An In Vitro Model for Conditioning Lesion Effect
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.
KeywordsConditioning In vitro Neuron culture Axon regeneration Degeneration Leukemia inhibitory factor LIF
This study was supported by Yüzüncü Yıl University, Directorate of Scientific Research Projects.
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.
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