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ORIGINAL ARTICLE
In Vivo Imaging of Human and Mouse Skin
with a Handheld Dual-Axis Confocal Fluorescence
Microscope
Hyejun Ra1,2,9, Wibool Piyawattanametha1,3,4,9, Emilio Gonzalez-Gonzalez1, Michael J. Mandella1,2,
Gordon S. Kino2, Olav Solgaard2, Devin Leake5, Roger L. Kaspar1,6, Anthony Oro7
and Christopher H. Contag1,8
Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that
can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the
presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC)
microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin.
Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical
administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have
utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets
the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated
indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with
nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with
reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye
and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These
results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the
efficacy of experimental molecular therapeutics.
Journal of Investigative Dermatology advance online publication, 30 December 2010; doi:10.1038/jid.2010.401
INTRODUCTION
In vivo imaging has the potential to monitor and assess the
effectiveness of skin therapeutics and augment the current
state-of-the-art biopsy procedure by providing image guidance and the power of repeated measures. As biopsies are
painful and may complicate interpretation of treatment
regimens because of their invasiveness and resulting tissue
1
James H. Clark Center for Biomedical Engineering & Sciences, Department
of Pediatrics and the Molecular Imaging Program, Stanford University,
Stanford, California, USA; 2Edward L. Ginzton Laboratory, Department of
Electrical Engineering, Stanford University, Stanford, California, USA;
3
National Electronics and Computer Technology Center, Pathumthani,
Thailand; 4Advanced Imaging Research Center, Faculty of Medicine,
Chulalongkorn University, Pathumwan, Thailand; 5Dharmacon Products,
Thermo Fisher Scientific, Lafayette, Colorado, USA; 6TransDerm, Santa Cruz,
California, USA; 7Department of Dermatology, Stanford University School of
Medicine, Stanford, California, USA and 8Departments of Radiology and
Microbiology & Immunology, Stanford University, Stanford, California, USA
9
These authors contributed equally to this work.
Correspondence: Hyejun Ra, Department of Pediatrics, Stanford University,
Stanford, California 94305, USA. E mail: [email protected]
Abbreviations: DAC, dual-axis confocal; ICG, indocyanine green; K, keratin
protein; MEMS, microelectromechanical systems; NIR, near infrared
PC, pachyonychia congenita; siRNA, small interfering RNA
Received 26 August 2010; revised 27 October 2010; accepted 1 November
2010
& 2010 The Society for Investigative Dermatology
damage, the use of noninvasive microscopy to guide tissue
sampling and increase the number of data points can refine
the study, and possibly reduce or replace biopsies. Our group
has developed a handheld dual-axis confocal (DAC) fluorescence microscope that is capable of detecting individual
keratinocytes expressing green fluorescent protein in mouse
skin in vivo (Gonzalez-Gonzalez et al., 2010a). This in vivo
microscope has also been used to show the effects of small
interfering RNA (siRNA) on target gene expression in the skin
after intradermal injection (Gonzalez-Gonzalez et al., 2009).
This approach provided quantitative data that revealed the
temporal inhibition patterns of reporter gene expression in a
living transgenic mouse model (Ra et al., 2010).
The ability to monitor noninvasively, at single cell
resolution, the effectiveness of siRNA-based or other experimental medicines in the skin would offer an informative
measure of therapeutic outcome that would accelerate and
refine the development of new treatment strategies for skin
disorders, and in the management of these diseases. Recently,
the first clinical trial to test the effectiveness of siRNA in skin
was completed for the rare skin disorder, pachyonychia
congenita (PC), with promising results (Leachman et al.,
2010). PC is an uncommon inherited skin disorder caused by
dominant-negative mutations in the differentiation-specific
keratins, keratin (K) 6a, K6b, K16, or K17, which disrupt
www.jidonline.org
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H Ra et al.
In Vivo Skin Imaging
RESULTS
DAC microscopy can visualize single cells in living mouse skin
The DAC system was used to first demonstrate microanatomic imaging in mouse and human skin using a topical
dye formulation, and later to show functional imaging of
siRNA delivery in murine skin. NIR fluorescent dye (IRDye
800CW) was topically applied to mouse footpad skin to test
tissue uptake in vivo. Figure 1 shows an en face image at
52 mm depth and a side view of the acquired threedimensional (3D) tissue volume after topical application of
IRDye 800CW in GeneCream formulation. This formulation
penetrates through the stratum corneum into lower layers of
the epidermis and into the dermis (to a depth of 100 mm), and
the IRDye appears to penetrate cells and localize within the
cytoplasm, as shown in Figure 1a, where dark nuclei of
epidermal keratinocytes are clearly visualized. The use of
contrast agents such as IRDye 800CW and ICG enable
visualization of tissue microanatomy, and may provide a
useful measure of epithelial cell fragility and the resulting
reorganization of the ultrastructure of the skin.
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Journal of Investigative Dermatology
50 µm
Skin depth (µm)
intermediate filament function and lead to epithelial cell
fragility (McLean and Lane, 1995; Leachman et al., 2005).
The symptoms, resulting from defective keratin filament
formation in a specific subset of epithelial tissues, include
thickened dystrophic nails, painful palmar and plantar
hyperkeratosis, leukokeratosis, cysts, and follicular hyperkeratosis (Leachman et al., 2005; Smith et al., 2005). Therapies
based on siRNAs have been developed that target the genes
and genetic mutations responsible for PC (Hickerson et al.,
2008; Leachman et al., 2008; Smith et al., 2008), and one of
these, K6a_513a.12 (formulated Good Manufacturing
Practice version is called TD101), which targets a single
nucleotide mutation (p.Arg171Lys) in the KRT6A gene, was
tested in a phase 1b clinical trial (Hickerson et al., 2008;
Leachman et al., 2010). The PC patient from the published
clinical trial (Leachman et al., 2010) was the subject of the
imaging studies presented here.
In this study we demonstrate the capacity of the DAC
system to visualize cellular architecture in mouse skin, and
then test its ability to evaluate anatomic differences in
human skin at different places in the body following topical
delivery of near-infrared (NIR) dyes and, finally, to evaluate
the ability of NIR dye-conjugated siRNA to penetrate the
stratum corneum barrier when applied topically to mouse
skin. This is done with the goal of using this technology
to noninvasively evaluate siRNA delivery and effectiveness in
patient skin, specifically PC skin. In mouse skin, the NIR dye
utilized was IRDye 800CW (LI-COR Biosciences, Lincoln,
NE)—this agent is being evaluated for clinical use (Marshall
et al., 2010). For the human skin studies, a clinically
approved dye, indocyanine green (ICG, IC-GREEN; Akorn,
Buffalo Grove, IL), was used. Formulated IC-GREEN more
readily penetrated callused or PC skin when compared with
calf skin, suggesting that the skin barrier properties are
compromised in diseased or callused skin and that the dye
and DAC microscope may comprise a useful measure of
therapeutic outcome.
0
50
100
Figure 1. Visualization of individual keratinocytes in mouse footpad skin by
noninvasive in vivo imaging. Dual-axis confocal (DAC) intravital image stacks
were obtained 30–60 minutes after topical application of IRDye 800CW
(GeneCream formulation) to mouse footpad skin (full movie file is provided in
Supplementary Materials online). (a) En face image taken at a depth of 52 mm.
Scale bar ¼ 50 mm. (b) Side view of the full three-dimensional (3D) volume.
The volume dimensions for b are 420 155 100 mm3.
Imaging of normal human skin
In order to evaluate the DAC system for imaging human skin
in vivo and to test the ability of the GeneCream formulation
to penetrate normal and diseased skin, the NIR handheld
DAC microscope and topical application of cream-formulated IC-GREEN was used to noninvasively image the
microanatomy of healthy skin. A normal volunteer was
imaged at two distinct anatomic sites with differing microanatomy; a site on the calf of the leg and callused plantar
region of the foot (Figure 2). Figure 2a and b show en face
images of the smooth thin skin of the calf taken at depths of
27 and 55 mm, whereas Figure 2c shows the side view of the
full 3D image volume. The dye was observed to penetrate
down to a depth of 80 mm in most regions. Beyond the
stratum corneum, the cream seemed to consistently penetrate
deeper layers of the normal epidermis in an uneven manner
(see Figure 2b). Images of a callus region of the foot are
shown in Figure 2d–f. En face images at 27 and 55 mm
demonstrate that the dye, in comparison with the smooth skin
of the calf, is distributed more uniformly throughout the
callus area, as shown in Figure 2d and e. Penetration of
the dye down to 160 mm depth was observed in some areas
of the callus, which was substantially greater than that
observed in the calf (Figure 2f).
DAC analysis of PC patient epidermis
Similar imaging procedures were performed with the PC
patient who had recently completed a clinical trial with
TD101 and vehicle control (Leachman et al., 2010). The
siRNA (TD101)-treated foot callus is shown in Figure 3.
En face images at 25 and 43 mm depth are presented in Figure
3a and b, whereas a perspective side view of the 3D image
stack is shown in Figure 3c. The fluorescence signal was
H Ra et al.
In Vivo Skin Imaging
Skin depth (µm)
0
75
25 µm
43 µm
25 µm
80 µm
0
50
100
150
27 µm
Skin depth (µm)
55 µm
55 µm
0
75
150
Figure 2. In vivo imaging demonstrates enhanced topical delivery of nearinfrared (NIR) dye to live layers of human plantar callused skin when
compared with smooth calf skin in a volunteer. Dual-axis confocal (DAC)
image stacks were generated (full movie files are provided in Supplementary
Materials online) from volunteer skin after topical application of GeneCreamformulated IC-GREEN NIR imaging dye. (a, b) En face calf skin images
at depths of 27 and 55 mm. (c) Side view of the full three-dimensional
(3D) volume. (d, e) En face images of callused skin taken at depths of 27 and
55 mm. (f) Side view of the full 3D volume. The 3D volume dimensions are
650 320 150 mm3. Scale bar ¼ 50 mm.
imaged down to a depth of 50 mm, and individual keratinocytes can be observed, probably within the granular layer of
the epidermis (Figure 3a and b). Most of the dye remained in
the top epidermal layers of the treated skin, and signal was
barely detected below a depth of 70 mm. This resembles the
nonlesional calf skin of the same patient (data not shown),
which was similar to images acquired in the calf skin of the
normal volunteer (Figure 2a–c). Images of vehicle-treated
plantar callus are also shown in Figure 3. En face images at
depths of 25 and 80 mm are presented in Figure 3d and e,
whereas a side view of the 3D image stack is shown in Figure
3f. The dye readily penetrated beyond 110 mm in PC
symptomatic skin (vehicle treated), sometimes showing a
strong fluorescence signal at depths 470 mm (Figure 3e–f).
After initial imaging, a region of callus from the vehicletreated foot was trimmed in order to investigate differences in
dye uptake and penetration after a reduction in the amount of
callus present. Following debridement, more cells were
observed that resembled keratinocytes, such as those shown
in dashed circles in Figure 4a, which were located at a depth
of 13 mm. The signal was relatively high down to 150 mm in
depth, whereas diffuse fluorescence was detected beyond
200 mm in depth. The TD101-treated foot skin was not pared
Skin depth (µm)
Skin depth (µm)
27 µm
0
50
100
Figure 3. In vivo imaging of small interfering RNA (siRNA)- and vehicletreated pachyonychia congenita (PC) patient skin reveals differences in dye
penetration and tissue morphology. Dual-axis confocal (DAC) image stacks
were generated (full movie files are provided in Supplementary Materials
online) from patient skin 30–60 minutes following topical application of
GeneCream-formulated IC-GREEN imaging dye. (a, b) En face images of
siRNA-treated callused skin at depths of 25 and 43 mm. (c) Side view of
the full three-dimensional (3D) volume. (d, e) En face images of
vehicle-treated callus at depths of 25 and 80 mm. (f) Side view of the
full 3D volume. The 3D volume dimensions are 650 320 100 mm3.
Scale bar ¼ 50 mm.
as there was no appreciable callus to trim at the treatment site
(Leachman et al., 2010).
DAC imaging of cutaneous siRNA uptake in vivo
DAC microscopy has the potential to also follow the delivery
of therapeutic agents into the skin by using a fluorescent tag
on the agent and imaging its distribution. However, these
studies cannot be conducted in humans. Therefore, to test the
ability of the DAC system to directly visualize delivery of
siRNA in skin, we returned to a mouse model. Cream
formulated NIR dye-labeled siRNA was applied to murine
back skin and the depth of penetration of the therapeutic
nucleic acid was assessed. Figure 5 shows in vivo images at
3 hours after topical application. The NIR dye used
for conjugation is DyLight 800 (Thermo Fisher Scientific,
Rockford, IL), which has similar fluorescence properties to
IRDye 800CW. Keratinocytes can be seen in optical sections
taken with the DAC microscope, such as inside the dashed
circles of Figure 5a at a depth of 20 mm. Unlike the use of the
more general contrast agents (ICG and IRDye 800CW) that
are intended to stain a substantial amount of tissue to reveal
microanatomic changes, imaging of labeled therapeutics
should reveal localization of the agent. Figure 5b shows the
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H Ra et al.
In Vivo Skin Imaging
50 µm
0
100
200
Figure 4. In vivo imaging following trimming of pachyonychia congenita
(PC) patient callus and topical application of near-infrared (NIR) dye reveals
individual skin cells. Dual-axis confocal (DAC) image stacks were
acquired 20–60 minutes after topical application of GeneCream-formulated
IC-GREEN imaging agent (complete movie file is provided in Supplementary
Materials online). (a) En face image of epidermal cells (inside dashed circles)
at a depth of 13 mm. These cells are likely keratinocytes based on morphology
and depth. Scale bar ¼ 50 mm. (b) Side view of the full three-dimensional (3D)
volume of 650 320 200 mm3.
side view of the 3D tissue volume, where a uniform signal is
observed down to 50 mm. This suggested that the labeled
siRNA penetrated the stratum corneum and was in association with epidermal keratinocytes.
DISCUSSION
The NIR DAC system features a miniaturized handheld
microscope that acquires cellular-resolution images in real
time, which is enabled by the dual-axis architecture (Wang
et al., 2003) and microelectromechanical systems (MEMS)
technology (Ra et al., 2007). The NIR wavelength of the
laser and superior rejection of scattered light allow for deep
imaging in tissue that is relevant not only for normal skin but
also for a variety of skin pathologies. In addition, the 3D
imaging capability of the microscope provides perspective
views of tissue volumes that are otherwise difficult to attain
from traditional tissue sections. These characteristics of the
DAC system in its miniaturized package make in vivo
imaging of cellular and molecular changes possible through
the range of preclinical studies to clinical applications,
especially in areas that are difficult to reach with larger,
static imaging systems.
In this study, we demonstrated that topically applied
fluorescent agents to mouse and human skin allow intravital
visualization of individual cells and measurement of the
penetration depths of contrast agents with the DAC microscope, and this can be used to assess transdermal delivery.
IRDye 800CW delivered with the GeneCream formulation
exhibits cytoplasmic localization in mouse skin, whereas
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Journal of Investigative Dermatology
Skin depth (µm)
Skin depth (µm)
50 µm
0
35
70
Figure 5. Intravital imaging demonstrates penetration of small interfering
RNA (siRNA) through the murine stratum corneum barrier following topical
application. Dual-axis confocal (DAC) image stacks (complete movie file is
provided in Supplementary Materials online) were generated 3 hours after
topical application of DyLight 800-conjugated siRNA to mouse back skin.
(a) En face image at a depth of 20 mm; epidermal cells (likely keratinocytes
based on morphology and depth) can be visualized inside the dashed circles.
Scale bar ¼ 50 mm. (b) Side view of the full three-dimensional (3D) volume
with dimensions of 405 235 70 mm3.
Food and Drug Administration (FDA)-approved IC-GREEN
was used in place of IRDye 800, which has similar
fluorescence attributes in the NIR region of the spectrum,
for imaging patient skin. We observed that nonaffected skin
in a PC patient and noncallused skin in a normal volunteer
(e.g., calf skin) were more impervious to dye delivery and
therefore are more difficult to access via topical application.
The dye distribution was generally nonuniform, and some of
the dye may have penetrated through compromised regions
of the skin. This pattern continued down to a depth of 80 mm,
whereas at greater depths, only a diffuse signal was observed
(see Figure 2b and c). In contrast, in vehicle-treated, callused
PC skin or calluses of a normal volunteer, the GeneCream
formulation readily penetrated the stratum corneum and
delivered IC-GREEN to greater depths. This may be because
of a ‘‘loosening’’ of the ‘‘morter’’ and/or tight junctions
between stratum corneum keratinocytes, resulting in a
compromised barrier function, allowing some of the cargo
to penetrate further. These results are consistent with previous
reports where fluorescently labeled oligonucleotides showed
greater penetration through regions of severely impaired
stratum corneum formation in ex vivo psoriatic skin,
demonstrating nuclear localization in parakeratotic cells, as
well as other keratinocytes (White et al., 2002). In contrast,
those agents remained confined to the stratum corneum of
normal skin. In the PC study shown here, the vehicle-treated,
callused skin of the PC patient showed more structural
disorganization of the callus, likely because of the expression
of filament-disrupting mutant K6a protein (see Hickerson
H Ra et al.
In Vivo Skin Imaging
et al., this issue). This can be observed in Figure 3d–f, where
the dye appeared to have been transported through fissures
within the epidermis and became more widely distributed in
the deeper layers of the skin.
Imaging of siRNA-treated PC skin revealed changes suggestive of reversion to healthy nonaffected skin when compared
with skin treated with vehicle alone. siRNA-treated PC skin
showed comparable dye penetration patterns with healthy skin
from a normal volunteer (compare Figure 3a–c with Figure
2a–c). Additionally, DAC imaging of the vehicle-treated foot
after trimming of the callus region also revealed live cells of the
epidermis (see Figure 4a), although the cells are not as clear as
in Figure 1, where IRDye 800CW was utilized. This may be
because of lower uptake of the IC-GREEN dye compared with
IRDye 800CW, where similar results were observed in mouse
skin experiments (data not shown), or it may be explained by
the greater quantum efficiency of the IRDye 800CW relative to
IC-GREEN. The increased resolution may also be due, at least in
part, to differential cellular localization of the dyes; the IRDye
800CW does not appear to enter the nucleus, allowing better
resolution of subcellular structures. The nonuniform dye uptake
pattern of the trimmed callus region is similar to that of healthy
skin of the volunteer, where the majority of signal is
concentrated at the upper layers and more diffuse in deeper
skin layers. It should also be noted that the in vivo DAC imaging
of human skin was a proof-of-principle study with limitations of
a small subject group consisting of one control and one patient.
All of the experiments above were focused on in vivo
imaging of skin with topically applied NIR dyes to observe
and assess differences in tissue structure. As a next step, we
have demonstrated that topical cream application of DyLight
800-conjugated siRNA allows for penetration through the
murine stratum corneum barrier. The distribution of the dyeconjugated siRNA was mostly uniform throughout the upper
layers of the epidermis, which is desirable for clinical
translation. Keratinocytes in the upper layers of the epidermis
can be observed (see Figure 5a), although little, if any, of
the labeled siRNA appeared to enter the cells, as appears to
occur with NIR dyes alone (compare with Figure 1). This is
likely because of the higher molecular weight and polyanionic nature of siRNA. In recent studies, we have shown
that unmodified siRNA is not readily taken up by cells in the
absence of pressure and that the so-called ‘‘self-delivery’’
modified siRNAs may facilitate keratinocyte uptake (Gonzalez-Gonzalez et al., 2010a, b). Future experiments using NIR
dye-labeled siRNAs that have been modified to enhance
cellular uptake, such as the Accell technology from Thermo
Scientific/Dharmacon (www.dharmacon.com) (GonzalezGonzalez et al., 2010b) or self-delivery inhibitors being
developed by RXi Pharmaceuticals (www.rxipharma.com),
may be highly informative. In this context, the DAC system
can be used to study various siRNA delivery methodologies
using fluorescently tagged siRNAs, which will not only show
whether penetration through the stratum corneum barrier is
achieved, but also give an idea of whether the siRNA is being
taken up by keratinocytes.
In our study of in vivo fluorescence microscopy in the skin
with the DAC system, we conclude that 3D cellular imaging
with a microscope conducive to preclinical and clinical
settings brings value to assessing siRNA uptake and/or
differences in tissue structure. This supports in vivo imaging
with the DAC microscope as an effective clinical end point in
evaluating the efficacy of molecular therapeutics of the skin.
MATERIALS AND METHODS
In vivo DAC microscopy system
The specifications of the handheld DAC microscope system for
in vivo imaging have been previously reported (Ra et al., 2008;
Piyawattanametha et al., 2009). The microscope operates at the NIR
wavelength of 785 nm, which allows for noninvasive imaging in skin
down to a depth of 200 mm. The dual-axis optical configuration
(Wang et al., 2003) and MEMS technology (Ra et al., 2007) allow
miniaturization of a confocal microscope into a 10-mm-diameter
handheld unit that is amenable for intravital imaging as well
as clinical imaging in skin. The transverse and axial resolutions are
5 and 7 mm, respectively. The MEMS device continuously scans
for real-time en face imaging at 5 frames per second, whereas a
piezoelectric actuator translates the MEMS scanner in the depth
direction to acquire image stacks. All image stacks start at 10 mm
below the skin surface. Full 3D volumes are reconstructed using
Amira (Visage Imaging, San Diego, CA) software. The demonstration
of the DAC system in animal studies has shown stability and
reproducibility in in vivo fluorescence imaging (Ra et al., 2010).
Preparation of GeneCream topical formulations
For the IRDye 800CW formulation (550 ml), 0.5 mg of IRDye 800CW
N-hydroxysuccinimide Ester Infrared Dye (LI-COR Biosciences)
was dissolved in 30 ml of DMSO, and 10 ml of this solution was
mixed with 500 ml of a lipid/alcohol-based cream (‘‘GeneCream’’)
(Takanashi et al., 2009) to give a final concentration of 0.3 mg ml–1.
For the ICG topical formulation, clinical grade IC-GREEN (Akorn)
was dissolved in 10 mM Tris (pH 7.5) at 10 mg ml–1 and formulated to
a final concentration of 2 mg ml–1 in GeneCream. CBL3 siRNA
(targets click beetle luciferase (Gonzalez-Gonzalez et al., 2009))
was synthesized with a 50 N6 modification, a primary amine for the
N-hydroxysuccinimide ester reaction, for conjugating the DyLight
800 N-hydroxysuccinimide Ester Infrared Dye (Thermo Fisher
Scientific). Following conjugation, excess dye was removed using
an anion exchange column, and molecular weight was verified by
mass spectrometry. The resulting conjugated compound was used to
image siRNA treatment with the DAC microscope.
Cream application and imaging of mice
Before cream application, the skin was cleaned with alcohol (70%
isopropanol) swabs (BD, Franklin Lakes, NJ) and allowed to dry for
5 minutes. Mice were anesthetized using 2% isofluorane, and approximately 50 ml of cream was topically applied to the mouse footpad or back
skin that was closely shaved with a razor blade (Personna, Hauppauge,
NY) 2 hours before cream application. Excess cream was removed from
the treated area 30 minutes after application with alcohol swabs and the
mice were imaged with the DAC microscope under the same anesthesia
conditions. Gel (Vidal Sassoon hair gel, Beverly Hills, CA) was applied to
the region of skin as an index-matching optical coupling agent for
imaging with the DAC microscope. All animal work was carried out
under strict adherence to institutional guidelines for animal care of both
the National Institutes of Health and Stanford University.
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H Ra et al.
In Vivo Skin Imaging
Cream application and imaging of human skin
The patient and volunteer treatment and imaging were performed
with Institutional Review Board approval (Stanford IRB Protocol no.
14823). The study was conducted according to the Declaration of
Helsinki Principles, and participants gave their written informed
consent. Approximately 200 ml of the IC-GREEN cream formulation
was applied to callused and noncallused skin regions (previously
cleaned with alcohol swabs) of a normal volunteer or PC lesions
(nonaffected, siRNA-treated, or vehicle-treated regions) of a patient
who had completed siRNA therapy (Leachman et al., 2010). The
patient imaging occurred 48 days after the last siRNA treatment, at
which time there was a clear difference in callus thickness between
the siRNA-treated and vehicle-treated sites. As a control, the cream
was also applied to clinically nonaffected calf skin of the PC patient.
Plantar callus of the normal volunteer was imaged as a model for the
callus in PC patients, whereas noncallused calf skin of the volunteer
was imaged as a model for normal healthy skin. Excess cream was
removed with cotton pads 15–30 minutes after topical application.
Next, gel (Vidal Sassoon hair gel) was applied to the skin as an optical
coupling agent and imaging was performed with the DAC microscope. To facilitate imaging of live skin layers, callused regions were
groomed by 3 to 4 mm in depth by the patient using a scalpel,
stopping just before penetrating the underlying, innervated, vascularized dermis. Formulated IC-GREEN was subsequently applied and the
skin was imaged as described above. Multiple sites of the skin were
imaged with the DAC microscope in each area. Representative
images are presented in the figures.
CONFLICT OF INTEREST
RLK has intellectual property in the area of using siRNAs to treat skin disorders
and topical siRNA delivery and is an employee of TransDerm. MJM and GSK
are founders of and consultants to Optical Biopsy Technologies (OBTI) with
financial interests in OBTI. DL is an employee of Dharmacon Products,
Thermo Fisher Scientific.
ACKNOWLEDGMENTS
We thank the PC patient and volunteer for their participation in this study and
Irwin McLean, University of Dundee, UK, and Sancy Leachman, University of
Utah, for editing and commenting on the manuscript. This work was supported
by the NIH grants R44AR055881-02 (to RLK) and U54 CA105296 (to CHC).
EGG is the recipient of a Pachyonychia Congenita Project fellowship.
SUPPLEMENTARY MATERIAL
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