controls receiving the vehicle. To examine possible mechanisms for NRG-1 action, we examined its effects on vascular smooth muscle cell (VSMC) function. Rat VSMC cultures were pretreated with NRG-1 for 24 h and then stimulated with platelet-derived growth factor. NRG-1 significantly decreased platelet-derived growth factor-stimulated VSMC proliferation and migration. These findings suggest that NRG-1 may be a novel therapeutic candidate for the treatment of restenosis and atherosclerosis.

Neuroprotection by neuregulin-1 following focal stroke is associated with the attenuation of ischemia-induced pro-inflammatory and stress gene expression.

Neuregulins are a family of growth factors with potent neuroprotective properties. We recently demonstrated that neuregulin-1 blocked delayed neuronal death following focal ischemic stroke in the rat. Focal ischemia results in the release of pro-inflammatory cytokines that produce profound changes in gene expression and contribute to cell death associated with stroke. Inflammatory and stress mediators are involved in the pathogenesis of focal ischemic brain damage. We examined whether neuregulin-1 can influence inflammatory and stress gene expression in the rat brain following transient middle cerebral artery occlusion (MCAO). In this study, we compared gene expression profiles in animals treated with neuregulin-1beta (NRG-1) or vehicle followed by MCAO. We used the Affymetrix GeneChip system to analyze gene expression in focal ischemia of the rat brain. Several inflammatory and stress genes were significantly induced following MCAO compared to sham controls including heat shock protein-70 (HSP70), interleukin-1beta, and macrophage chemotattractant protein-1 (JE/MCP-1). Treatment with NRG-1 attenuated the expression of many of these genes by 50% or more. In vitro studies demonstrated that NRG-1 suppressed inflammatory gene expression in activated macrophages. NRG-1 also prevented neuronal death induced by oxygen-glucose deprivation in a rat neuroblastoma cell line, suggesting that NRG-1 may have both direct and indirect neuroprotective capacity. These results demonstrate that NRG-1 can regulate inflammatory and stress gene expression and may give new insight to the molecular mechanisms involved in the neuroprotective role of neuregulins in stroke.