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Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. A devastating element of TBI is the diffuse and widespread injury of axons in a process known as traumatic axonal injury (TAI). TAI is a difficult entity to study as gross protein or molecular analyses have been largely precluded due to the requisite for specifically localizing protein changes to axons rather than the supporting glia or neuronal soma. As such, much of the mechanistic insight into TAI pathogenesis thusfar has come through histochemical and immunohistochemical analyses. In order to address the next generation of questions in TAI pathogenesis it has become critical to develop methodologies that allow for direct examination of protein-protein interactions in relation to sites of TAI. In the current communication, we report on a modified method of multiphoton Fluorescent Resonance Energy Transfer (FRET) microscopy that allows for the direct assessment of protein-protein interactions in aldehyde fixed tissue sections through the use of a conventional dual-label immunofluorescent approach. In the utilization of this technique, we explored whether the bcl-2-related proteins BAD and Bcl-xL heterodimerize in a pro-apoptotic fashion within traumatically injured axons following TBI.
Adult SD rats were subjected to impact acceleration TBI and euthanized at multiple time points. Vibratome sections derived from adult SD rats that had previously undergone impact acceleration TBI were processed for immunohistochemical double labeling with anti-BAD 1° antibody/Alexa 488 2° antibody, followed by anti-Bcl-xL 1° antibody/Alexa 555 2° antibody. Images were processed for spectral bleedthrough, and efficiency/distance calculations were performed. BAD/Bcl-xL heterodimerization was examined in relation to cytochrome c release and caspase-3 activation, by employing a third immunofluorescent label visualized with an Alexa 647 dye. Multiphoton FRET microscopy was carried out upon 40 micron thick sections. Further, to determine if FRET analysis could be performed in thick tissue specimens, 100 and 200 micron thick sections were examined as well.
At 6h postinjury, swollen axons in medial lemniscus demonstrated a mean energy transfer efficiency greater than 20% indicating formation of Bad-Bcl-xL complexes. Thick tissue specimens up to 200 microns thick likewise demonstrated FRET efficiencies greater than 20%. Specimens positively labeled for either cytochrome c or caspase-3 demonstrated FRET efficiencies greater than 10%.
The current investigation demonstrates FRET microscopy can be employed to assess protein-protein interactions in aldehyde-fixed tissue sections through multi-label immunofluorescent methodologies. It is believed that the current approach will have widespread applicability as examination of protein-protein interactions within in vivo systems may now be assessed on the cellular and subcellular level within aldehyde fixed tissue sections.
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