Towards an automated chemical screen for modulators of brain repair in zebrafish

Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom

Traumatic Brain Injury (TBI) is a leading cause of death around the world and occurs when an external physical force damages the brain (1). After the initial trauma of the primary injury, damage continues to spread from the wound site, exacerbated by noxious substances released from cells that die through the initial insult (2). Currently, there exists no pharmacological method of intervention to aid removal of these dead cells in the hours following injury.

Zebrafish have the ability to regenerate their brains following injury. One of the key processes that aids this is the clearance of dead cells, which in zebrafish larvae occurs in the first 48 hours following injury. The larval zebrafish's transparency enables us to monitor this process in vivo, and together with the advantages of using zebrafish as cost-effective vertebrate model for drug screening, makes performing a screen for modulators of dead cell removal an exciting proposition.

Here we present a screening setup that we established using the Vertebrate Automated Screening Platform (VAST), which will allow us to test potentially therapeutic compounds in an semi-automated high-content manner (3). Zebrafish larvae are injured manually and then incubated in small molecules for six hours, upon which they are automatically imaged using the VAST system linked to a spinning disc confocal microscope. The VAST system allows us to produce high-quality images showing the extent of tissue damage using the fluorescent nuclear report line Tg(H2A:GFP) and the extent of necrotic cell death using propidium iodide.

We have successfully established an assay which measures the number of dead cells following brain injury in zebrafish, and have validated our assay using a small molecule that blocks the removal of dead cells. This high-content screening assay will allow us to perform a medium-throughput chemical screen to identify modulators of dead cell clearance.

References

1. Abelson-Mitchell N. Epidemiology and prevention of head injuries: Literature review. J Clin Nurs. 2008;17(1):46-57.
2. Park E, Bell JD, Baker AJ. Traumatic brain injury: Can the consequences be stopped? Cmaj. 2008;178(9):1163-1170.
3. Pardo-Martin C, Chang T-Y, Koo BK, Gilleland CL, Wasserman SC, Yanik MF. High-throughput in vivo vertebrate screening. Nat Methods. 2010;7(8):634-636.

Funded by:  Rosetrees Trust, The Carnegie Trust