Effect of ketamine on apoptosis by energy deprivation in astroglioma cells using flow cytometry system

Abstract

Apoptosis is a programmed, physiologic mode of cell death that plays an important role in tissue homeostasis. As for the central nervous system, ischemic insults can induce pathophysiologic cascade of apoptosis in neurophils. Impairment of astrocyte functions during brain ischemia can critically influence neuron survival by neuronglia interactions. We aimed to elucidate the protective effect of ketamine on apoptosis by energy deprivation in astrocytes. Ischemic insults was induced with iodoacetate/ carbonylcyanide m-chlorophenylhydrazone (IAA/CCCP) 1.5 mM/20 microm or 150 microm/2 microm for 1 hr in the HTB-15 and CRL-1690 astrocytoma cells. Then these cells were reperfused with normal media or ketamine (0.1 mM) containing media for 1 hr or 24 hr. FITC-annexin-V staining and propidium iodide binding were determined by using flow cytometry. Cell size and granularity were measured by forward and side light scattering properties of flow cytometry system, respectively. An addition of ketamine during reperfusion increased the proportion of viable cells. Ketamine alleviated cell shrinkage and increased granularity during the early period, and ameliorated cell swelling during the late reperfusion period. Ketamine may have a valuable effect on amelioration of early and late apoptosis in the astrocytoma cells, even though the exact mechanism remains to be verified.

Fig. 1

Bivariate PI/annexin-V analysis of the CRL-1690 cells during the reperfusion for 1 hr after IAA/CCCP treatment. Quadrant % gated in this assay identify the different cell populations, i.e. region UL: PI-positive/ annexin-V-negative, UR: PI-positive/ annexin-V-positive, LL: PI-negative/annexin-V-negative, LR: PI-negative/ annexin-V-positive. (A) and (B) indicate the effect of different concentrations of IAA/CCCP (150 µM/2 µM vs. 1.5 mM/20 µM) on the cell apoptosis.

Fig. 2

The effect of ketamine on the apoptosis of the CRL-1690 cells during the reperfusion for 1 hr Ketamine 0.1 mM addition during the reperfusion increased cell viability after both low (A) and high concentration (B) IAA/CCCP treatment.

Fig. 3

Bivariate PI/annexin V analysis of the CRL-1690 cells during the reperfusion for 24 hr. A, B, C indicate cells in normal media for 24 hr, in reperfusion for 24 hr after IAA/CCCP (150 µM/2 µM) treatment for 1 hr and in reperfusion with ketamine 0.1 mM containing media after IAA/CCCP treatment, respectively. (A) and (B) are examples of many experiments.

Fig. 4

The effect of ketamine on the changes of size and granularity of the HTB-15 cells. Forward light scattering properties measured the size of cells in the normal media for 1 hr (1), in reperfusion for 1 hr after IAA/CCCP (150 µM/2 µM) treatment for 1 hr (2), and in reperfusion with ketamine 0.1 mM containing media for 1 hr after IAA/CCCP treatment (3), respectively (A). Side light scattering properties measured the granularity of cells (B) in the normal media for 1 hr (1), in reperfusion for 1 hr after IAA/CCCP (150 µM/2 µM) treatment for 1 hr (2), and in reperfusion with ketamine 0.1 mM containing media for 1 hr after IAA/CCCP treatment (3).