Gunja K Pathak1, Helim Aranda-Espinoza1, Sameer B Shah2. 1. Fischell Department of Bioengineering, University of Maryland, College Park, MD, United States. 2. Fischell Department of Bioengineering, University of Maryland, College Park, MD, United States; Departments of Orthopaedic Surgery and Bioengineering, University of California, San Diego, La Jolla, CA, United States. Electronic address: sbshah@ucsd.edu.
Abstract
BACKGROUND: Studies of neuronal regeneration require examination of axons independently of their cell bodies. Several effective strategies have been deployed to compartmentalize long axons of the peripheral nervous system (PNS). However, current strategies to compartmentalize axons of the central nervous system (CNS) may be limited by physical damage to cells during tissue dissociation or slicing, perturbation of three-dimensional tissue architecture, or insufficient axonal tissue for biological analysis. NEW METHODS: We developed a novel mouse neonate whole-hippocampus explant culture system, to probe neuronal regeneration in the central nervous system. This system enables imaging, biological, and biophysical analysis of isolated axons. RESULTS: We validated this model by isolating pure axonal populations. Additionally, cells within the explant were viable and amenable to transfection. We implemented the explant system to characterize axonal outgrowth following crush injury to the explant at the time of harvest, and also a secondary axonal transection injury 2 days post-culture. The initial crush injury delayed axonal outgrowth; however, axotomy did not alter rates of outgrowth up to 1h post-injury, with or without initial tissue crush injury. COMPARISON WITH EXISTING METHODS: Our explant system addresses shortcomings of other strategies developed to compartmentalize CNS axons. It provides a simple method to examine axonal activity and function without requiring additional equipment to slice tissue or segregate axons. CONCLUSION: Our hippocampal explant model may be used to study axonal response to injury. We have demonstrated the feasibility of probing axonal biology, biochemistry, and outgrowth free from confounding effects of neuronal cell bodies.
BACKGROUND: Studies of neuronal regeneration require examination of axons independently of their cell bodies. Several effective strategies have been deployed to compartmentalize long axons of the peripheral nervous system (PNS). However, current strategies to compartmentalize axons of the central nervous system (CNS) may be limited by physical damage to cells during tissue dissociation or slicing, perturbation of three-dimensional tissue architecture, or insufficient axonal tissue for biological analysis. NEW METHODS: We developed a novel mouse neonate whole-hippocampus explant culture system, to probe neuronal regeneration in the central nervous system. This system enables imaging, biological, and biophysical analysis of isolated axons. RESULTS: We validated this model by isolating pure axonal populations. Additionally, cells within the explant were viable and amenable to transfection. We implemented the explant system to characterize axonal outgrowth following crush injury to the explant at the time of harvest, and also a secondary axonal transection injury 2 days post-culture. The initial crush injury delayed axonal outgrowth; however, axotomy did not alter rates of outgrowth up to 1h post-injury, with or without initial tissue crush injury. COMPARISON WITH EXISTING METHODS: Our explant system addresses shortcomings of other strategies developed to compartmentalize CNS axons. It provides a simple method to examine axonal activity and function without requiring additional equipment to slice tissue or segregate axons. CONCLUSION: Our hippocampal explant model may be used to study axonal response to injury. We have demonstrated the feasibility of probing axonal biology, biochemistry, and outgrowth free from confounding effects of neuronal cell bodies.