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

Abstract

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.

Figure 1

NRG-1 treatment reduces pMCAO-induced brain infarction. Representative TTC stained brain sections are shown where rats were injected with vehicle (a; n = 5), NRG-1 (b; n = 5) before pMCAO. Animals were killed 24 hours later and the brains were sliced into 2 mm sections and stained with 2,3,5-triphenyltetrazolium chloride (TTC). Infarct volumes in brains from vehicle and NRG-1 treated animals are shown in the graph (c). Values are presented as mean ± SD; * denotes significantly different from respective vehicle treated animals. (P< 0.01).

Figure 2

NRG-1 treatment reduces pMCAO-induced brain infarction. Infarct volumes in brains from vehicle and NRG-1 treated animals are shown in the graph. Values are presented as mean ± SD; * denotes significantly different from respective vehicle treated animals. (P< 0.01).

Figure 3

The percentage of cortex and stratum with infarction after treatment with NRG-1 or vehicle are shown in the graph. Values are presented as mean ± SD; * denotes significantly different from respective vehicle treated animals. (P< 0.01).

Figure 4

Combination treatment with NRG-1 and MK-801 results in enhanced neuroprotection following pMCAO. The percentage of cortex and stratum with infarction are shown in the graph (a) for control animals (n = 5), and animals treated with NRG-1 (n = 5), MK-801 (n = 5) or NRG + MK-801 (n = 5). Values are presented as mean ± SD; * denotes significantly different from respective vehicle treated animals. (P< 0.01); # denotes significantly treatment with NRG-1 or MK-801 alone. (P< 0.05). Representative TTC stained brain sections are shown where rats were injected with NRG-1 + MK-801 (b) before MCAO.

Figure 5

NRG-1 increases survival after ischemia in vitro. B-35 cells were subjected to 60 minutes of OGD 23 hours of reoxygenation. Cultures were treated with NRG-1, MK-801, NRG-1 + MK-801 or the vehicle control (CTRL) immediately before reoxygenation. Viability was measured using the live/dead assay. The graph illustrates the quantitative analysis of cell survival following the treatments. Each experiment was performed three or more times in triplicate. Values are represented as means ± SD; * denotes significantly different from control, ** denotes significantly different from OGD, ## denotes significantly different from OGD+NRG1, (p<0.05).