291790 Biomaterial Models of Spinal Cord Injury Substrates Elicit Astrocyte Morphology Change

Monday, October 29, 2012
Hall B (Convention Center )
Karl Schaettle, Chemical, Biological, and Materials Engineering, University of Oklahoma, Norman, OK

At spinal cord injury (SCI) sites, neuronal cells called astrocytes become reactive. These cells become enlarged, mobilize toward the injury. Eventually, SCI results in a glial scar, formed by reactive astrocytes releasing chondroitin sulfate proteoglycans (CSPG) into the injury environment. Axons are unable to elongate past the scar, so the injury results in permanently disrupted signal transduction, although it does prevent infiltration from immune cells and prevent infections.

This research focused on an in vitro model of astrocyte response to acute SCI. Microlesions of various sizes and orientations were modeled using electrospun fibers on top of poly-L-lactic acid (PLLA) films.

Small, rectangular gaps in the electrospun fiber mat were created by airbrushing each sample with chloroform. To create consistent gaps, two glass microscope slides were affixed, parallel to one another, and held in place on top of each film while chloroform was sprayed over them. Using this technique, two different gap widths were tested. The gap on each sample ran either parallel or perpendicular to the overall direction of the aligned fibers.

10 μL of PDL was dissolved per 1 mL of DIH2O. 500 μL of this solution was pipetted onto each 15 x15 mm fiber substrate, and this was allowed to coat for one hour. Each film was then washed twice with DIH2O before seeding of astrocytes. The astrocytes were then fixed and stained with anti-GFAP 1:700 (DAKO) and DAPI (Invitrogen) after a four day incubation. Alexafluor 488 anti-rabbit secondary antibodies were used at a concentration of 1:1000. All imaging was done using an Olympus IX81 Spinning Disk Fluorescence Microscope. Results of the astrocytic response to each type of material interface are presented.

Preliminary Conclusions:

  • Small gaps between fibers can be spanned by a single astrocyte
  • Apparent astrocyte morphology can vary throughout cell body
  • More reactive/randomly oriented astrocytes apparent in large gap samples
  • Sufficiently small lesions likely to reduce glial scarring

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