SEXI: Sciatic Expression and Identification of Differential Proteins in Traumatized Versus Nontraumatized Nerves



Status:Active, not recruiting
Conditions:Chronic Pain
Therapuetic Areas:Musculoskeletal
Healthy:No
Age Range:18 - 89
Updated:2/16/2017
Start Date:July 2011
End Date:January 2019

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SEXI: Sciatic Expression and Identification of Differential Proteins in Traumatized Versus Non-traumatized Nerves

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.

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)

Inclusion Criteria:

- Males and females 18 years and older, capable of providing informed consent
indicating awareness of the investigational nature of the study, in keeping with
institutional policy

- Written informed consent must be obtained from each subject prior to entering the
study

- Traumatic lower extremity (above knee or below knee) amputation that occurred as a
result of exposure to improvised explosive device (IED) blast, gunshot, or vehicle
rollover during combat in support of OIF/OND or OEF.

- Scheduled final amputation revision to occur during the initial hospital admission at
Walter Reed National Military Medical Center

Exclusion Criteria:

- Inability to provide informed consent

- Traumatic or surgical foot amputations

- Amputation occurring at Walter Reed Army Medical Center or another continental United
States (CONUS) hospital after failed limb salvage

- Nerve specimen less than 5cm in length
We found this trial at
1
site
8901 Rockville Pike
Bethesda, Maryland 20889
(301) 295-4000
Walter Reed National Military Medical Center The Walter Reed National Military Medical Center is one...
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from
Bethesda, MD
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