Subjects scheduled for final amputation revision and closure(s) will be approached to participate in the study. On the day of the study participant's planned amputation revision, research staff will liaise with surgical staff to coordinate nerve specimen retrieval. If a patient is to have an above the knee amputation, a sciatic nerve specimen will be obtained, if a patient is to have a below the knee or through knee amputation, a tibial nerve specimen will be obtained (the major component of the sciatic nerve after it divides into the tibial and peroneal nerves in the popliteal fossa of the knee). The surgeon will place a suture in the proximal end of the specimen to indicate the non-traumatized end of the nerve, as well as provide an estimate of the longitudinal location of the nerve sampled (i.e. 10cm distal to the knee joint line, 15 cm proximal to the knee at the knee joint level). Each sample will be excised in the same manner - using a scalpel. Once the tissue is excised by the surgeon, it will be placed in a plastic test tube that is labeled with the subjects' study identification number (with the suffix "A" or "B" if two samples are being retrieved from that subject), transferred to a member of the research staff and immediately frozen in liquid nitrogen. The frozen specimen will then be delivered to the Department of Research Programs laboratory where it will be stored at -80°C prior to analysis. The promixal, middle, and distal samples will be obtained at the DRP lab and the remaining segments of nerve will be replaced in the -80 freezer for future analysis. Samples will then be sent to Duke University for processing and analysis via a secure air mail carrier. The task of processing samples will be performed immediately prior to mass spectroscopy analysis by colleagues at Duke University. Collaborator Dr Moseley's lab responsibilities include both protein extraction and assay. The number of extractions per sample will vary depending on its length, from 2 to about 50. Collaborator Dr Van de ven's lab responsibilities include sample handling, pathologic examination, removing thin slices for histologic examination and segment homogenization for RNA,DNA and protein extraction.
Individual segments will be thawed on ice and homogenized immediately upon thawing. Homogenization methods will be optimized to produce the highest dissolved protein content. Methods to be trialed will include sonication, mechanical disruption by laboratory blender, mechanical disruption by manual chopping followed by Potter device, and freeze thawing.
Buffer and solvent system variables to be trialed include salt concentration, pH and buffer type, chaotrope concentration (urea and thiourea), presence of organic co-solvents such as DMSO, and the presence of nonionic detergent. All homogenization will be performed between 0 and 4°C and in the presence of protease inhibitors.
For proteomic analysis, High-performance liquid chromatography/electrospray mass spectrometry will be employed as a complementary route to discovering differences in protein expression between healthy and traumatized tissue. Following solid-phase extraction of soluble and solubilized proteins, we will optimize conditions for separation of protein peaks detected by UV absorbance and/or total ion current and may employ more than one set of conditions in order to focus on different sets of extracted proteins. We will use high-porosity C-18 reversed-phase columns to separate cytosolic proteins, and C-4 for membrane proteins. Standard methods of ionization, fragmentation, and detection will be used that are consistent with the Shimadzu LCMS-IT-TOF mass spectrometer at the Research Operations Service (ROS).
For RNA analysis, extracted RNA will be brought to the Duke Center for Genomic and Computational Biology Genomic Technologies core. There, RNA quality will be determined using an Agilent 2100bioanalyzer followed by Affymetrix GeneChip analysis. David Corcoran, the biostatistician who works at the Duke Center for Genomic and Computational Biology, will perform data analysis.
In this study, no changes to the standard of care will be implemented. The sciatic nerve specimens would otherwise be discarded as human tissue; there are no other ongoing investigations using these specimens.
Following the analysis of the sciatic nerve, we will collect data - demographic, injury, and pain treatment information, as well as pre-operative pain scores - from subjects' charts to determine secondary outcome variables. (See Appendix B - Case Report Forms)
