title: Improved method to track and precisely count Schwann cells post-transplantation in a peripheral nerve injury model.
abstract: 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.
  
  
  
  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.
  
  
  
  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).
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Clinical Neurosciences and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: Hardeep S
authors.initials: HS
authors.lastname: Gambhir
authors.affiliation: Department of Comparative Biology and Experimental Medicine and Alberta Children Hospital Research Institute, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada.
authors.forename: Eko
authors.initials: E
authors.lastname: Raharjo
authors.affiliation: Department of Clinical Neurosciences and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: Joanne
authors.initials: J
authors.lastname: Forden
authors.affiliation: Department of Clinical Neurosciences and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada; Department of Comparative Biology and Experimental Medicine and Alberta Children Hospital Research Institute, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada.
authors.forename: Ranjan
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Department of Clinical Neurosciences and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: Chinmaya
authors.initials: C
authors.lastname: Mishra
authors.affiliation: Department of Clinical Neurosciences and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: Gui Fang
authors.initials: GF
authors.lastname: Guo
authors.affiliation: Department of Clinical Neurosciences and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: Joey
authors.initials: J
authors.lastname: Grochmal
authors.affiliation: Department of Clinical Neurosciences and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Shapira
authors.affiliation: Department of Clinical Neurosciences and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Canada. Electronic address: rajiv.midha@ahs.ca.
authors.forename: Rajiv
authors.initials: R
authors.lastname: Midha
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002452:Cell Count; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D019913:Nuclear Localization Signals / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011917:Rats, Inbred Lew; D012583:Schwann Cells / Q000187:drug effects / Q000378:metabolism* / Q000637:transplantation*; D020426:Sciatic Neuropathy / Q000601:surgery*; D014161:Transduction, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D000097573:Red Fluorescent Protein
keywords: Fluorescent cell labeling; Neuregulin-1 β; Schwann cell; Sciatic nerve