M. Eldeiry1, K. Yamanaka1, L. S. Foley1, X. Meng1, M. Aftab1, M. J. Weyant1, J. C. Cleveland1, D. A. Fullerton1, T. B. Reece1 1University Of Colorado Denver,Aurora, CO, USA
Introduction:
Spinal cord ischemia is a dreaded complication of complex aortic repairs that can result in paraplegia in up to 10-20% of cases. Erythropoietin plays an important, but still unclear role in protection of the spinal cord during ischemic injury. This role is elucidated by heterodimerization of the erythropoietin receptor (EpoR) with the beta common receptor (βCR) as previously demonstrated in a murine model of spinal cord ischemia-reperfusion injury. However, the cell-specific role and mechanism of βCR and EpoR upregulation and signaling have yet to be elucidated. The current model of neuronal injury employs oxygen and glucose starvation along with B27 neuronal supplement deprivation from the cell culture media. We wanted to evaluate the effects of glucose starvation and B27 deprivation on the support cell in the neuronal network, the astrocytes.
Methods:
Spinal cords were isolated from 3 day old neonatal mice. Following enzymatic tissue digestion and cell separation utilizing a density gradient, astrocytes were seeded onto a tissue culture until reaching confluence. The cells were subsequently enzymatically separated and re-seeded onto individual wells in a 24-well plate. After reaching maturity in 4-5 days the cells were treated with media that was either glucose depleted, deprived of B-27 neuronal supplement, or lacking both for 1, 2, and 4 hours. Cells were lysed and Western Blot analysis was used to evaluate the βCR and EpoR expression in the lysate.
Results:
Glucose starvation with or without B-27 deprivation led to a time-dependent increase in βCR expression with a peak at 2 hours (1.8 ± 0.7 and 1.9 ± 0.6 vs. 1.0 ± 0.1, p < 0.05, Figure a). Alternatively, glucose starvation alone did not seem to affect EpoR expression (0.8 ± 0.2, 1.0 ± 0.2, 1.0 ± 0.3 vs. 1.0 ± 0.2 , p > 0.99). However, B27 deprivation caused EpoR expression to be undetectable whether glucose was present during the treatment (Figure b).
Conclusion:
Creating in vitro models to gain a better understanding of the cellular biology of spinal cord ischemia reperfusion requires certain assumptions to be made. In this study, we evaluated the role of glucose starvation along with B27 supplement deprivation. Two patterns were observed from this study. Glucose starvation causes a time-dependent increase in βCR expression without affecting EpoR expression. On the other hand, B27 deprivation doesn’t affect βCR expression but does seem to cause EpoR expression to be undetectable. These data give insight into the model of neuronal injury and its effect on the astrocytes that can be used to further guide studying of this complex neuronal network.
