Gene delivery to overcome astrocyte inhibition of axonal growth: An in vitro Model of the glial scar

• Published in: Biotechnology and bioengineering 2013-03, Vol.110 (3), p.947-957
• Main Authors: Tuinstra, Hannah M., Ducommun, Melissa M., Briley, William E., Shea, Lonnie D.
• Format: Article
• Language: English
• Subjects: Animals; astrocyte interface; astrocyte/neuron co-culture; Astrocytes - physiology; Cell Line; Chondroitin Sulfate Proteoglycans - antagonists & inhibitors; Chondroitinases and Chondroitin Lyases - metabolism; Cicatrix - physiopathology; CSPG inhibition; Enzymes; gene delivery; Gene Silencing; Gene Transfer Techniques; Genes; Genetic Vectors; Intervention; Lentivirus - enzymology; Lentivirus - genetics; Models, Theoretical; Nervous system; Neurons - physiology; Rats; Ribonucleic acid; RNA; RNA, Small Interfering - genetics; Scars; Transduction, Genetic; Transformation, Genetic
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.24750

After injury to the central nervous system, a glial scar develops that physically and biochemically inhibits axon growth. In the scar, activated astrocytes secrete inhibitory extracellular matrix, of which chondroitin sulfate proteoglycans (CSPGs) are considered the major inhibitory component. An inhibitory interface of CSPGs forms around the lesion and prevents axons from traversing the injury, and decreasing CSPGs can enhance axon growth. In this report, we established an in vitro interface model of activated astrocytes and subsequently investigated gene delivery as a means to reduce CSPG levels and enhance axon growth. In the model, a continuous interface of CSPG producing astrocytes was created with neurons seeded opposite the astrocytes, and neurite crossing, stopping, and turning were evaluated as they approached the interface. We investigated the efficacy of lentiviral delivery to degrade or prevent the synthesis of CSPGs, thereby removing CSPG inhibition of neurite growth. Lentiviral delivery of RNAi targeting two key CSPG synthesis enzymes, chondroitin polymerizing factor and chondroitin synthase‐1, decreased CSPGs, and reduced inhibition by the interface. Degradation of CSPGs by lentiviral delivery of chondroitinase also resulted in less inhibition and more neurites crossing the interface. These results indicate that the interface model provides a tool to investigate interventions that reduce inhibition by CSPGs, and that gene delivery can be effective in promoting neurite growth across an interface of CSPG producing astrocytes. Biotechnol. Bioeng. 2013; 110: 947–957. © 2012 Wiley Periodicals, Inc. An in vitro neuronal co‐culture model was developed to simulate the glial scar observed in vivo, which physically and biochemically inhibits axon growth. The authors investigated gene therapy approaches to overcome this inhibition, which included miRNA to reduce deposition of chondroitin sulfate proteoglycans and expression of the enzyme chondroitinase to degrade the inhibitory ECM.