TY - JOUR
T1 - In vitro and in situ visualization of cytoskeletal deformation under load
T2 - Traumatic axonal injury
AU - Fournier, Adam J.
AU - Rajbhandari, Labchan
AU - Shrestha, Shiva
AU - Venkatesan, Arun
AU - Ramesh, K. T.
N1 - Publisher Copyright:
© FASEB.
PY - 2014/12/1
Y1 - 2014/12/1
N2 - It is difficult to obtain insight into the mechanisms occurring within live cells during mechanical loading, because this complex environment is dynamic and evolving. This is a particular challenge from a subcellular mechanics perspective, where temporal and spatial information on the evolving cytoskeletal structures is required under loading. Using fluorescently labeled proteins, we visualize 3-dimensional live subcellular cytoskeletal populations under mechanical loading using a high-resolution confocal microscope. The mechanical forces are determined using a computational (finite element) model that is validated by integrating instrumentation into the testing platform. Transfected microtubules and neurofilaments of E17 rat neuronal axons are imaged before, during, and after loading. Comparisons between unloaded and loaded live cells demonstrate both spatial and temporal changes for cytoskeletal populations within the imaged volume. NF signal decreases by 24%, yet the microtubule signal exhibits no significant change 20-35 s after loading. Transmission electron microscopy assesses cytoskeletal structure spatial distribution for undeformed and deformed axons. While cytoskeletal degeneration occurs at prolonged time intervals following loads, our data provides insights into real time cytoskeletal evolution occurring in situ. Our findings suggest that, for axons undergoing traumatic injury in response to applied mechanical loads, changes at the substructural level of neurofilaments may precede microtubule rupture and degeneration.
AB - It is difficult to obtain insight into the mechanisms occurring within live cells during mechanical loading, because this complex environment is dynamic and evolving. This is a particular challenge from a subcellular mechanics perspective, where temporal and spatial information on the evolving cytoskeletal structures is required under loading. Using fluorescently labeled proteins, we visualize 3-dimensional live subcellular cytoskeletal populations under mechanical loading using a high-resolution confocal microscope. The mechanical forces are determined using a computational (finite element) model that is validated by integrating instrumentation into the testing platform. Transfected microtubules and neurofilaments of E17 rat neuronal axons are imaged before, during, and after loading. Comparisons between unloaded and loaded live cells demonstrate both spatial and temporal changes for cytoskeletal populations within the imaged volume. NF signal decreases by 24%, yet the microtubule signal exhibits no significant change 20-35 s after loading. Transmission electron microscopy assesses cytoskeletal structure spatial distribution for undeformed and deformed axons. While cytoskeletal degeneration occurs at prolonged time intervals following loads, our data provides insights into real time cytoskeletal evolution occurring in situ. Our findings suggest that, for axons undergoing traumatic injury in response to applied mechanical loads, changes at the substructural level of neurofilaments may precede microtubule rupture and degeneration.
KW - Confocal
KW - Cytoskeleton micromechanics
KW - Microfluidics
KW - Spinal cord injury
KW - Traumatic brain injury
UR - http://www.scopus.com/inward/record.url?scp=84919832789&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84919832789&partnerID=8YFLogxK
U2 - 10.1096/fj.14-251942
DO - 10.1096/fj.14-251942
M3 - Article
C2 - 25205740
AN - SCOPUS:84919832789
SN - 0892-6638
VL - 28
SP - 5277
EP - 5287
JO - FASEB Journal
JF - FASEB Journal
IS - 12
ER -