Recent studies have demonstrated the potential for endogenous and transplanted neural stem/progenitor cells (NSPCs) to ameliorate the structural and behavioral deficits associated with cerebral ischemia in animal models
, providing a potential therapy for ischemic stroke. However, poor NSPC survival within the ischemic core and peri-infarct regions following stroke has hampered the benefits and applications of cell-based therapies
[18, 19]. Many factors are involved in the regulation of the biological behaviors of NSCs, including genetics, growth factors, neurotransmitters, stress, hormones, and environmental factors like hypoxia. Recent studies have shown that the availability of glucose, but not of oxygen, is a restricting factor for NSC survival and proliferation following hypoxic/ischemic damage
. Furthermore, the proliferation of certain developmental stage-specific cells, such as embryonic and postnatal NSCs, has been proven to be dependent on the glucose concentration under physiological and pathological conditions such as diabetes
[20–22]. It is increasingly evident that post-stroke hyperglycemia is associated with poor outcome, and seems to particularly affect outcome in patients without diabetes
[23, 24]. With regard to cerebral ischemia/reperfusion pathophysiology, it is reported that hyperglycemia exacerbates brain injury due to poor blood flow to the ischemic penumbra, accumulation of lactate and intracellular acidosis in the ischemic brain
[25–27], and enhancement of the inflammatory response
. Whether the harmful effects of hyperglycemia are mediated by exacerbating the ischemic injury in NSCs or NPCs is unclear. So far, little is known about the effect of high glucose on the proliferation of adult neural stem cells following in vitro ischemia. In this study, we found that exposure to high glucose induced apoptosis of NSCs in a dose-dependent manner and inhibited the viability and proliferation of NSCs following OGD in vitro. Furthermore, we observed prolonged activation of JNK/p38 MAPK, suppressed ERK1/2 activity, and an increased percentage of cells in G0/G1-phase in NSCs treated with high glucose. In conclusion, our results indicate that high glucose induces the apoptosis and inhibits the proliferation of NSCs following OGD in vitro, which may be associated with a prolonged activation of JNK/p38 MAPK pathways and a delay of the cell G1-S transition.
Since glucose concentrations can be controlled and the actions of extrinsic factors can be delineated in an in vitro culture system, we used immortalized adult NSCs to investigate the effects of high glucose on the proliferation of NSCs using a well-characterized in vitro OGD model. The NSCs were isolated from the hippocampus of adult Fisher 344 rats, widely used for a variety of research applications including drug development, studies of neurotoxicity, neurogenesis, electrophysiology, neurotransmitter and receptor functions, and CNS disorders. In NSC cultures, the majority of cells kept their neural stem and/or progenitor status during the different passages. Most in vitro models of ischemia using neuronal cultures have used OGD to mimic the reduced intracellular energy state that occurs in neuronal cells following permanent and transient cerebral ischemia
[28–30]. These models have been used to assess whether agents exacerbate or reduce in vitro neuronal ischemic injury
[31, 32]. However, the duration of OGD that was required to induce NSC ischemic injury was reported to vary in different in vitro models of ischemia. Additionally, it is common practice to culture cells in a sealed hypoxia chamber to mimic anoxic conditions, in which the O2 level is usually approximately 0%
, while the O2 level observed in the anoxic environment often remains unchanged between 0% and 1%. In our study, we used a tri-gas incubator to adjust the O2 level in the cultures to a constant level (1%), and determined appropriate anaerobic incubation times by modifying and incorporating features used in neuronal in vitro models of ischemia
. We thus determined that 6 h of OGD incubation mimicked cerebral hypoxic-ischemic injury.
In vitro systems used to study neuronal responses to changes in ambient glucose concentrations must consider that the glucose levels in vitro should be of practical relevance to the brain in vivo[35, 36]. The physiological or normal blood glucose concentration in vivo can range from 5.5–7.0 mM. Thus, 5.5 mM is usually recognized as “englycemic” in vitro culture conditions for CNS research. However, it is not applied to the in vitro NSC culture media that are usually used (e.g. DMEM/F12), which normally contain 17.5 mM glucose, a level perhaps seen in the plasma of obese ob/ob mice. At the beginning of our study, we were puzzled as to why the NSCs must be grown in media with such a high glucose concentration, rather than in media with lower glucose concentrations, such as 7.0 mM and 5.5 mM. To address this question, we conducted initial experiments using 7.0 mM and 5.5 mM glucose to mimic diabetic and physiological glucose levels in vivo, respectively. The viability of NSCs exposed to 5.5, 7.0, and 17.5 mM glucose medium for 24, 48 or 72 h was examined by MTS assay (Figure S1, see Additional file
1, available online). The relative increase in the number of NSCs in each group was represented by the ratio of 72 h viability to 24 h viability (Figure S2, see Additional file
1). We found that the NSCs could not be grown in the medium with 5.5 or 7.0 mM glucose, but grew well in the medium with 17.5 mM glucose. To evaluate the effects of high glucose on the survival and proliferation of NSCs following in vitro ischemia, 17.5 mM glucose was chosen as the control, and higher concentrations of glucose were contained in the experimental medium. Thus, we used in vitro concentrations of 27.75, 41.75, and 83.75 mM glucose, which are similar to in vivo levels of glucose under “diabetes mellitus”, “diabetic ketoacidosis”, and “hyperglycemia hyperosmolar status” conditions, respectively.
High glucose concentrations are known to have detrimental effects on many cell types, by impairing cellular functions and inducing cell apoptosis. High glucose has been shown to inhibit the proliferation, migration and in vitro angiogenic capacity of bone marrow-derived endothelial progenitor cells
 and to alter the regenerative potential of mesenchymal stem cells
. Furthermore, hyperglycemic conditions affect the proliferation and apoptosis of NPCs in the developing spinal neural tube, leading to abnormal development
. If elevated levels of glucose are detrimental to neuronal survival during ischemia, does high glucose (up to 40 mM) damage neurons and NPCs? In the present study, exposure to high glucose (up to 41.75 mM for 24 h) decreased viability and proliferation and increased apoptosis in NSCs following in vitro ischemia. Our results are consistent with the studies reported above, but we used different concentrations of glucose. Meanwhile, our study also showed that high glucose treatment consistently suppressed DNA duplication and cell division of NSCs following in vitro ischemia by blocking the G1-S transition of the cell cycle.
We further examined the regulatory effect of high glucose on the activation of signaling molecules from the MAPK pathways. MAPKs include three major families: extracellular signal-regulated kinases 1/2 (ERK 1/2), c-Jun N-terminal kinases (JNK), and p38 MAPKs (p38). Upon their activation by the phosphorylation of Thr and Tyr residues, MAPKs regulate cellular processes such as proliferation, survival/apoptosis, differentiation, development, adherence, motility, metabolism, and gene regulation
. In the central nervous system, MAPKs are relatively highly expressed. Previous studies suggested that the expression or phosphorylation levels of MAPKs drastically changed in post-ischemic brain tissues, and that the inhibition of MAPK cascades could alter the outcome of ischemic brain injury in in vitro and in vivo experimental models
[41, 42]. Therefore, we examined the phosphorylation levels of ERK1/2, JNK and p38 in NSCs exposed to different concentrations of glucose after OGD. We found that the level of p-ERK2 decreased, while the levels of p-p38 and p-JNK2 increased in the cells treated with the three higher glucose concentrations (27.75, 41.75, and 83.75 mM glucose) compared with the control. It has been reported that the role of ERK1/2 in ischemia-mediated neuronal death is disputable
. Despite the volume of evidence supporting that the elevation of p-ERK1/2 after ischemic injury is a detrimental effect essential for oxidative stress and inflammation-related cell death, numerous studies have demonstrated that ERK1/2 activation contributes to the protective effects of many neuroprotectants. Our results showed that high glucose decreased ERK2 phosphorylation in OGD NSCs, resulting in less proliferation. Because JNK2 and p38 are generally activated by the same stress signals, such as osmotic shock and heart shock, they are referred to as stress-activated protein kinases (SAPKs). Phosphorylation of the p38 pathway can induce cell apoptosis and inhibition of p38 with SB203580 can reduce cell death
[44, 45]. In addition, JNK also stimulates cell apoptosis and inhibits cell proliferation when it is activated by cell stress
. The increased levels of p-JNK2/p-p38 and the decreased level of p-ERK2 observed in our experiments may reflect a new balance between cell growth and cell death after cells are exposed to high glucose treatment following in vitro ischemia.