Cellular and Molecular Neurobiology

, Volume 39, Issue 1, pp 61–71 | Cite as

An In Vitro Model for Conditioning Lesion Effect

  • Elif Kaval Oğuz
  • Gürkan ÖztürkEmail author
Original Research


Axons of a peripheral nerve grow faster after an axotomy if it attains a prior injury a few days earlier. This is called conditioning lesion effect (CLE) and very much valued since it may provide new insights into neuron biology and axonal regeneration. There are established in vivo experimental paradigms to study CLE, however, there is a need to have an in vitro conditioning technique where CLE occurs in a maximally controlled environment. Mouse primary sensory neurons were isolated from lumbar 4–5 dorsal root ganglia and incubated at 37 °C on a silicon-coated watch glass that prevents cell attachment. After this conditioning period they were transferred to laminin coated culture dishes. Similar cultures were set up with freshly isolated neurons from control animals and from the animals that received a sciatic nerve cut 3 days earlier. All preparations were placed on a live cell imaging microscopy providing physiological conditions and photographed for 48 h. Axonal regeneration and neuronal survival was assessed. During the conditioning incubation period neurons remained in suspended aggregates and did not grow axons. The regeneration rate of the in vitro conditioned neurons was much higher than the in vivo conditioned and control preparations during the first day of normal incubation. However, higher regeneration rates were compromised by progressive substantial neuronal death in both types of conditioned cultures but not in the control preparations. By using neutralizing antibodies, we demonstrated that activity of endogenous leukemia inhibitory factor is essential for induction of CLE in this model.


Conditioning In vitro Neuron culture Axon regeneration Degeneration Leukemia inhibitory factor LIF 



This study was supported by Yüzüncü Yıl University, Directorate of Scientific Research Projects.

Author Contributions

Elif Kaval Oğuz: Conducting experiments, image analysis, drafting the manuscript. Gürkan Öztürk: Designing the experiments, statistics, revision of the manuscript. Both authors have reviewed the final version of the manuscript and approved it for publication.

Compliance with Ethical Standards

Conflict of interest

Authors declare that they have adhered to ethical standards in this study and that they have no conflict of interest to disclose.


  1. Banner LR, Patterson PH (1994) Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia. Proc Natl Acad Sci USA 91:7109–7113CrossRefGoogle Scholar
  2. Bauer S et al (2003) Leukemia inhibitory factor is a key signal for injury-induced neurogenesis in the adult mouse olfactory epithelium. J Neurosci 23:1792–1803CrossRefGoogle Scholar
  3. Bisby MA (1985) Enhancement of the conditioning lesion effect in rat sciatic motor axons after superimposition of conditioning and test lesions. Exp Neurol 90:385–394CrossRefGoogle Scholar
  4. Blesch A et al (2012) Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration: superiority to camp-mediated effects. Exp Neurol 235:162–173. CrossRefGoogle Scholar
  5. Bontioti EN, Kanje M, Dahlin LB (2003) Regeneration and functional recovery in the upper extremity of rats after various types of nerve injuries. J Peripher Nerv Syst 8:159–168CrossRefGoogle Scholar
  6. Cafferty WB et al (2001) Leukemia inhibitory factor determines the growth status of injured adult sensory neurons. J Neurosci 21:7161–7170CrossRefGoogle Scholar
  7. Cafferty WB, Gardiner NJ, Das P, Qiu J, McMahon SB, Thompson SW (2004) Conditioning injury-induced spinal axon regeneration fails in interleukin-6 knock-out mice. J Neurosci 24:4432–4443CrossRefGoogle Scholar
  8. Cengiz N, Ozturk G, Erdogan E, Him A, Oguz EK (2012) Consequences of neurite transection in vitro. J Neurotrauma 29:2465–2474. CrossRefGoogle Scholar
  9. Chen W et al (2016) Rapamycin-resistant mTOR activity is required for sensory axon regeneration induced by a conditioning lesion. eNeuro. Google Scholar
  10. Curtis R et al (1994) Retrograde axonal transport of LIF is increased by peripheral nerve injury: correlation with increased LIF expression in distal nerve. Neuron 12:191–204CrossRefGoogle Scholar
  11. Davoust J, Gruenberg J, Howell KE (1987) Two threshold values of low pH block endocytosis at different stages. EMBO J 6:3601–3609CrossRefGoogle Scholar
  12. Dowsing BJ, Morrison WA, Nicola NA, Starkey GP, Bucci T, Kilpatrick TJ (1999) Leukemia inhibitory factor is an autocrine survival factor for Schwann cells. J Neurochem 73:96–104CrossRefGoogle Scholar
  13. Dowsing BJ, Romeo R, Morrison WA (2001) Expression of leukemia inhibitory factor in human nerve following injury. J Neurotrauma 18:1279–1287CrossRefGoogle Scholar
  14. Ekstrom PA, Mayer U, Panjwani A, Pountney D, Pizzey J, Tonge DA (2003) Involvement of alpha7beta1 integrin in the conditioning-lesion effect on sensory axon regeneration. Mol Cell Neurosci 22:383–395CrossRefGoogle Scholar
  15. Forman DS, McQuarrie IG, Labore FW, Wood DK, Stone LS, Braddock CH, Fuchs DA (1980) Time course of the conditioning lesion effect on axonal regeneration. Brain Res 182:180–185CrossRefGoogle Scholar
  16. Franz CK et al (2009) A conditioning lesion provides selective protection in a rat model of amyotrophic lateral sclerosis. PLoS ONE 4:e7357. CrossRefGoogle Scholar
  17. Frey E, Valakh V, Karney-Grobe S, Shi Y, Milbrandt J, DiAntonio A (2015) An in vitro assay to study induction of the regenerative state in sensory neurons. Exp Neurol 263:350–363. CrossRefGoogle Scholar
  18. Gardiner NJ, Cafferty WB, Slack SE, Thompson SW (2002) Expression of gp130 and leukaemia inhibitory factor receptor subunits in adult rat sensory neurones: regulation by nerve injury. J Neurochem 83:100–109CrossRefGoogle Scholar
  19. Gardiner NJ, Moffatt S, Fernyhough P, Humphries MJ, Streuli CH, Tomlinson DR (2007) Preconditioning injury-induced neurite outgrowth of adult rat sensory neurons on fibronectin is mediated by mobilisation of axonal alpha5 integrin. Mol Cell Neurosci 35:249–260CrossRefGoogle Scholar
  20. Han PJ, Shukla S, Subramanian PS, Hoffman PN (2004) Cyclic AMP elevates tubulin expression without increasing intrinsic axon growth capacity. Exp Neurol 189:293–302CrossRefGoogle Scholar
  21. Hollis ER 2nd, Ishiko N, Tolentino K, Doherty E, Rodriguez MJ, Calcutt NA, Zou Y (2015) A novel and robust conditioning lesion induced by ethidium bromide. Exp Neurol 265:30–39. CrossRefGoogle Scholar
  22. Hu G, Huang K, Hu Y, Du G, Xue Z, Zhu X, Fan G (2016) Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons. Sci Rep 6:31851. CrossRefGoogle Scholar
  23. Hyatt Sachs H, Rohrer H, Zigmond RE (2010) The conditioning lesion effect on sympathetic neurite outgrowth is dependent on gp130 cytokines. Exp Neurol 223:516–522. CrossRefGoogle Scholar
  24. Ito Y et al (1998) Differential temporal expression of mRNAs for ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and their receptors (CNTFR alpha, LIFR beta, IL-6R alpha and gp130) in injured peripheral nerves. Brain Res 793:321–327CrossRefGoogle Scholar
  25. Jacob JM, McQuarrie IG (1993) Acceleration of axonal outgrowth in rat sciatic nerve at one week after axotomy. J Neurobiol 24:356–367CrossRefGoogle Scholar
  26. Kalous A, Keast JR (2010) Conditioning lesions enhance growth state only in sensory neurons lacking calcitonin gene-related peptide and isolectin B4-binding. Neuroscience 166:107–121. CrossRefGoogle Scholar
  27. Knott EP, Assi M, Pearse DD (2014) Cyclic AMP signaling: a molecular determinant of peripheral nerve regeneration. Biomed Res Int 2014:651625. CrossRefGoogle Scholar
  28. Kwon MJ et al (2013) Contribution of macrophages to enhanced regenerative capacity of dorsal root ganglia sensory neurons by conditioning injury. J Neurosci 33:15095–15108. CrossRefGoogle Scholar
  29. Kwon MJ et al (2015) CCL2 Mediates neuron-macrophage interactions to drive proregenerative macrophage activation following preconditioning injury. J Neurosci 35:15934–15947. CrossRefGoogle Scholar
  30. Kwon MJ, Yoon HJ, Kim BG (2016) Regeneration-associated macrophages: a novel approach to boost intrinsic regenerative capacity for axon regeneration. Neural Regen Res 11:1368–1371. Google Scholar
  31. Lankford KL, Waxman SG, Kocsis JD (1998) Mechanisms of enhancement of neurite regeneration in vitro following a conditioning sciatic nerve lesion. J Comp Neurol 391:11–29CrossRefGoogle Scholar
  32. Lu X, Richardson PM (1991) Inflammation near the nerve cell body enhances axonal regeneration. J Neurosci 11:972–978CrossRefGoogle Scholar
  33. Mar FM et al (2014) CNS axons globally increase axonal transport after peripheral conditioning. J Neurosci 34:5965–5970. CrossRefGoogle Scholar
  34. McQuarrie IG (1986) Structural protein transport in elongating motor axons after sciatic nerve crush. Effect of a conditioning lesion. Neurochem Pathol 5:153–164CrossRefGoogle Scholar
  35. McQuarrie IG, Grafstein B, Gershon MD (1977) Axonal regeneration in the rat sciatic nerve: effect of a conditioning lesion and of dbcAMP. Brain Res 132:443–453CrossRefGoogle Scholar
  36. Murphy M, Reid K, Hilton DJ, Bartlett PF (1991) Generation of sensory neurons is stimulated by leukemia inhibitory factor. Proc Natl Acad Sci USA 88:3498–3501CrossRefGoogle Scholar
  37. Navarro X, Kennedy WR (1990) The effect of a conditioning lesion on sudomotor axon regeneration. Brain Res 509:232–236CrossRefGoogle Scholar
  38. Niemi JP, DeFrancesco-Lisowitz A, Roldan-Hernandez L, Lindborg JA, Mandell D, Zigmond RE (2013) A critical role for macrophages near axotomized neuronal cell bodies in stimulating nerve regeneration. J Neurosci 33:16236–16248. CrossRefGoogle Scholar
  39. Niemi JP, DeFrancesco-Lisowitz A, Cregg JM, Howarth M, Zigmond RE (2016) Overexpression of the monocyte chemokine CCL2 in dorsal root ganglion neurons causes a conditioning-like increase in neurite outgrowth and does so via a STAT3 dependent mechanism. Exp Neurol 275(Pt 1):25–37. CrossRefGoogle Scholar
  40. Ozturk G, Erdogan E (2004) Multidimensional long-term time-lapse microscopy of in vitro peripheral nerve regeneration. Microsc Res Tech 64:228–242. CrossRefGoogle Scholar
  41. Ozturk G, Tonge DA (2001) Effects of leukemia inhibitory factor on galanin expression and on axonal growth in adult dorsal root ganglion neurons in vitro. Exp Neurol 169:376–385. CrossRefGoogle Scholar
  42. Qiu J, Cai D, Dai H, McAtee M, Hoffman PN, Bregman BS, Filbin MT (2002) Spinal axon regeneration induced by elevation of cyclic. AMP Neuron 34:895–903CrossRefGoogle Scholar
  43. Redshaw JD, Bisby MA (1987) Proteins of fast axonal transport in regenerating rat sciatic sensory axons: a conditioning lesion does not amplify the characteristic response to axotomy. Exp Neurol 98:212–221CrossRefGoogle Scholar
  44. Rigaud M, Gemes G, Barabas ME, Chernoff DI, Abram SE, Stucky CL, Hogan QH (2008) Species and strain differences in rodent sciatic nerve anatomy: implications for studies of neuropathic pain. Pain 136:188–201. CrossRefGoogle Scholar
  45. Sachs HH, Wynick D, Zigmond RE (2007) Galanin plays a role in the conditioning lesion effect in sensory neurons. Neuroreport 18:1729–1733CrossRefGoogle Scholar
  46. Saijilafu FQZ (2012) Genetic study of axon regeneration with cultured adult dorsal root ganglion neurons. J Vis Exp. Google Scholar
  47. Saijilafu EMH, Liu CM, Jiao Z, Xu WL, Zhou FQ (2013) PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1. Nat Commun 4:2690. CrossRefGoogle Scholar
  48. Salegio EA, Pollard AN, Smith M, Zhou XF (2011) Macrophage presence is essential for the regeneration of ascending afferent fibres following a conditioning sciatic nerve lesion in adult rats. BMC Neurosci 12:11. CrossRefGoogle Scholar
  49. Savastano LE, Laurito SR, Fitt MR, Rasmussen JA, Gonzalez Polo V, Patterson SI (2014) Sciatic nerve injury: a simple and subtle model for investigating many aspects of nervous system damage and recovery. J Neurosci Methods 227:166–180. CrossRefGoogle Scholar
  50. Scott RL, Gurusinghe AD, Rudvosky AA, Kozlakivsky V, Murray SS, Satoh M, Cheema SS (2000) Expression of leukemia inhibitory factor receptor mRNA in sensory dorsal root ganglion and spinal motor neurons of the neonatal rat. Neurosci Lett 295:49–53CrossRefGoogle Scholar
  51. Sjoberg J, Kanje M (1990a) Effects of repetitive conditioning crush lesions on regeneration of the rat sciatic nerve. Brain Res 530:167–169CrossRefGoogle Scholar
  52. Sjoberg J, Kanje M (1990b) The initial period of peripheral nerve regeneration and the importance of the local environment for the conditioning lesion effect. Brain Res 529:79–84CrossRefGoogle Scholar
  53. Smith DS, Skene JH (1997) A transcription-dependent switch controls competence of adult neurons for distinct modes of axon growth. J Neurosci 17:646–658CrossRefGoogle Scholar
  54. Tetzlaff W, Leonard C, Krekoski CA, Parhad IM, Bisby MA (1996) Reductions in motoneuronal neurofilament synthesis by successive axotomies: a possible explanation for the conditioning lesion effect on axon regeneration. Exp Neurol 139:95–106CrossRefGoogle Scholar
  55. Thomas PK (1970) The cellular response to nerve injury. 3. The effect of repeated crush injuries. J Anat 106:463–470Google Scholar
  56. Torigoe K, Hashimoto K, Lundborg G (1999) A role of migratory Schwann cells in a conditioning effect of peripheral nerve regeneration. Exp Neurol 160:99–108CrossRefGoogle Scholar
  57. Wu D et al (2007) Actions of neuropoietic cytokines and cyclic AMP in regenerative conditioning of rat primary sensory neurons. Exp Neurol 204:66–76CrossRefGoogle Scholar
  58. Zou H, Ho C, Wong K, Tessier-Lavigne M (2009) Axotomy-induced Smad1 activation promotes axonal growth in adult sensory neurons. J Neurosci 29:7116–7123. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of EducationYüzüncü Yıl UniversityVanTurkey
  2. 2.Physiology Department, International School of MedicineIstanbul Medipol UniversityIstanbulTurkey
  3. 3.Regenerative and Restorative Medicine Research Center (REMER)Istanbul Medipol UniversityIstanbulTurkey

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