Establishment of a Reproducible Ischemic Stroke Model in Nestin-GFP Mice with High Survival Rates.

An accumulation of evidence shows that endogenous neural stem/progenitor cells (NSPCs) are activated following brain injury such as that suffered during ischemic stroke. To understand the expression patterns of these cells, researchers have developed mice that express an NSPC marker, Nestin, which is detectable by specific reporters such as green fluorescent protein (GFP), i.e., Nestin-GFP mice. However, the genetic background of most transgenic mice, including Nestin-GFP mice, comes from the C57BL/6 strain. Because mice from this background strain have many cerebral arterial branches and collateral vessels, they are accompanied by several major problems including variable ischemic areas and high mortality when subjected to ischemic stroke by occluding the middle cerebral artery (MCA). In contrast, CB-17 wild-type mice are free from these problems. Therefore, with the aim of overcoming the aforementioned defects, we first crossed Nestin-GFP mice (C57BL/6 background) with CB-17 wild-type mice and then developed Nestin-GFP mice (CB-17 background) by further backcrossing the generated hybrid mice with CB-17 wild-type mice. Subsequently, we investigated the phenotypes of the established Nestin-GFP mice (CB-17 background) following MCA occlusion; these mice had fewer blood vessels around the MCA compared with the number of blood vessels in Nestin-GFP mice (C57BL/6 background). In addition, TTC staining showed that infarcted volume was variable in Nestin-GFP mice (C57BL/6 background) but highly reproducible in Nestin-GFP mice (CB-17 background). In a further investigation of mice survival rates up to 28 days after MCA occlusion, all Nestin-GFP mice (CB-17 background) survived the period, whereas Nestin-GFP mice (C57BL/6 background) frequently died within 1 week and exhibited a higher mortality rate. Immunohistochemistry analysis of Nestin-GFP mice (CB-17 background) showed that GFP+ cells were mainly obverted in not only conventional neurogenic areas, including the subventricular zone (SVZ), but also ischemic areas. In vitro, cells isolated from the ischemic areas and the SVZ formed GFP+ neurosphere-like cell clusters that gave rise to various neural lineages including neurons, astrocytes, and oligodendrocytes. However, microarray analysis of these cells and genetic mapping experiments by Nestin-CreERT2 Line4 mice crossed with yellow fluorescent protein (YFP) reporter mice (Nestin promoter-driven YFP-expressing mice) indicated that cells with NSPC activities in the ischemic areas and the SVZ had different characteristics and origins. These results show that the expression patterns and fate of GFP+ cells with NSPC activities can be precisely investigated over a long period in Nestin-GFP mice (CB-17 background), which is not necessarily possible with Nestin-GFP mice (C57BL/6 background). Thus, Nestin-GFP mice (CB-17 background) could become a useful tool with which to investigate the mechanism of neurogenesis via the aforementioned cells under pathological conditions such as following ischemic stroke.

Post-weaning mice fed exclusively milk have deficits in induction of long-term depression in the CA1 hippocampal region and spatial learning and memory.

Previously, we have found that post-weaning mice fed exclusively milk display low-frequency exploratory behavior compared to mice fed a food pellet diet (Ishii et al., 2005a). Because cognitive functions play a key role in animal exploration, in the present study we examined the effect of an exclusively milk formula diet on spatial learning and memory in a water maze and also on induction of long-term potentiation (LTP) and long-term depression (LTD) at the Schaffer collateral-CA1 synapse in the hippocampus. Exclusively milk-fed mice exhibited slower learning and memory deficits in hidden water maze tests as compared with pellet-fed mice. Moreover, milk-fed mice showed a significant inhibition of LTD but a normal induction of LTP. Despite these functional deficits, adult neurogenesis in the dentate gyrus of the hippocampus, which has been proposed to have a causal relationship to spatial memory, was stimulated in milk-fed mice. These result suggest that an exclusively milk formula diet after weaning leads to a stimulation of hippocampal neurogenesis but causes deficits in the induction of LTD in the CA1 hippocampal region and impairment of spatial learning and memory.