1. K R I S T E N T R I P I C C H I O
STUDY OF NITROGEN MUSTARD VESICANT
INJURY IN BASAL KERATINOCYTES USING THE
SCRATCH WOUND MODEL
2. INTRODUCTION: MUSTARDS AS VESICANTS
• Vesicant chemical warfare agent – causes blisters to the
eyes, skin, and mucosal membranes
• Structure & analogs
• Alkylating agent via the carbenium ion6
Sulfur mustard Nitrogen mustard 2-chloroethyl ethyl
sulfide (CEES)
3. INTRODUCTION: MUSTARDS AS VESICANTS
• Blistering occurs in a delayed fashion – apoptotic death6
• Injury occurs because the epidermis basal keratinocytes
detach from the basement membrane or dermal layer6
• Delayed reepithelialization2
4. INTRODUCTION: WOUND HEALING &
LAMININ 332
• Basal keratinocytes are anchored to the basement membrane via
hemidesmosomes7
• Anchoring molecule - holds cells via integrins to collagen fibers in the BM4,8
• Trimeric protein – previous studies on γ2 chain of the trimer1,2
5. THE SCRATCH WOUND ASSAY
• “Simple, reproducible assay commonly used to measure basic cell
migration parameters such as speed, persistence, and polarity.”3
• Different approaches to analysis: change in length, area, track one
particular cell
Make scratch in
each well
Seed cells into plate
Take images at regular
intervals
Data
Analysis
6. SCRATCH WOUND: PARAMETERS TESTED
• Initial seeding concentration – % confluency
• What seeding number will lead to consistent results and will lead to a
nice monolayer after 24 hrs of growth?
• Passage number
• Does passage number have an effect on the migration rate of the cells?
• Human variation in seeding & scratching
• If the same cells are seeded at the same density, does it matter who
seeds or scratches?
7. SEEDING CONCENTRATION/CONFLUENCY
• Wells were plated with 1.25x10⁵, 2.5x10⁵, 3.75x10⁵, & 5x10⁵ cells/mL
• Wells with 3.75x10⁵ & 2.5x10⁵ saw significantly less variation in migration
rate than the higher seeded 5x10⁵ cells/mL concentration
• Wells plated with 2.5x10 ⁵ cells/mL did not result in a complete monolayer
• Therefore, 3.75x10 ⁵ cells/mL was chosen as the ideal seeding conc.
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10
WoundClosure(%)
Time (hrs)
Average Area Migrated vs. Time (5x10⁵
cells/mL)
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10
WoundClosure(%)
Time (hrs)
Average Area Migrated vs. Time (3.75x10⁵
cells/mL)
2.5x10 ⁵ cells/mL 3.75x10 ⁵ cells/mL
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
WoundClosure(%)
Time (hrs)
Control
A1B
A1T
A2B
A2T
A3B
A3T
8. PASSAGE NUMBER
• Compared PAM212 cells
with a passage number of
p5 & p23
• Similar rate of migration
and amount of variation
among samples
• Average rates of migration
were similar (5.5 &
6.3%/hr) & not statistically
significant when including
the std. deviations
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8
Percent Area Migrated (p5)
A1B
A1T
A2B
A2T
A3B
A3T
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8
Percent Area Migrated (p23)
B1B
B1T
B2B
B2T
B3B
B3T
y = 0.0551x + 0.0105
R² = 0.9964
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8
p5 Avg Migration Rate
y = 0.0633x + 0.0004
R² = 0.9979
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8
p23 Avg Migration Rate
9. HUMAN VARIATION: SEEDING & SCRATCHING
• Lab tech, Amy, and I each seeded our own 6-well plates
• We then scratched half of each plate
• No notable difference
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10
WoundClosure(%)
Time (hrs)
Average Migration Rates (Scratch
comparison)
Amy
scratch
KT
scratch
y = 0.0151x + 0.0051
y = 0.0172x + 0.0064
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10
Woundclosure(%)
Time (hrs)
Average migration rates (Seeding
comparison)
Amy
seeding
KT
seeding
10. EXPERIMENTAL SETUP
Control
0.1 µM NM
10 µM NM
1 µM NM
Non-Coated Coated
(2 µg/mL laminin 332)
Control
0.1 µM NM
1 µM NM
10 µM NM
NitrogenMustardDoseStudy
HYPOTHESIS: As dose increases, migration will decrease. Coated wells will decrease
migration.
11. RESULTS
0
0.2
0.4
0.6
0.8
1
Control 0.1 1 10
WoundClosure(%)
Dose of NM (µM)
Average Wound Closure after 9 hrs for coated and
non-coated wells
Non-coated
Coated
• Chose 9-hr time
point because this
was the longest
time point prior to
the wounds closing
• Overall, coated
wells have slower
closure rate
• 10 µM non-coated
average changed
the trend…
12. RESULTS
• Compared to the 1 µM
data, there was a lot of
variation present resulting
in a large std deviation
• Effect of scratch size
• 3.4% deviation vs. 38.5%!
• Redoing the results with
the two wells with similar
sizes resulted in…
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Noncoated 10 µM NM
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Noncoated 1 µM NM
14. DISCUSSION/CONCLUSIONS
• Preliminary study
• Validated assay – confluency & similar scratch sizes are critical
• Increasing dose of NM resulted in increased migration rate –
implications, what does this mean?
• The coated wells resulted in a decreased migration rate – this
may better represent how the tissue acts in vivo
15. FUTURE EXPERIMENTS
• Perform experiment with higher doses of NM
• Add different parts of the laminin trimer to the media to see if
they result in faster migration
• Coat plate with other EM proteins: fibronectin, collagen, etc.
• Develop assay to be high throughput & efficient – 96-well
plates (triplicate resulted in large std devs)
16. ACKNOWLEDGEMENTS
• I would like to thank Dr. Chang, Amy, and Dr. Gerecke for offering endless
guidance over the last few weeks and being quick to answer any questions
• I would also like to thank Dr. Aleksunes, Erin, and Jorge for putting in all of
your time and effort to making the SURF program so successful
• Lastly, thanks to the NIH Graduation School of Biomedical Sciences for
funding my research experience this summer.
17. BIBLIOGRAPHY
1. Chang et al. Upregulation of Gamma-2 Laminin 332 in the mouse ear vesicant wound
model. J Biochem Molecular Toxicology, 2009, 23, p. 172-184.
2. Chang et al. Sulfur mustard induces an endoplasmic reticulum stress response in the
mouse ear vesicant model. Toxicology and Applied Pharmacology, 2013, 268, p. 178-187.
3. Cory, Giles. Scratch-Wound Assay. Chapter 2 in Methods in Molecular Biology, 2011, 769,
p. 25-30.
4. Decline, F; Rousselle, P. Keratinocyte migration requires α2β1 integrin-mediated
interaction with the laminin 5 γ2 chain. Journal of Cell Science, 2001, 114, p 811-823.
5. Jourdan, MM et al. Laminin 332 deposition is diminished in irradiated skin in an animal
model of combined radiation and wound skin injury. Radiation Research, 2011, 176, p.
636-648.
6. Kehe, K; Balszuweit, F; Steinritz, D; Thiermann, H. Molecular toxicology of SM-induced
cutaneous inflammation and blistering. Toxicology, 2009, 263, p. 12-19.
7. Kirfel, G; Herzog, V. Migration of epidermal keratinocytes: mechanisms, regulation, and
biological significance. Protoplasma, 2004, 223, p 67-78.
8. Nguyen et al. Deposition of laminin 5 in epidermal wounds regulates integrin signaling and
adhesion. Current Opinion in Cell Biology, 2000, 12, p. 554-562.
I know this is a mouthful of a title. But my research here this summer has essentially taken on two roles: the first was to develop the scratch wound assay in order to establish basic parameters to result in consistent data. The second was a telling experiment where I performed a scratch wound assay that was both a dose-dependent study & took a look at possible treatment to vesicant injury.
Nitrogen mustard dose study on migration in mouse keratinocytes using the scratch wound model.
-Causes blistering primarily due to the keratinocytes detaching from the basement membrane
Oftne wounds aren’t able to reepithelialize meaning have new healthy cells migrate into the wound bed or “heal”
Apoptotic cell death leads since symptoms of SM damage occur
Need pictures of dermis detaching from the epidermis
Separation of the epidermis from dermis is evident, prolonged inflammation, delayed wound repair and carring – is known to crosslink DNA and other cellular components which discrupts tissue structure yet the specific mechanisms underlying blistering have still not been determined
Laminin 332 is cleaved after secretion by the cell – studies have shown that the unprocessed laminin 5 induces migration and impedes the assembly of HD’s – after cleavage – review = kirfel, herzog 2004, Nguyen et al 2000, Zhang and Kramer 1996
Contradicting stuff – lam332 in irradiated skin says that lam332 needs to be processed to become migratory whereas above source says that the unprocessed form is what helps with migration
Use of gamma2 as scattering factor in cancer
Since a better way of promoting healing in SM injury cells is via migration into the wound bed, the scratch wound assay (a simple assay used in wound healing models, epidermylosis bullosa symplex, and cancer) is an easy, inexpensive model to measure success of treatments for SM damage.
If a drug can promote “healing” or faster migration, than it can potentially help to heal victims of SM poisoning
Here’s how it works..
Before I was able to do that experiment, however, I had to make sure that the assay resulted in reliable, consistent data.
In the past, many students have attempted the scratch wound assay and have gotten nonconsistent results after performing the experiment. My job was to test factors to see what was causing this variation.
Graphs that show that there is less deviation in the trials with the lower concentration
Show the one outlier in the control experiment demonstrating how confluency is very important.
Now going back to the main experiment, here is a reminder of what the setup was.
And here is a summary of the results that I received. Coating the wells does change the data… therefore we should probably always coat the wells to create a better model that is more representative of the tissue in vivo.