Effect of BDNF and Other Potential Survival Factors in Models of In Vitro Oxidative Stress on Adult Spinal Cord–Derived Neural Stem/Progenitor Cells

Abstract

Transplantation of neural stem/progenitor cells (NSPCs) is a promising strategy in spinal cord injury (SCI). However, poor survival of transplanted stem cells remains a major limitation of this therapy due to the hostile environment of the injured cord. Oxidative stress is a hallmark in the pathogenesis of SCI; however, its effects on NSPCs from the adult spinal cord have yet to be examined. We therefore developed in vitro models of mild and severe oxidative stress of adult spinal cord–derived NSPCs and used these models to examine potential cell survival factors. NSPCs harvested from the adult rat spinal cord were treated with hydrogen peroxide (H2O2) in vitro to induce oxidative stress. A mild 4 h exposure to H2O2 (500 μM) significantly increased the level of intracellular reactive oxygen species with minimal effect on viability. In contrast, 24 h of oxidative stress led to a marked reduction in cell survival. Pretreatment with brain-derived neurotrophic factor (BDNF) for 48 h attenuated the increase in intracellular reactive oxygen species and enhanced survival. This survival effect was associated with a significant reduction in the number of apoptotic cells and a significant increase in the activity of the antioxidant enzymes glutathione reductase and superoxide dismutase. BDNF treatment had no effect on NSPC differentiation or proliferation. In contrast, cyclosporin A and thyrotropin-releasing hormone had minimal or no effect on NSPC survival. Thus, these models of in vitro oxidative stress may be useful for screening neuroprotective factors administered prior to transplantation to enhance survival of stem cell transplants.

Glutamate Increases In Vitro Survival and Proliferation and Attenuates Oxidative Stress-Induced Cell Death in Adult Spinal Cord-Derived Neural Stem/Progenitor Cells via Non-NMDA Ionotropic Glutamate Receptors

“…Despite this, little is known about the effect of glutamate and its various receptors on neural stem cells derived from the adult spinal cord. [13,14] A thorough investigation is necessary given the potential age and region-specific differences in stem cell responses to glutamate. Also, the potential for glutamate to enhance survival of neural stem cells makes it of great interest to examine the effect of glutamate in the setting of other components of the secondary injury of SCI that are known to impair NSPC survival such as oxidative stress.…”

“…Also, the potential for glutamate to enhance survival of neural stem cells makes it of great interest to examine the effect of glutamate in the setting of other components of the secondary injury of SCI that are known to impair NSPC survival such as oxidative stress. [13] The present study examined the effects of glutamate and various glutamate receptor agonists on adult rat spinal cord-derived neural stem/progenitor cells both alone and in the setting of oxidative stress in vitro. We show for the first time that: (1) glutamate increases survival and proliferation of adult spinal cord-derived NSPCs via AMPA/kainate receptors, (2) concurrent glutamate exposure attenuates oxidative stressinduced cell death in adult NSPCs, (3) glutamate-mediated protection against oxidative stress is dependent on AMPA/kainate receptors.…”

Evaluation of the effects of riluzole on adult spinal cord‐derived neural stem/progenitor cells in vitro and in vivo

“…Riluzole (Sigma) was dissolved in 30% (w/v) of 2‐hydroxypropyl‐b‐cyclodextrin (HBC; Sigma) to reach a final concentration of 8 mg/mL and filtered using a 0.22 μm syringe as previously described (Wu et al, 2010). In order to examine the effects of riluzole on oxidative stress, an in vitro model of oxidative stress using H 2 O 2 that was previously developed by our laboratory was used (Hachem et al, 2015). For the glutamate studies, l ‐glutamic acid (Sigma) was dissolved in water and filtered prior to adding to each well.…”

“…The generation of intracellular ROS was measured using dihydroethidium (DHE) staining and cell viability was assessed using the live/dead assay (Invitrogen), as we have previously described (Hachem et al, 2015). Membrane integrity was assessed using the lactate dehydrogenase (LDH) assay (Roche) and absorbance was measured using an ultraviolet plate reader.…”

Unlocking the paradoxical endogenous stem cell response after spinal cord injury

“…Previous studies have demonstrated that ependymal NSPCs are capable of producing BDNF and furthermore respond positively to exogenous BDNF treatment. 36 In light of these findings, it is possible that AMPA receptor-mediated proliferation and survival of NSPCs relies on a BDNF-dependent autocrine mechanism. However, further work is necessary to delineate the exact downstream pathways involved in this response.…”