Cocaine affects astrocyte activation and iNOS expression

Abstract

It is estimated that there are more than two million people addicted to cocaine in the United States. How cocaine affects neuronal functions has been extensively studied. Astrocytes, an important member of the neurovascular unit, also play significant roles in brain pathology. However, their role in cocaine use disorder is unclear. Astrocytes are glial cells that support the neurons and maintain the blood‐brain barrier. Astrocytes can express inducible nitric oxide synthase (iNOS) that is responsible for the transient production of nitric oxide (NO). Both iNOS and NO have been reported to be actively involved in cocaine addiction. We hypothesize that cocaine can cause astrocyte activation and inflammation, resulting in iNOS upregulation, which can in turn, affect both neuronal and brain microvascular endothelial functions. To test this hypothesis, an in vitro astrocyte culture model and an in vivo chronic cocaine mouse model were used. In the in vitro model, human astrocytes from the cerebrocortex were treated with cocaine (10μM) (or lipopolysaccharide at 2μg/mL as the positive control) for 24 hours. iNOS expression was measured using solid‐phase ELISA. Astrocyte activation was assessed by immunofluorescence microscopy for Glial Fibrillary Acidic Protein (GFAP) expression. Changes in astrocyte morphology were quantified using a Sholl analysis. In the in vivo model, wild type C57BL/6 mice were treated with cocaine (30 mg/kg per day through intraperitoneal injection) for two weeks. Mouse brain tissue, especially in the prefrontal cortex, was collected and fixed with paraformaldehyde before slicing. Astrocyte activation and number were quantified using immunofluorescence microscopy (of GFAP). Astrocyte iNOS expression was measured using an iNOS ELISA kit (using collected mouse brain homogenate). The results demonstrated that lipopolysaccharide (LPS, the endotoxin) significantly increased iNOS expression after 24hr treatment. Cocaine, in comparison, had a more significant impact in iNOS upregulation in early passages of cultured astrocytes. Sholl analysis demonstrated that cocaine and LPS both induced significant morphological changes and reduced the number of astrocyte processes. Results obtained from the in vivo studies indicated that chronic cocaine treatment significantly decreased the number of astrocytes in the mouse prefrontal cortex, while increasing iNOS expression. In summary, astrocytes can respond to cocaine stimulation in vitro and in vivo, likely through iNOS‐medicated inflammatory responses. Therefore, investigation in the role of astrocytes in cocaine addiction may bring insight to new therapeutic target to treat cocaine use disorder.