The Response of Dorsal Root Ganglion Axons to Nerve Growth Factor Gradients Depends on Spinal Level Irina Vetter, 1, * Zac Pujic, 1, * and Geoffrey J. Goodhill 1,2 Abstract Directed sensory axon regeneration has the potential to promote functional recovery after peripheral nerve injury. Using a novel guidance assay to generate precisely controllable nerve growth factor gradients, we show for the first time that the guidance and outgrowth response of rat dorsal root ganglion neurons to identical nerve growth factor gradients depends on the rostrocaudal origin of the dorsal root ganglion explant. These findings have implications for the study of peripheral nerve regeneration in response to exogenous neurotrophins such as nerve growth factor, and provide new insight into the clinical potential of nerve growth factor in the treatment of nerve injury. Key words: axon guidance; dorsal root ganglion; nerve growth factor; regeneration; spinal level Introduction N erve injury is associated with significant morbidity due to the inability of neurons to appropriately re- connect with their targets. A therapeutic aim after peripheral nerve injury is thus to re-establish connectivity by aiding in axonal regeneration and correct axonal guidance. Neuro- trophic factors such as nerve growth factor (NGF) are of clinical interest, as they promote neuronal survival and pro- vide trophic support to prenatal and adult neurons (Verge et al., 1996). Furthermore, when present in gradients, NGF functions as a neuronal guidance cue, and may thus be useful in promoting regrowth of peripheral axons to the correct targets (Gundersen and Barrett, 1979; Letourneau, 1978; Moore et al., 2006; Rosoff et al., 2004). In the treatment of peripheral nerve injury, administration of NGF increases nerve regeneration and prevents axotomy-induced neuronal changes, highlighting the therapeutic potential of neuro- trophins for enhancement of functional recovery (Savignat et al., 2008; Verge et al., 1996). The level of the spinal cord at which injury occurs is crucial in determining functional losses and physiological effects. However, the effect of spinal level on NGF-mediated neurite regeneration and the guidance response of peripheral neurons to gradients of NGF have not been systematically assessed. Such information is important for the in vitro study of pe- ripheral nerve regeneration in response to NGF, and also for assessing the therapeutic potential of NGF based on the level of the spinal cord at which injury occurred. Limitations of available guidance assays (Pujic et al., 2009), such as the dif- ficulty of precisely controlling gradient steepness and con- centration at the explant, have precluded systematic assessment to date. We recently introduced a novel guidance assay allowing generation of precisely controlled gradient conditions (Mortimer et al., 2009; Rosoff et al., 2005, 2004). Using this assay we have shown that, averaging over dorsal root ganglia (DRG) from several spinal levels, the effect of exquisitely small differences in gradient parameters can be seen in differential guidance responses of DRGs to NGF gra- dients (Mortimer et al., 2009). Using this assay we now show that for identical gradient parameters, the guidance and out- growth response of DRG explants to NGF gradients varies significantly depending on spinal level. This has important implications not only for the in vitro study of NGF in pe- ripheral nerve regeneration, but also for the application of NGF gradients to the treatment and prognosis of nerve injury in vivo. Methods Neurite guidance assay Experiments involving animals were conducted in accor- dance with the Animal Care and Protection Act Qld (2002), and the Australian Code of Practice for the Care and Use of Ani- mals for Scientific Purposes, 7th edition (2004). Ethics approval was obtained from the University of Queensland Ethics Committee. 1 The Queensland Brain Institute, and 2 School of Mathematics and Physics, The University of Queensland, Brisbane, Queensland, Australia. *These authors contributed equally to this work. JOURNAL OF NEUROTRAUMA 27:1379–1386 (August 2010) ª Mary Ann Liebert, Inc. DOI: 10.1089/neu.2010.1279 1379