A two-compartment organotypic model of mammalian peripheral nerve repair.
BACKGROUND
Schwann cells in the distal stump of transected nerve upregulate growth factors that support regeneration on a modality-specific basis. It is unclear, however, which of these preferentially support motor axon regeneration. Identification of these factors will require a model that can isolate growth factor effects to growing axons while reproducing the complex three-dimensional structure of peripheral nerve.


METHODS
A two-compartment PDMS base is topped by a collagen-coated membrane that supports a spinal cord cross-section above one compartment. Fluorescent motoneurons in this section reinnervate a segment of peripheral nerve that directs axons through a water-tight barrier to the second compartment, where nerve repair is performed.


RESULTS
Motoneurons remain healthy for several weeks. The axons they project through the water-tight barrier survive transection and cross a nerve repair in substantial numbers to reinnervate an additional nerve segment. Fluidic isolation of the two compartments was confirmed with a dye leakage test, and the physiologic integrity of the system was tested by retrograde labeling of only those motor neurons to which tracer was exposed and by limitation of toxin effects to a single compartment.


METHODS
Nerve repair cannot be modeled in monolayer cell culture. Our previous organotypic model accurately modeled nerve repair, but did not allow individual control of motoneuron and growth cone environments.


CONCLUSIONS
This model isolates treatment effects to growing axons while reproducing the complex three-dimensional structure of peripheral nerve. Additionally, it facilitates surgical manipulation of tissues and high-resolution imaging.
Department of Biomedical Engineering, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
Rezina
R
Siddique
Department of Orthopaedic Surgery, Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA.
Alka
A
Vyas
Department of Biomedical Engineering, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.|SINAPSE Institute, National University of Singapore, Singapore.
Nitish
N
Thakor
Department of Orthopaedic Surgery, Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA.|Department of Neurology, Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA.
Thomas M
TM
Brushart
D000818:Animals; D000831:Animals, Newborn; D019771:Axotomy; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003094:Collagen; D003911:Dextrans; D015194:Diffusion Chambers, Culture; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009046:Motor Neurons / Q000187:drug effects / Q000502:physiology*; D009416:Nerve Regeneration / Q000187:drug effects / Q000502:physiology*; D015739:Nocodazole / Q000494:pharmacology; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods; D012235:Rhodamines; D013116:Spinal Cord / Q000166:cytology*; D050257:Tubulin Modulators / Q000494:pharmacology
D001426:Bacterial Proteins; D003911:Dextrans; C067661:Fluoro-Ruby; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D012235:Rhodamines; D050257:Tubulin Modulators; C054562:yellow fluorescent protein, Bacteria; D003094:Collagen; D015739:Nocodazole
Organotypic; Peripheral nerve; Regeneration; Spinal cord; YFP