Cocaine Increases Glycolysis Activity in Microglia in Vitro and in Vivo
Poster #: 170
Session/Time: A
Author:
Kelly Carter, BS
Mentor:
Ming-Lei Guo, MD, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
Cocaine, one of the most abused drugs throughout the world, is capable of activating microglia (Mg) in vitro and in vivo. However, the detailed mechanisms underlying cocaine-mediated Mg activation remain much elusive. Recently, accumulating evidence showed that glycolysis is inherently involved in immune responses in Mg (immunometabolism). Increase in anaerobic glycolysis activity could promote Mg activation under various stimuli. Whether cocaine can modulate glycolysis activity in Mg has never been explored. In this investigation, we explored the effects of cocaine on glycolysis activity by determining the expression levels of glucose transports (Glut) 1, 3, 5 and hexokinase (HK) 1/2 in BV2 cells and primary microglia (PM). Also, we determined the effects of cocaine on Glut1 in Mg in vivo.
METHODS:
BV2 cells were maintained and cultured in vitro. PM were isolated from the new-born mouse pups (24 hours) and cultured in vitro for 10 days. BV2 and PM were treated with cocaine (10 µM) for varying time periods or for 24 hours with different doses (1, 10, 100 µM). After the treatments, the cells were collected for protein extraction. The samples were prepared to determine the expression of Glut1, 3, 5 and HK and hypoxia inducible factor 1 alpha (HIF1a) by Western blots. Wild type (C57BL/6) mice (3-month-old, male) were injected with saline or cocaine (20 mg/Kg, daily) for three weeks and sacrificed for brain removal. Followed, the brain cryosections were prepared for double immunostaining of Iba1 (Mg marker) and Glut1. The immunofluorescent images were captured by the Zeiss inverted fluorescent microscopy and processed/analyzed by Zenlite software or ImagJ. The results were analyzed by one-way ANNOVA and * p < 0.05 were considered significantly different among groups.
RESULTS:
Our data shows that cocaine can increase Glut1 and 5 in BV2 cells and PM in both time-course and dose- dependent manners. Cocaine also increases HK2 expression in vitro. For in vivo experiments, cocaine significantly increases Glut1 levels in Mg.
CONCLUSION:
Our findings demonstrate that cocaine can increase glycolysis activity in Mg implying glycolysis is implicated in cocaine-mediated Mg activation. For further experiments, we will use Glut1 specific inhibitor or siRNA-Glult1 to knock down Glut1 to explore whether Glut1 inhibition can mitigate cocaine-mediated Mg activation.
Cocaine, one of the most abused drugs throughout the world, is capable of activating microglia (Mg) in vitro and in vivo. However, the detailed mechanisms underlying cocaine-mediated Mg activation remain much elusive. Recently, accumulating evidence showed that glycolysis is inherently involved in immune responses in Mg (immunometabolism). Increase in anaerobic glycolysis activity could promote Mg activation under various stimuli. Whether cocaine can modulate glycolysis activity in Mg has never been explored. In this investigation, we explored the effects of cocaine on glycolysis activity by determining the expression levels of glucose transports (Glut) 1, 3, 5 and hexokinase (HK) 1/2 in BV2 cells and primary microglia (PM). Also, we determined the effects of cocaine on Glut1 in Mg in vivo.
METHODS:
BV2 cells were maintained and cultured in vitro. PM were isolated from the new-born mouse pups (24 hours) and cultured in vitro for 10 days. BV2 and PM were treated with cocaine (10 µM) for varying time periods or for 24 hours with different doses (1, 10, 100 µM). After the treatments, the cells were collected for protein extraction. The samples were prepared to determine the expression of Glut1, 3, 5 and HK and hypoxia inducible factor 1 alpha (HIF1a) by Western blots. Wild type (C57BL/6) mice (3-month-old, male) were injected with saline or cocaine (20 mg/Kg, daily) for three weeks and sacrificed for brain removal. Followed, the brain cryosections were prepared for double immunostaining of Iba1 (Mg marker) and Glut1. The immunofluorescent images were captured by the Zeiss inverted fluorescent microscopy and processed/analyzed by Zenlite software or ImagJ. The results were analyzed by one-way ANNOVA and * p < 0.05 were considered significantly different among groups.
RESULTS:
Our data shows that cocaine can increase Glut1 and 5 in BV2 cells and PM in both time-course and dose- dependent manners. Cocaine also increases HK2 expression in vitro. For in vivo experiments, cocaine significantly increases Glut1 levels in Mg.
CONCLUSION:
Our findings demonstrate that cocaine can increase glycolysis activity in Mg implying glycolysis is implicated in cocaine-mediated Mg activation. For further experiments, we will use Glut1 specific inhibitor or siRNA-Glult1 to knock down Glut1 to explore whether Glut1 inhibition can mitigate cocaine-mediated Mg activation.