Clinical Trial Details
— Status: Active, not recruiting
Administrative data
| NCT number |
NCT01710618 |
| Other study ID # |
359367-12 |
| Secondary ID |
|
| Status |
Active, not recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
July 2011 |
| Est. completion date |
March 2025 |
Study information
| Verified date |
April 2023 |
| Source |
Defense and Veterans Center for Integrative Pain Management |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
The purpose of the study is to elucidate the different proteins that may appear on the
"injured" end of the sciatic nerve and compare it to the other end of the cut nerve, which is
"uninjured". By comparing the "normal" versus "traumatized" ends of the cut sciatic nerve,
the investigators hope to identify proteins that may be specifically related to pain. For
instance, if the non-traumatized end of the nerve has none of Protein X but the traumatized
end of the nerve has a lot of Protein X, then Protein X would be a protein that the
investigators will want to study further and see if it plays any role in the sensation of
pain.
Other studies have shown that there are specific pain-related proteins and they may be
increased or decreased after traumatic injury. If the investigators are able to identify
specific proteins that are related to pain, then that may eventually lead to the development
of medications that will block those proteins and decrease the sensation of pain.
Description:
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)
