Abstract
BackgroundTissue injury has been employed to study diverse biological processes such as regeneration and inflammation. In addition to physical or surgical based methods for tissue injury, current protocols for localized tissue damage include laser and two-photon wounding, which allow a high degree of accuracy, but are expensive and difficult to apply. In contrast, electrical injury is a simple and inexpensive technique, which allows reproducible and localized cell or tissue damage in a variety of contexts.ResultsWe describe a novel technique that combines the advantages of zebrafish for in vivo visualization of cells with those of electrical injury methods in a simple and versatile protocol which allows the study of regeneration and inflammation. The source of the electrical pulse is a microelectrode that can be placed with precision adjacent to specific cells expressing fluorescent proteins. We demonstrate the use of this technique in zebrafish larvae by damaging different cell types and structures. Neurectomy can be carried out in peripheral nerves or in the spinal cord allowing the study of degeneration and regeneration of nerve fibers. We also apply this method for the ablation of single lateral line mechanosensory neuromasts, showing the utility of this approach as a tool for the study of organ regeneration. In addition, we show that electrical injury induces immune cell recruitment to damaged tissues, allowing in vivo studies of leukocyte dynamics during inflammation within a confined and localized injury. Finally, we show that it is possible to apply electroablation as a method of tissue injury and inflammation induction in adult fish.ConclusionsElectrical injury using a fine microelectrode can be used for axotomy of neurons, as a general tissue ablation tool and as a method to induce a powerful inflammatory response. We demonstrate its utility to studies in both larvae and in adult zebrafish but we expect that this technique can be readily applied to other organisms as well. We have called this method of electrical based tissue ablation, electroablation.
Highlights
Tissue injury has been employed to study diverse biological processes such as regeneration and inflammation
A simple methodology for electrical tissue injury Our aim was to develop a simple protocol for inducing localized tissue damage for the study of inflammation and regeneration of different cell types in zebrafish
We have taken advantage of the numerous transgenic lines available that label organs and tissues with fluorescent proteins, and that these can be readily visualized in the live zebrafish due to the optical transparency of the embryos and larvae
Summary
Tissue injury has been employed to study diverse biological processes such as regeneration and inflammation. In addition to physical or surgical based methods for tissue injury, current protocols for localized tissue damage include laser and two-photon wounding, which allow a high degree of accuracy, but are expensive and difficult to apply. Electrical injury is a simple and inexpensive technique, which allows reproducible and localized cell or tissue damage in a variety of contexts. Electrical injury provides a simple and inexpensive method for tissue injury, which allows modulating the extent of damage produced by modifying current intensity and number of pulses. Strong electrical fields alter the conformation of membrane proteins, as they undergo conformational changes in order to orientate their dipole moment in the direction of the field [13] This effect, called electroconformational denaturation, is especially strong in voltage gated ion channels [17]. Since the force of an electric field drops off quadratically with distance from the focal point, electrical injury is a suitable approach circumscribing the damage to a small area of tissue [18,19,20,21]
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