Journal of neuroscience methods · 2016
Improved method to track and precisely count Schwann cells post-transplantation in a peripheral nerve injury model.
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Gambhir Hardeep S, Raharjo Eko, Forden Joanne, Kumar Ranjan, Mishra Chinmaya, Guo Gui Fang, Grochmal Joey, Shapira Yuval, Midha Rajiv
Аннотация
BACKGROUND: To optimize survival evaluation of Schwann cells (SCs) in vivo, we tested fluorescent labeling of the nucleus as an improved method of tracking and counting the transplanted SCs at sciatic nerve injury sites in rodents. We also investigated if co-administering cells with the glial growth factor Neuregulin-1 β (NRG1β) improves in vivo survival.
NEW METHOD: We transduced SCs using a Lentiviral vector with a nuclear localization signal (NLS) fused with mCherry and transplanted them in the sciatic nerve of rat post-crush injury (bilateral) either in the presence or absence of NRG1β in the injectate media. For comparison, in a separate group of similar injury, GFP-labeled cells were transplanted. After 10 days, nerves were harvested and sections (14μm) were counterstained with Hoechst and imaged. Cells showing co-localization with Hoechst and GFP or mCherry were exhaustively counted and data analyzed.
RESULTS: Percentage cells counted in with- and without-NRG condition in both the groups were 0.83±0.13% and 0.06±0.04% (Group 1) & 2.83*±1.95% and 0.23*±0.29% (Group 2).
COMPARISON TO EXISTING METHOD: We are introducing fluorescent labeling of the nucleus as a reliable and efficient technique to perform survival assessments in Schwann cell based treatment studies in animal model. This method can overcome the challenges and limitations of the existing method that could result in underestimation of the therapeutic outcome.
CONCLUSIONS: Nucleus-restricted fluorescent labeling technique offer improved method of tracking as well as accurately counting transplanted SCs in vivo while NRG1β in the injectate media can improve survival.
NEW METHOD: We transduced SCs using a Lentiviral vector with a nuclear localization signal (NLS) fused with mCherry and transplanted them in the sciatic nerve of rat post-crush injury (bilateral) either in the presence or absence of NRG1β in the injectate media. For comparison, in a separate group of similar injury, GFP-labeled cells were transplanted. After 10 days, nerves were harvested and sections (14μm) were counterstained with Hoechst and imaged. Cells showing co-localization with Hoechst and GFP or mCherry were exhaustively counted and data analyzed.
RESULTS: Percentage cells counted in with- and without-NRG condition in both the groups were 0.83±0.13% and 0.06±0.04% (Group 1) & 2.83*±1.95% and 0.23*±0.29% (Group 2).
COMPARISON TO EXISTING METHOD: We are introducing fluorescent labeling of the nucleus as a reliable and efficient technique to perform survival assessments in Schwann cell based treatment studies in animal model. This method can overcome the challenges and limitations of the existing method that could result in underestimation of the therapeutic outcome.
CONCLUSIONS: Nucleus-restricted fluorescent labeling technique offer improved method of tracking as well as accurately counting transplanted SCs in vivo while NRG1β in the injectate media can improve survival.