

































Intradermal Injection of Bleomycin 
Shows Preliminary Evidence of 

Dermal Fibrosis in a Mouse Model
!arun Potluri, Cameron S. D'Orio, BS, Lauren T. Mo"att, 

PhD, Je"rey W. Shupp, MD, Bonnie C. Carney, PhD

Volume Four
Edition One
Spring 2024

 
GEORGETOWN SCIENTIFIC
RESEARCH JOURNAL

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1 Department of Human Science, School of Health, Georgetown University, Washington DC, USA
2 Firefighters’ Burn and Surgical Research Laboratory, MedStar Health Research Institute, Washington DC, 
USA 
3 Departments of Surgery and Biochemistry and Molecular & Cellular Biology, Georgetown University School of 
Medicine, Washington DC, USA 
4 Department of Plastic Surgery, Georgetown University School of Medicine, Washington DC, USA 
5 Department of Surgery, The Burn Center, MedStar Washington Hospital Center, Washington DC, USA
Email: tp600@georgetown.edu 
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Burn wounds can result in the development of hypertrophic scar (HTS). A problematic characteristic of 
HTS is dyschromia, which is often distressing to patients and has been shown to impact quality of life. 
There are currently no viable, non-surgical treatment methods for dyschromia, and the mechanisms of its 
etiology are not well understood. We aimed to test if the fibrotic microenvironment of the dermis plays a 
contributing role in the development of epidermal dyschromia. To do this, we focused on reproducing a 
previously published model of bleomycin-induced dermal fibrosis to add to the replicability of previous 
findings and establish a reliable model to serve as a basis for understanding post-burn dyschromia 
development. By treating C57BL/6 mice with intradermal injections of three varying doses of bleomycin 
for two weeks and conducting histological and molecular analyses of collected data, we were able to assess 
the adequacy of dermal fibrosis development. Collected data showed evidence of hair follicle obliteration, 
dermal thickening, and other characteristics of fibrotic architecture within all three bleomycin-treated 
groups, bust mostly profoundly in the high-dose group. However, qRT-PCR data did not reveal any 
significant trends in differential expression of genes of interest. In future work, additional qRT-PCR 
analysis, immunofluorescence visualization, and other molecular assays should be conducted to obtain 
further molecular confirmation of bleomycin-induced dermal fibrosis before we can attempt to understand 
the mechanistic relationship between dermal fibrosis and epidermal dyschromia. 

Keywords: hypertrophic scar, dyschromia, fibrosis, bleomycin

11..  IInnttrroodduuccttiioonn  
Recent advances in the management and 

treatment of burn-related injuries have led to a 
significant decrease in mortality rates, especially 
for large total body surface area full-thickness 
burns.1 With an increasing patient survival rate, 
attention is now being turned to managing the 

after-effects of burn injury. Burn wounds and 
other cutaneous trauma can result in the 
development of hypertrophic scar (HTS), among 
other physiological complications such as muscle 
wasting, contractures, infection, and more.2 HTS 
is typically raised, thick, non-pliable, 
erythematous, pruritic, and painful.3 Irregular 
inflammatory processes in the dermis contribute to 

21



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HTS pathophysiology. HTS forms as a result of 
excess collagen deposition and abnormal fibroblast 
proliferation. The abnormal functioning of these 
fibroblasts results in excess amounts of 
extracellular matrix components that cause 
elevated and stiff skin characteristic of HTS.4 The 
bulk of HTS tissue is composed of the dermis layer 
which is primarily made up of type 1 collagen, 
fibroblasts, and endothelial cells.5 A number of 
proteins, such as collagen 1 alpha 1 (COL1A1), 
COL1A3, transforming growth factor beta-1 
(TGFB1), and galectin-1 (LGALS1), have been 
shown to be differentially overexpressed in fibrotic 
skin characteristic of HTS.6 Additionally, HTS 
typically has thickened dermis and epidermis layers 
and a change in collagen architecture that causes 
the increased skin stiffness.   

In addition to the physical impacts of post-
burn HTS development, patients with HTS 
frequently suffer from the psychosocial impacts of 
altered appearance and restricted range of motion 
that often results due to HTS.7 Almost all HTSs 
display dyschromia, which appears as hypo- or 
hyperpigmentation of the skin. More severe scars 
with extreme dermal pathology (increased 
thickness, hyper-vasculature, hyper-cellularity, 
non-pliability, contracture) often exhibit the most 
profound dyschromia. Dyschromic HTS, which 
develops pervasively amongst patients with skin of 
color, is often distressing to patients and has been 
shown to impact quality of life metrics such as 
stigmatization, sexual health, and physiological 
health.8-12 While some therapies have led to 
modest improvements in HTS symptoms for some 
patients, HTS and its psychosocial impact 
continues to be challenging to treat. Finding 
viable, non-surgical treatment methods for 
dyschromia could significantly improve the quality 
of life for patients with HTS.  

Before testing treatment methods, however, 
the mechanistic processes behind dyschromia must 
first be understood. There is currently a large gap 
in knowledge as to how post-burn dyschromia 
actually develops, which is underscored by the 
paucity of literature surrounding the topic. One 
hypothesis is that dyschromia develops due to 
abnormalities in the paracrine signaling between 

melanocytes and surrounding cell types.13 In 
fibrotic skin, fibroblasts are in an altered state of 
inflammation that could influence melanocyte 
function, which can be quantifiably assessed by 
melanin index.14 However, it is also possible that 
fibroblasts do not contribute to dyschromia at all, 
and that keratinocytes play a more significant role. 
Understanding the cellular abnormalities that 
cause dyschromia is a crucial first step in the effort 
to develop treatments for dyschromic HTS. Thus, 
a reliable and repeatable model for HTS must be 
established by which dyschromia can be studied.   

Not only does skin fibrosis develop as a result 
of burn wounds (HTS), but it can also occur due 
to various disease processes. A common disease 
that results in skin fibrosis is systemic sclerosis.15 
Patients with this disease have thickened skin that 
looks similar to burn scars and often exhibits 
dyschromia classified as “salt-and-pepper” skin.16 
An approach that has previously been taken by 
other researchers is to study non-HTS dermal 
fibrosis by synthetically inducing fibrosis with a 
commonly used antineoplastic drug called 
bleomycin. A study conducted by B!yszczuk et al. 
showed the efficacy of bleomycin-induced dermal 
fibrosis in a mouse model through regular 
injections of bleomycin.14  

This study aims to reproduce this published 
model of bleomycin-induced dermal fibrosis in an 
effort to establish the utility of this model for the 
study of HTS dyschromia. 
22..  MMeetthhooddss  

In this study, two groups of mice were tested. 
Following the completion of Group 1 mice, data 
was collected and analyzed before starting the next 
group. Using lessons learned from the Group 1 
mice, Group 2 mice had a refined experimental 
procedure in an effort to produce more conclusive 
results.  

All animal work was approved by the MedStar 
Health Research Institute’s Institutional Animal 
Care and Use Committee. Facility standard 
operating procedures under an animal care and use 
program accredited by the Association for 
Assessment and Accreditation of Laboratory 
Animal Care International were strictly followed 

22



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for handling and care of all the animals used in this 
study.  
22..11  GGrroouupp  SSeelleeccttiioonn,,  AAnneesstthheessiiaa,,  aanndd  
PPrreettrreeaattmmeenntt  PPrroocceedduurreess  

FFiigguurree  11..  Dorsal demarcation of the injection site. 
The 1cm x 1cm demarcated square is where all 
injections occurred. This square was drawn 3cm 
below the midline of the ears on each mouse for 
standardization. A 2cm x 2cm square was 
demarcated as a “buffer zone” to account for 
spreading of bleomycin or saline control outside of 
the 1cm x 1cm injection square. 

Female C57-BL/6 mice (22-45±6.59g) were 
used in Group 1 (n=3 in control, high-dose, mid-
dose, n=4 in low-dose groups). In Group 2, female 
C57-BL/6 mice (19-23±1.11g) were used (n=5 in 
control, n=4 in high-dose, mid-dose, low-dose 
groups). Anesthesia was induced by placing the 
mice into a box with 3-5% isoflurane. Once a plane 
of anesthesia was established by confirming 
absence of corneal and pedal reflexes, anesthesia 
was continued via nose cone administration of 1-
4% isoflurane. Ophthalmic ointment was used in 
cases of prolonged anesthesia. Thermoregulation 
was achieved with a warming blanket during 
anesthesia procedures. During the pre-injury 
timepoint, the dorsum of the mouse was shaved 
using standard veterinary clippers and depilated 
using a commercial agent for 90 seconds with 
massage of the agent into the skin with a cotton 
swab (Nair, Church & Dwight Co., Ewing, NJ). 
In Group 1 mice, a 1 cm x 1 cm square was 
demarcated 3 cm below the midline of the ears 
using a surgical marker (Figure 1). In Group 2 
mice, a 2 cm x 2 cm square was demarcated around 

the 1 cm x 1 cm square (Figure 1). In addition to 
baseline weights, non-invasive skin probe 
measurements (elasticity, trans-epidermal water 
loss, erythema, melanin) (Delfin Technologies, 
Kuopio, Finland) were also taken within the 
demarcated area. Mice were placed on their sides 
with their hind legs facing the same direction 
during probe measurements to reduce the pressure 
of the probe on the spine. 
 
22..22  BBlleeoommyycciinn  IInnjjeeccttiioonn  aanndd  MMoonniittoorriinngg  
 Lyophilized bleomycin sulphate (Sigma-
Aldrich, St. Louis, MO) was dissolved in normal 
saline. 100 µl of bleomycin or saline control was 
injected on days 1, 2, 4, 5, 8, 9, 11, and 12 for 
Group 1, and days 1, 2, 5, 6, 8, 9, 12, and 13 for 
Group 2. Injections were administered 
intradermally into the 1 cm x 1 cm square at 
different positions on different days (Figure 2). On 
day 1, injections were performed at position 1, on 
day 2 at position 2, continuing in this fashion 
throughout the time course. After position 5, 
injections were further continued cyclically starting 
again at position 1. This injection pattern, which 
was based off of previously published literature, 
ensured that the bleomycin was distributed equally 
across the entire demarcated treatment zone.14  

Three different dosages of bleomycin were 
tested in each group. In Group 1 mice, low-dose 
(0.5 U/mL), mid-dose (1.0 U/mL), and high-dose 
(5 U/mL) bleomycin was used. In Group 2, the 
high-dose concentration was adjusted to 2.5 
U/mL. After injection, mice were removed from 
anesthesia and monitored until ambulatory. Non-
invasive skin probe measurements of the treated 
zone and normal skin were taken periodically 
throughout the study and were collected in 
triplicate for each probe. Animal checks were 
conducted twice a day at minimum and mice 
weights were obtained each day. Mice with weight 
loss of 5% compared to their initial weight or with 
obvious signs of distress were given a 1 mL 
intraperitoneal injection of lactated ringers daily in 
addition to DietGel dietary supplements 
(ClearH2O, Westbrook, ME). In Group 2 mice, 
all mice were given DietGels on day 1 

23



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prophylactically. The mice were never subjected to 
more than two days of back-to-back isoflurane 
anesthesia to prevent a negative reaction to 
cumulative anesthesia.   
22..33  TTeerrmmiinnaall  TTiimmeeppooiinntt  
 On day 15, all mice were placed under 
anesthesia and euthanized via exsanguination and 
cervical dislocation. Terminal weights and non-
invasive probe measurements were obtained prior 
to euthanasia. Treated zone skin and normal skin 
were collected and preserved in Allprotect Tissue 
Reagent (Qiagen, Germantown, MD) and 10% 
neutral buffered formalin (NBF). Blood was 
collected and placed in RNAprotect tubes 
(Qiagen, Germantown, MD) and K2EDTA 
tubes, and was spun down to isolate plasma. Lungs 
were dissected and preserved in Allprotect Tissue 
Reagent (Qiagen, Germantown, MD) and NBF. 
  
  
  
  
  
  
FFiigguurree  22..  Injection pattern for intradermal 
injection of bleomycin or saline control. Injections 
were administered from positions 1 to 5 
throughout the time course. After position 5, 
injections were further continued cyclically starting 
again at position 1. 
2.4 HHiissttoollooggiiccaall  AAnnaallyyssiiss 
 Formalin-fixed skin and lung samples collected 
at the terminal timepoint were paraffin embedded, 
sectioned at 4 µm thickness onto slides, and 
stained with hematoxylin and eosin (H&E) as 
previously described.17 Tile images were taken at 
10X magnification to assess dermal architecture, 
dermal cellularity, and dermal and epidermal 
thickness using an Axio imager M2 microscope 
with motorized stage (Carl Zeiss Inc., 
Oberkochen, Germany) and an Axiocam 208 color 

camera. Zen blue software was used to export 
images (Carl Zeiss Inc., Oberkochen, Germany). 
ImageJ software was used to quantify dermal and 
epidermal thickness and 10 replicates were taken 
at different positions across the biopsy for each 
sample.  
22..55  CCoonnffiirrmmaattoorryy  qqRRTT--PPCCRR  AAnnaallyyssiiss  
 RNA from collected skin biopsies was isolated 
and purified for molecular assays using an 
automated protocol for the RNeasy fibrous tissue 
mini kit (Qiagen, Germantown, MD) with the 
Qiacube. RNA quality and quantity determined by 
spectro-photometry (Nandrop2000, 
ThermoFisher, Waltham, MA) and all samples 
were diluted to 1 ng/uL prior to running. 
Confirmatory qRT-PCR was conducted using a 
one-step reverse transcription and SYBR green kit 
(BioRad Laboratories Inc., Hercules, CA) to 
measure differential expression of COL1A1, 
COL1A3, TGFB1, and LGALS1 as previously 
described.17 Glyceraldehyde 3-phosphate 
dehydrogenase (GAPDH) and ribosomal protein 
L13A (RPL13A) were used as housekeeping 
genes. Fold changed was assessed using 
!!Ct analysis.  
22..66  SSttaattiissttiiccss  
 For non-invasive probe measurements and 
PCR data, a two-way ANOVA with multiple 
comparisons was run to compare differences across 
treatment groups and time points. Data was also 
analyzed as fold change at various time points to 
compare treatment to control at similar time 
points, or as fold change over time compared to the 
day of treatment initiation. For all analyses, p<0.05 
was considered statistically significant. For qRT-
PCR, fold change > or < 2 was established as the 
significance threshold. Outliers were excluded 
using Grubb’s outlier test. GraphPad Prism 10 was 
used for data visualization.  
33..  RReessuullttss  
33..11  BBlleeoommyycciinn  TTrreeaattmmeenntt  RReessuulltteedd  iinn  DDeeccrreeaasseedd  
SSkkiinn  EEllaassttiicciittyy  

The high-dose bleomycin group showed a 
significant decrease in skin elasticity at the 

24



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terminal timepoint (day 15) compared to baseline 
measurements (day 1-4), indicating decreased skin 
pliability (58±22.9 vs 135.8±57.9 N/m) (Figure 
3A). The low-dose group showed a similar 
decrease in skin elasticity over time (50.1±17.9 vs 
91.1±34.1 N/m). However, there was no 
significant difference in skin elasticity over time in 
the mid-dose and control groups.  
33..22  MMeellaanniinn  IInnddeexx  IInnccrreeaasseedd  TThhrroouugghhoouutt  tthhee  
DDuurraattiioonn  ooff  tthhee  SSttuuddyy  iinn  AAllll  GGrroouuppss  
 Non-invasive probe data showed a 4-fold 
increase in melanin at the terminal timepoint (day 
15) compared to baseline measurements (day 1-4) 
for the control group (628.7±42.4 vs 882.9±81.8) 
(Figure 3B). Similar trends were present for the 
low-dose group (648.4±60.6 vs 850.2±56.5) and 
mid-dose group (658.2±116.5 vs 867.2±52.6). The 
high-dose group did not show any significant 
difference in melanin index (746.6±132 vs 
806.9±108.7).  
 
 
  
  
  
  
  
  
  
  
  
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FFiigguurree  33..  Elasticity and melanin measurements 
using non-invasive skin probes. (A) In the low-
dose and high-dose groups, there was a significant 
increase in stiffness between days 1-4 and day 15 
(P < 0.05). (B) Control, low-dose, and mid-dose 
groups showed significant increase in melanin 

index between days 1-4 and day 15. Values 
presented as mean ± SEM. 
33..33  HHiissttoollooggiiccaall  AAnnaallyyssiiss  SShhoowweedd  CChhaannggeess  ttoo  
DDeerrmmaall  AArrcchhiitteeccttuurree 

Analysis of H&E-stained biopsies of the 
normal and treated zone in high-dose mice showed 
areas of dermal and epidermal thickening, hair 
follicle obliteration, and evidence of fibrotic 
architecture (Figure 4A). Mid- and low-dose mice 
showed similar dermal thickening, however 
changes to epidermal thickness and hair follicle 
architecture were less prominent than in the high 
dose group. Control mice treated zone skin 
appeared the same as normal skin, as expected.  

Quantitative assessment of epidermal 
thickness (Figure 4B) and dermal thickness 
(Figure 4C) confirmed thickening of the epidermis 
(14.1±0.19 vs 38.8±4.83 µm) and dermis 
(104.7±11.1 vs 231±52.5 µm) in treated zone skin 
compared to normal skin for high-dose mice, 
however this difference did not reach significance 
(p > 0.05). Control, low-dose, and mid-dose skin 
not show any significant difference in epidermal or 
dermal thickness in the treated zone skin 
compared to normal skin. 

 
  
  
  

FFiigguurree  44..  Effects of bleomycin treatment on 
dermal and epidermal architecture in a mouse 
model. (A) Biopsies of normal skin and treatment 
zone skin taken at the terminal timepoint were 
sectioned and H&E stained to be assessed for 
dermal thickness (red arrows), epidermal thickness 
(yellow arrow), hair follicle presence (blue arrow), 

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and other changes to dermal architecture. 
Quantitative measurements of (B) epidermal and 
(C) dermal thickness analyzed from H&E stained 
images. Values presented as mean ± SEM. 
33..44  PPCCRR  AAnnaallyyssiiss  DDiidd  NNoott  SShhooww  SSiiggnniiffiiccaanntt  
DDiiffffeerreennttiiaall  EExxpprreessssiioonn  ooff  GGeenneess  ooff  IInntteerreesstt 
 Analysis of COL1A1, COL3A1, and TGFB1 
qRT-PCR data between the normal skin and 
treated zone skin for each group did not show any 
significant differential expression (Figure 5A-C). 
Though there was an increase in expression of 
LGALS1 in the treated zone skin compared to 
normal skin in the low-dose, mid-dose, and high-
dose mice, none of the comparisons reached 
significance (Figure 5D).  
  
  
  
  
  
  
  
  
  
  
FFiigguurree  55..  COL1A1 (A), COL3A1 (B), TGFB1 
(C), and LGALS1 (D) expression in normal skin 
compared to treated zone skin for each group. 
RNA was isolated and purified from skin biopsies 
for confirmatory qRT-PCR analysis. Fold change 
was assessed using !!Ct analysis. P < 0.05 is 
considered statistically significant.  
33..55  BBlleeoommyycciinn  TTrreeaattmmeenntt  RReessuulltteedd  iinn  AArreeaass  ooff  
HHaaiirr  LLoossss  WWiitthhiinn  tthhee  TTrreeaatteedd  ZZoonnee  
 Images taken at the terminal timepoint for 
each mouse revealed a trend of hair loss within the 
demarcated treated zone for the high-dose mice 
(Figure 6). A similar pattern of hair loss was also 
present in the mid- and low-dose groups, though 
not as prominent and widespread. Control mice 
did not show any evidence of hair loss within the 
treated zone.  
 
  

  
  
  
  
  
  
  
  
  
  
  
  
  
  
FFiigguurree  66..  Representative images of the dorsal 
surface of high-dose, mid-dose, low-dose, and 
control mice at the terminal timepoint. Red arrows 
indicate areas of hair loss within the demarcated 
treated zone. 
33..66  HHiigghh--DDoossee  BBlleeoommyycciinn  NNeeggaattiivveellyy  AAffffeeccttss  
MMiiccee  WWeeiigghhttss 
 All mice used in Groups 1 and 2 were weighed 
daily throughout the duration of the study. Group 
1 high-dose mice lost a large amount of weight 
throughout the study duration, ending with a 
percent difference of -23.6% at the terminal 
timepoint compared to baseline measurements 
(Figure 7A). Group 1 control mice also dropped 
weight, though not as severely (-13.4%) at the 
terminal timepoint). Low-and mid-dose mice 
weights stayed relatively consistent. Group 2 
control, low-, and mid-dose mice weights stayed 
relatively consistent, however high-dose mice lost 
a large amount of weight (-7.8% at the terminal 
timepoint) (Figure 7B). 
 
 
 
 
 
  
  

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FFiigguurree  77..  Mice weights for each group throughout 
the study duration. (A) Group 1 mice weights. (B) 
Group 2 mice weights. Mice weights were 
obtained each day. Percent difference calculations 
were obtained by comparing to initial mice weights 
on day 1. Mice with weight loss of 5% compared 
to their initial weight or with obvious signs of 
distress were given a 1 mL intraperitoneal injection 
of lactated ringers daily in addition to DietGel 
dietary supplements. All Group 2 mice were given 
DietGels on day 1 prophylactically. Values 
presented as mean ± SEM. 
44..  DDiissccuussssiioonn  
 This study aimed to confirm the experimental 
methods of a previously proven model of 
bleomycin-induced dermal fibrosis in mice within 
our laboratory. The eventual goal of being able to 
synthetically induce dermal fibrosis is to ascertain 
if there is a causal relationship between dermal 
fibrosis and epidermal dyschromia. Following 
regular injections of three different bleomycin 
doses and saline control into the dorsal skin of 
C57-BL/6 mice, data from non-invasive skin 
probes, histological analysis, and qRT-PCR 
analysis were used to determine if bleomycin 
injection induced dermal fibrosis.  

One of the more promising markers of skin 
fibrosis is known to be skin elasticity.17 Elasticity 
measurements were collected by non-invasive skin 
probes throughout the duration of this study. 
Analysis of probe data showed a significant 
decrease in skin elasticity at the terminal timepoint 
compared to baseline measurements for high-dose 
and low-dose groups, suggesting an increased skin 
thickness characteristic of dermal fibrosis. 
Interestingly, the mid-dose group did not show 
any significant difference in elasticity. One 
explanation for this finding is that the mid-dose 
mice had an increased presence of self-mutilated 
skin (caused by stress, irritation, etc.) which could 
have affected elasticity measurements.   Imaging of 
H&E-stained skin biopsies showed clear 
epidermal and dermal thickening indicative of 
fibrotic architecture in the high-dose, mid-dose, 
and low-dose mice, though less prominent in the 
latter two groups. Differences in treated zone 
versus normal skin thickness might not have 

reached significance due to improper collection of 
normal skin. Normal skin was collected directly 
outside of the 2cm x 2cm demarcated outer square. 
As a result, some of the bleomycin could have 
spread outside of the demarcated injection site and 
caused changes to the skin outside of the treated 
zone, potentially influencing skin thickness 
measurements. Collection of normal skin further 
away from the treated zone should be performed in 
future studies to account for this problem. High-
dose skin biopsies showed clear obliteration of hair 
follicles in the dermis caused by the bleomycin 
treatment, providing further evidence supporting 
the efficacy of the drug. Hair loss within the 
treated zone could also be seen on the images taken 
of the dorsal surface of the bleomycin-treated 
groups. 
 Collected qRT-PCR data did not show any 
statistically significant differences in differential 
expression in the treated zone skin compared to 
the normal skin for genes of interest. A potential 
explanation for this lack of significance is that the 
bleomycin destroyed tissue architecture and 
cellular function to such an extent that cells were 
unable to undergo gene transcription. However, an 
increase in expression of LGALS1 in the low-, 
mid-, and high-dose mice was observed. Increased 
presence of LGALS1 provided some molecular 
evidence of fibrotic development within the 
treated zone, though perhaps other genes should 
be assessed for further confirmation of fibrosis.18 In 
future work, immunofluorescence visualization of 
�-SMA-positive myofibroblasts should be 
conducted and assessed for further molecular 
confirmation.19 

In addition to affecting the dermal and 
epidermal histoarchitecture of the mice, bleomycin 
also seemed to have systemic effects on the mice as 
observed by changes in their weights. Group 1 
high-dose mice were injected with a 5 U/mL 
concentration of bleomycin, whereas Group 2 
high-dose mice were injected with a 2.5 U/mL 
concentration. These dosages were arbitrarily 
chosen and were not based on previously proven 
models as the literature did not report specific 
dosage values for study replication. This problem 
was the main issue we faced when attempting to 

27



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replicate the previously proven model. The dosage 
of bleomycin was adjusted for Group 2 due to the 
significant weight loss that was observed in Group 
1 mice. Group 1 high-dose mice lost more weight 
overall than Group 2 high-dose mice. However, in 
addition to receiving a lower dosage of bleomycin, 
Group 2 mice were also prophylactically given 
Dietgels at the beginning of the study in an effort 
to mitigate weight loss. These two changes to the 
study protocol clearly helped reduce the amount of 
weight the mice lost throughout the study. In 
future work, RNA from lung biopsies and blood 
samples should be isolated and analyzed via qRT-
PCR to look for evidence of systemic impacts of 
bleomycin. 

The C57-BL/6 mice used in this study lack 
melanocytes within the skin, however melanin 
index significantly increased in control, low-dose, 
and mid-dose mice throughout the study duration. 
An explanation for this finding is that hair 
regrowth influenced melanin measurements in the 
mice. This would also explain why the high-dose 
mice did not show a significant difference in 
melanin index because the high-dose mice 
experienced hair loss as a result of bleomycin, 
limiting the extent to which melanin 
measurements were falsely influenced. The 
application of Nair during depilation seemed to 
induce hair follicle neogenesis or induction of the 
hair cycling pathway, which further affected 
melanin measurements. Further investigation 
needs to be conducted on the influence on 
depilating agents on hair follicle neogenesis and 
induction of cycling to better understand how to 
account for this problem in future work. 
Additionally, melanin content can be assessed via 
Fontana Masson staining techniques instead of 
non-invasive probes measurements for more 
accurate results. Fontana-Masson staining allows 
for visual-ization of melanin within the epidermis 
of the skin. 

Elasticity measurements and H&E-stained 
skin biopsies showed evidence of bleomycin-
induced dermal fibrosis in all three drug treatment 
groups, but most profoundly in the high-dose 
group. However, additional qRT-PCR analysis, 
immuno-fluorescence visualization, and other 

molecular assays should be conducted to obtain 
significant molecular confirmation of fibrosis. 
Further evidence is needed to confirm the efficacy 
of bleomycin-induced dermal fibrosis before 
attempting to understand the mechanistic 
relationship between dermal fibrosis and 
epidermal dyschromia. 
RReeffeerreenncceess  
1.�Aarabi, S., Longaker, M.T., & Gurtner, G.C. 

(2007). Hypertrophic scar formation following 
burns and trauma: new approaches to 
treatment. PLoS Med. 4(9), 1464-70. 
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