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Can red-light 5-aminolevulinic photodynamic therapy cure port wine stains on comb animal model?
Accepted Manuscript Title: Can red-light 5-aminolevulinic photodynamic therapy cure port wine stains on comb animal model? Authors: Yongxian Lai, Haiyan Zhang, Minglei Wei, Ji Jie, Lei Shi, Peiru Wang, Bo Wang, Zheng Huang, Xiuli Wang PII: DOI: Reference:
S1572-1000(17)30534-3 https://doi.org/10.1016/j.pdpdt.2018.04.017 PDPDT 1156
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
5-12-2017 23-3-2018 23-4-2018
Please cite this article as: Lai Y, Zhang H, Wei M, Ji J, Shi L, Wang P, Wang B, Huang Z, Wang X, Can red-light 5-aminolevulinic photodynamic therapy cure port wine stains on comb animal model?, Photodiagnosis and Photodynamic Therapy (2010), https://doi.org/10.1016/j.pdpdt.2018.04.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Can red-light 5-aminolevulinic photodynamic therapy cure port wine stains on comb animal model? Yongxian Lai1,2#, Haiyan Zhang2#, Minglei Wei2, Jie Ji2, Lei Shi2, Peiru Wang2, Bo
1 Shanghai skin Diseases clinical college of Anhui Medical University
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Wang2, Zheng Huang3, Xiuli Wang 2*
2 Institute of Photomedicine, Shanghai Skin Disease Hospital, Tongji University School of
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Medicine, Shanghai, China
3 MOE Key Laboratory of OptoElectronic Science and Technology for Medicine, Fujian Normal
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University, Fuzhou, China
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#Contributed equally to this work
Highlights
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*Corresponding author: [email protected];
Topical and systemic ALA was firstly used to study the curative effect of ALA-PDT
There is no curative effect of the topical red-light ALA-PDT on comb dermal
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to port wine stains(PWS) on comb animal model.
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capillaries. Systemic ALA-PDT could damage the dermal blood vessels, but it also has severe effect on the epidermis and other non-targeted parts.
Either topical or systemic red-light ALA-PDT is not suitable treatment methods for PWS.
Abstract Objective: To study the curative effect of red-light 5-Aminolevulinic photodynamic therapy(ALA-PDT) to port wine stains(PWS) on comb animal model. Methods: 160 male cocks were randomly divided into 16 groups.
Light only groups were
only given 630 nm red light irradiation with different light density.
ALA-PDT groups
were given red light after the application of topical or systemic ALA.
PDL group was
The distribution of fluorescence in tissue after topical or
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given PDL irradiation.
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group was given ALA only topical or systemic application.
The ALA only
systemic application of ALA was detected.
The morphological changes, the
pathological changes and the capillary reduction rate of the comb were observed after
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treatment for 0, 1, 3, 5, 7, 14 days.
In the topical ALA-PDT group at low light density 80J/cm2, the morphology
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ALA.
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Results: The PpIX fluorescence generated after topical and systemic application of
While under 160J/cm2 and 200J/cm2
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and the histopathology had no obvious change.
light density, severe erosion and thick scab appeared.
The immunohistochemistry showed that there was no
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epidermal necrosis and loss.
The histopathology showed
significant change in the number of capillaries under different light density (P>0.05). In the systemic ALA-PDT group under low light density 80J/cm2, only partial erosion
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and thin scab was observed on the treatment side. thick charred crust and even scar was observed.
With the increase of light density,
The histopathology showed that there
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were different degrees of damage to dermal and epidermal tissues.
And the
immunohistochemistry showed the capillary reduced significantly in the treatment side
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(P<0.01).
In control group, the comb is ruddy and plump.
Conclusion: These results suggest that either topical or systemic red-light ALA-PDT is not suitable treatment methods for PWS.
Keywords: 5-aminolevulinic acid; photodynamic therapy; red light; port wine stains； comb animal model
Introduction The nevus flammeus, also named port wine stains(PWS), is congenital venous malformation and common congenital papillary telangiectasia [1,2].
Now,
pulsed dye laser (PDL) is the ‘gold standard’ for the treatment of PWS [3].
But it is
For large area PWS, the curative effect is not ideal
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mainly used in superficial PWS.
the
due to the defects of careless operation, easy scarring and recurring.
With the
development of technology, photodynamic therapy(PDT) is used successfully to treat
for the treatment of PWS in large area.
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PWS. Compared with PDL, PDT has higher selectivity, which is especially suitable It has gradually become one of the preferred
methods for the treatment of PWS [4]. Currently, the method of intravenous injection
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of the photosensitizer hematoporphyrin monomethyl ether (HMME) is mainly used for
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PDT [5].
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5-aminolevulinic acid(ALA) is a kind of second generation porphyrin photosensitizer Under
normal circumstances, the amount of ALA in the cell is very low and non-toxic.
After
It is precursor of hemoglobin synthesis in vivo.
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developed in recent years.
the application of ALA, exogenous ALA can be selectively absorbed by proliferating cells and transform into protoporphyrin Ⅸ(PpIX) [6].
When exposed to light with the
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specific wavelength, photodynamic reaction happened [7].
Since the first report of
ALA-PDT by Kennedy in 1990, it is widely used to treat superficial skin cancer and
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precancerous lesions in the world [6].
It has been widely used in recent years.
Currently, ALA-PDT have been reported for a variety of skin diseases, including skin
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tumors, infectious skin diseases and inflammatory skin diseases [8-13].
Recently, some reports demonstrated that topical ALA-PDT on the PWS also has an effect on the treatment of PWS [14,15].
However, according to previous research, it
is found that the water solubility of ALA is too high to penetrate the dermis. And it
is only absorbed by epidermal and sebaceous gland cells [16-18].
Therefore, the
curative effect of the topical ALA-PDT on the capillaries of the dermis needs further investigation.
The comb has rich capillary network, whose pathological structure and blood
animal model of PWS [20].
Thus, it is usually used as the
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absorption characteristics are similar to PWS [19].
As the two sides of a comb are completely symmetrical,
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the untreated side of the same comb was used as control to explore the curative effect.
Here we used comb as animal model to investigate the curative effect of the red-light ALA-PDT to PWS on comb animal model by observing the changes of comb
The results will provide valuable guidance of the
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and systemic red-light ALA-PDT.
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morphology, histopathology and the number of blood vessels before and after topical
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designs and protocols for the clinical treatment of PWS.
Materials and methods
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1.Main materials and instruments
5-aminolevulinic acid (Shanghai Fudan Zhangjiang Biological Pharmaceutical Co., Ltd.); Cock derived VEGF antibody (a Research Industrial Co., Ltd. In Shanghai); Red
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light instrument (LED light source, Wuhan Yage Photoelectric Technology Co., Ltd., wavelength 630 nm, output power density 20~100mW/cm2); Vbeam laser therapeutic
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apparatus (CANDELA, USA, Wavelength 595nm, pulse width 1.5~40ms, transmitting frequency 1.5Hz); Canon PowerShot G16 digital camera (Canon Co, Japan); Curalux
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spectrum analyzer (Laser Institute, University of Munich, Germany).
2.Laboratory animal selection Six months male farm cock, a total of 170, weight 1.5~2 kg, with uniform ruddy color comb, no ulcers and necrosis.
The middle of the combs was used for this experiment.
The research was conducted in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the United National Institutes of Health.
All experimental protocols were approved by
the Review Committee for the Use of Human or Animal Subjects of Shanghai Skin Disease Hospital.
Ten of them were used to detect PpIX produced by topical or One side of the comb,
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systemic ALA, the remaining 160 for animal experiments.
mark a circular area with a diameter of 1cm using a waterproof marker pen as experimental area.
The laser penetration depth was less than 1mm. And the crest
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thickness was about 6~10mm. Thus, the non-treatment side of the comb was used as own control.
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3.Detecting the distribution of fluorescence in the comb
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The tissue absorption of ALA depends on the ALA concentration and incubation time.
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In the topical ALA-PDT group, 10% ALA cream was applied to the experimental area
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after cleaning the surface of the comb according to our previous experimental results. For the systemic ALA-PDT groups, different concentrations of ALA solution (25mg/kg,
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50mg/kg, 75mg/kg, 100mg/kg, 200mg/kg) were injected in the vein from the cock wing root, respectively.
After topical and systemic application of ALA, the curalux
spectrum analysis instrument was used to detect the protoporphyrin IX(PpIX)
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fluorescence intensity produced by ALA incubation 1 times per hour.
After incubated
for 0~7h in dark, the combs were observed by woods light for PpIX per hour.
After
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incubated for 0~28h in dark, the combs were observed by woods light for PpIX per hour.
In order to further explore the tissue absorption of ALA, the biopsy tissue of
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the comb was made for frozen section after the application of ALA topically for 4h and systematically for 6h.
The fluorescence in tissue was observed by fluorescence
microscope.
4.Grouping and therapeutic regimen of the cock
Before the experiment, 10% chloral hydrate intraperitoneal anesthesia was made on all the cocks.
160 male cocks were randomly divided into 16 groups: blank control group,
simple ALA group, red-light only 80 J/cm2 group, red-light only 120 J/cm2 group, redlight only 160 J/cm2, red-light only 200 J/cm2 group, 80 J/cm2 topical ALA-PDT group, 120 J/cm2 topical ALA-PDT group, 160 J/cm2 topical ALA-PDT, 200 J/cm2 topical
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ALA-PDT group, 80 J/cm2 systemic ALA-PDT group, 120 J/cm2 systemic ALA-PDT group, 160 J/cm2 systemic ALA-PDT, 200 J/cm2 systemic ALA-PDT group, and pulsed dye laser control group.
In the topical ALA-PDT groups, in freshly prepared
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10%ALA cream was applied to the experimental area incubation for 4h in dark
followed by the red-light irradiation at total light density 80 J/cm2, 120 J/cm2, 160 J/cm2 and 200 J/cm2, respectively.
irradiation.
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Other non-experimental areas were covered with black opaque film during In the systemic ALA-PDT groups, the ALA solution was freshly prepared
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5cm.
The distance of the red-light source to comb surface was
After incubation in dark for 6 h, the experimental area of the comb was
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root.
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with a concentration of 50mg/kg and then was injected in the vein from the cock wing
illuminated by red-light with the light density of 80J/cm2, 120J/cm2, 160J/cm2 and The light parameters were the same with those in the topical
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200J/cm2, respectively. ALA-PDT groups.
In the positive control group, the 595nm PDL was given only once
in the experiment area with the light density of 15J/cm2, the spot diameter of 7mm and The non-experimental areas were covered with black opaque
film during irradiation.
Negative control groups were divided into blank control
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the pulse width of 3ms.
The comb in the blank control group didn’t accept
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group and the red-light only group.
The comb in the red-light only group was only given the red-light
respectively.
The experimental cocks were placed avoiding light for at least 48h after
any treatment.
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irradiation with the light density of 80J/cm2, 120J/cm2, 160J/cm2 and 200J/cm2
treatment.
5.Morphological changes of the comb
Digital camera and dermatoscope were used to take photos of the morphological changes of the treated area of each comb before and after treatment.
6. Histopathological changes of the comb Six mm trephine was used to drill the whole layer of comb tissue (including the
side of experimental side was taken as control.
The experimental side was marked
with medical skin marker before the experiment.
While the control side was not
The biopsy tissues were fixed with 10% formaldehyde and embedded in
7. Immunohistochemical changes of the comb
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paraffin. Then slice sections were stained with routinely.
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marked.
The opposite
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experimental side and the contralateral side) before and after treatment.
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The biopsy tissues above were fixed with 10% formaldehyde and embedded in paraffin.
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Slice sections were immunohistochemically stained with VEGF routinely. Each slice
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randomly selected 5 magnifications; visual field (10 x 20), calculating average capillary
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number.
8.Statistical analysis
The program GraphPad Prism5.0 was used for analysis.
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presented by x ± s.
The vascular data were
The comparison between the surface fluorescence intensity
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before and after the application of ALA and the comparison of the number of blood vessels between the experimental side and the control side of the comb were detected P<0.01 was statistically significant.
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by t detection method.
Results 1. The distribution of fluorescence in comb The fluorescence intensity of the topical ALA group gradually increased with the incubation time and reached the peak at 4 h (P<0.01).
Then, the fluorescence intensity
decreased and tended to the initial level at 7h (Figure 1A).
Thus, 4h incubation time
were selected as the optimal parameters for the topical ALA-PDT treatment.
In systemic ALA groups, the fluorescence intensity gradually increased and reached the highest point at 6h (P<0.01) with the incubation time between 0~28h at the five The
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ALA concentration (25mg/kg, 50mg/kg, 75mg/kg, 100mg/kg, 200mg/kg). fluorescence intensity increased with the increase of the ALA concentration.
And
there is significant different between 50mg/kg ALA and 25mg/kg (P<0.01) (The results
And other parts of the cock also have strong
fluorescence under high ALA concentrations.
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did not provide in this paper).
It was found that the PpIX fluorescence
decreased after 6h and tended to the initial level at 22h (Figure 1B).
So, 50mg/kg
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ALA and 6h incubation time were selected as the optimal parameters for the systemic
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ALA-PDT treatment.
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Figure 1
Observed in dark field by Wood light, no fluorescence observed before topical and After the applying with ALA, the bright brick red
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systemic application of ALA. fluorescence was observed.
And the fluorescence gradually increased with the
prolongation of incubation time.
After topical application of ALA at 3 and 4h, the
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brick red fluorescence was the strongest. weakened.
The fluorescence disappeared 7h after topical application of ALA (Figure
No red fluorescence was observed on the control side.
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2A).
After 4h, the fluorescence gradually
disappeared after light illumination (Figure 2B).
After systemic application of ALA
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at 5 and 6h, the brick red fluorescence was the strongest. gradually weakened.
And the fluorescence
Then, the fluorescence
The fluorescence tended to the initial level 22h after systemic
application of ALA (Figure 2C). illumination (Figure 2D).
And the fluorescence disappeared after light
In addition, after intravenous injection of ALA, the
fluorescence was observed in both comb and the other parts of body (Figure 2E).
Figure 2
In order to further explore the tissue absorption of ALA, the biopsy tissue of the comb was made for frozen section after the application of ALA topically for 4h and systematically for 6h.
After topical application of ALA for 4h, bright brick red fluorescence
was observed in epidermis and no fluorescence in dermis.
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microscope.
The fluorescence in tissue was observed by fluorescence
In normal control groups,
there was no fluorescence in both epidermis and dermis (Figure 3A).
After systemic
and dermis (Figure 3B).
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application of ALA for 6h, diffuse brick red fluorescence was observed in epidermis
In normal control groups, there was also no fluorescence in
both epidermis and dermis.
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2.Morphological changes of the comb
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Figure 3
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All of the experimental areas showed mild swelling immediately after the light irradiation with the light density of 80J/cm2, 120J/cm2 and 160J/cm2 in the topical And the color was restored to normal after 10 min.
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ALA-PDT groups.
When the
light density was up to 200J/cm2, there was significant swelling in the experimental area immediately after the irradiation.
In the 80J/cm2 topical ALA-PDT group, there was no obvious change after
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30min.
And the color was restored to normal after
the topical ALA-PDT for 1,3, 5, 7 and 14 days.
In the 120J/cm2 topical ALA-PDT
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group, there was slight edema in the experimental area 1d after treatment. erosion and exudation was observed 5d after treatment.
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treatment.
And slight
Thin crust occurred 7d after
In the 160J/cm2 topical ALA-PDT group, edema appeared in the
experimental area 1d after treatment.
Then erosive exudation was observed 3d after
treatment and yellow callus 5d after treatment. observed 14d after treatment.
Partial epidermal necrosis was
In the 200J/cm2 topical ALA-PDT group, obvious
edema appeared 1d after treatment.
Then erosive exudation was observed 3d after
treatment, aggravated erosion 5d after treatment and yellow callus 7d after treatment. And crusts not shed with partial epidermal necrosis 14d after treatment (Figure 4A). There were no obvious changes in the control and no experimental areas in each group.
In the 80J/cm2 and 120J/cm2 systemic ALA-PDT groups, slight edema was observed
after treatment and partial shedding of eschar 14d after treatment.
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1d after treatment, slight erosion and exudation 3d after treatment, the thin crust 5d In the 160J/cm2
systemic ALA-PDT group, the experimental area appeared purple black color change
In the 200J/cm2 systemic
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1d after treatment and black crusts 3d~14d after treatment.
ALA-PDT group, purple change was observed the experiment area 1d after treatment, black crusts 3d after treatment and crust peeling off 14d after treatment (Figure 4B).
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There were no obvious changes in the control and no experimental areas in each group.
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In the PDL group, slight edema occurred in the experimental area1d after treatment.
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Thin layer of yellow and black scab was observed 1d after treatment.
And 14th day,
crust was peeling off and the experimental area showed white. There were no obvious
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changes in the control and no experimental areas in each group (Figure 4C).
The morphology of the comb was rosy and plump in negative control group.
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Figure 4
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3.Histopathological changes of the comb Comb tissue was drilled by trephine for biopsy and stained by HE after treatment.
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In the 80J/cm2 topical ALA-PDT group, there was no obvious change both in the epidermis and dermis compared with the non-treatment side.
In the 120J/cm2 topical
ALA-PDT group, slight injury, cell edema, slight exudation and edema was observed in the epidermis on the first day after treatment. more severe 3, 5, 7 or 14 days after treatment.
The changes of dermal tissue were
There was no significant change in the
epidermis and the dermal blood vessels compared with the control group and nontreatment side.
In the 160J/cm2 topical ALA-PDT group, there was a significant
change in the epidermis.
And the dermal tissue showed slight exudation and edema.
In the 200J/cm2 topical ALA-PDT group, there was more severe damage in the
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epidermis (Figure 5A).
In the 80J/cm2 systemic ALA-PDT group, the epidermal cells showed slight edema,
hyperplasia and exudation and edema of dermal tissue on the first day after treatment
Significant changes were observed in the
epidermis 3,5 ,7and 14 days after treatment.
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compared with the non-treatment side.
In the 120J/cm2 systemic ALA-PDT
group, epidermal cells showed edema and hyperplasia and dermal tissue exudation and The epidermis was crusted and the number of
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edema on the first day after treatment.
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the vessels decreased gradually 3,5 ,7 and 14 days after treatment.
In the 160J/cm2
The results of the histopathology showed epidermal cell
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epidermis at the 1st day.
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and 200J/cm2 systemic ALA-PDT group, significant changes were observed in the
edema, dermal tissue exudation, edema and dermal superficial capillary thrombosis The epidermis was black and yellow, with the exudation
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were observed on the 3rd day.
and edema of the dermis, the decrease of the number of capillaries in the superficial layer of the dermis and the decrease of the diameter of the blood vessels 5, 7, and 14
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days after treatment (Figure 5B).
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In the PDL group, the epidermal cells edematous, the dermal tissue exudative and edema, the number of capillaries in the superficial layer of the dermis decreased and
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the diameter of the vessels became smaller on the first day after treatment.
And 3, 5,
7, 14 days after treatment, tissue changes were more obvious and the epidermis was crusted (Figure 5C). There was no significant histopathological change in negative control group. Figure 5
4. Immunohistochemical changes of the comb Comb tissue was drilled by trephine for biopsy and stained by VEGF for the capillary vessels after treatment.
The results showed that there was no significant change in
the number of blood vessels between the treatment side and the non-treatment side in
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the topical ALA-PDT groups(P>0.05) (Figure 6A and Table 1).
In the systemic ALA-PDT groups, the number of blood vessels in the treatment side
And the vessels decreased
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decreased significantly on the 3rd day after treatment.
significantly 5, 7 and 14 days after treatment (P<0.01).
The number of vessels
decreased significantly with the increase of the light density (P<0.01) (Figure 6B and
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Table 2).
And the vessels decreased significantly 5, 7 and 14 days
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significantly on the 1st day.
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In the PDL group, results showed that the number of the blood vessels began to decrease
after treatment (P<0.01) (Figure 6C and Table 3).
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The immunohistochemistry showed that there were no significant changes in the number of blood vessels on both sides of the comb in negative control groups (P>0.05).
Table 1
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Table 2
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Figure 6
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Table 3
Discussion
The superficial dermis of cock combs has a rich capillary network, which has similarities with the histopathology of PWS [19].
And the optical adsorption
properties of cock blood are basically the same as that of human blood. Therefore, the comb tissue is usually used as the animal model for capillary hemangiomas by foreign research groups since the late 1960s [20]. PDL with a wavelength 585 or 595
patients [3].
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nm is now a gold standard for the treatment of PWS, which has a defined effect on But it is not suitable for the treatment of a large area of PWS [21].
In
recent years, PDT, with intravenous injection of photosensitizer HMME, is used for
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PWS for its selective specificity to target tissues [6].
Recently, some reports demonstrated that the topical ALA-PDT also have good curative Zhang Anli et al [22] used 10% ALA solution combined with topical
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effect on PWS.
The total
Liu et al [15] used 20% ALA gel combined with
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effective rate was up to 96.4%.
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application of 532nm PDL in treating 28 cases of patients with PWS.
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595nm PDL in the treatment of 35 cases of patients with PWS. 37.14% of patients improved moderately, while 28.57% had mild improvement.
effect on PWS.
However, single 532nm PDL or 595nmPDL have the therapeutic
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adverse reaction.
All patients had no
Thus, the curative effect of ALA-PDT still needs further confirmation.
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ALA is a small hydrophilic molecule, which is also a precursor of porphyrin in the synthesis of hemoglobin.
When absorbed by high proliferating cells, it will be
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converted into PpIX by enzymatic catalysis.
The results of this study and other reports
indicate that ALA only exists in the epidermis and sebaceous gland, not in the basement
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membrane [16-18].
Our experiment also confirmed that the absorption and
transformation of ALA was found only in the epidermis after the applying of ALA. And the fluorescence intensity reached the peak 4h after topical ALA application, but no red fluorescence was observed in the dermis (Figure 3A). absorbed by the blood vessels in the dermis.
That is, ALA was not
While the blood vessels closure is the
primary purpose of the treatment of PWS.
We also confirmed that PpIX fluorescence
can be observed in the dermis after systemic application of ALA. fluorescence intensity peaked at 6h.
And the
While we also detect the PpIX fluorescence in
other parts of the cock body as well as the epidermis of the comb (Figure 2E, 3B). Then, systemic ALA-PDT caused the severe damage of the other parts of the cock body,
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especially the epidermis.
The best absorption light source of ALA is about 410nm blue light and the red light
penetrability.
Clinicaly, red light is selected as ALA-PDT light source for its deeper
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around 630nm.
In this experiment, 4 light density of 80J/cm2, 120J/cm2, 160J/cm2 and
200J/cm2 were used to further explore the curative effect of topical ALA-PDT for PWS.
When the light density was 120J/cm2,
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the comb under low light density of 80J/cm2.
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The results showed that there was no change in the morphology and histopathology of
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the comb appeared slight exudation and thin crust.
And the reaction was more severe
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under the light density of 160J/cm2 and 200J/cm2 (Figure 4A). histopathology showed epidermal necrosis and loss (Figure 5A).
The results of Therefore, as the
severe.
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energy density increased, the damage of topical ALA-PDT on the epidermis was more VEGF is a marker of vascular endothelial cell.
We use VEGF
immunohistochemistry to observe the change in the number of vessels after ALA-PDT It was found that there was no statistical difference in the number of
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treatment [21].
vessels between the treatment side and the non-treatment side (Figure 6A, table 1).
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Thus, the results demonstrated that topical ALA-PDT has direct damage on the epidermis with increased energy density, while there is no damage on the blood vessels
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in the dermis.
So, the topical ALA-PDT had no curative effect on PWS.
In order to further explore the influence of ALA-PDT on the blood vessels of PWS, systemic injection of ALA combined with red light was used for the treatment of comb. Previously few studies on systemic ALA-PDT were reported.
Thus, we do some
experiments to explore the concentration ALA.
We found that the fluorescence
intensity increased with the increase of ALA concentration.
The fluorescence
intensity in 50mg/kg was significantly different from that of 25mg/kg (P<0.01). However, with the increasing of the ALA concentration, the other parts of the cock also had a strong fluorescence generation, which would cause severe side effects after
for our experiment.
Therefore, the ALA concentration of 50mg/kg was chosen
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illumination (Figure 1B).
And the same light density as the topical ALA-PDT was chosen.
It was found that there was only partial erosion and thin crust appeared under low light With the increase of light density, charred thick crust and scar was observed
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density.
on the treatment side of the comb (Figure 4B). Histopathological examination showed the dermal and epidermal tissues were damaged correspondingly with the increase of
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light density (Figure 5B). Immunohistochemistry showed that there was a significant
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reduction in capillaries of the treatment side (Figure 6B, Table 2).
Based on the above
But it also had severe damage to the epidermis.
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energy.
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results, it showed systemic ALA-PDT had the effect on blood vessels under certain And when the ALA in
vascular endothelial cells transformed into other tissues and epidermis, there would be
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adverse reactions on other non-targeted tissues.
And we also found that there was still
fluorescence 28h after the application of intravenous ALA.
Therefore, systemic
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ALA-PDT was not suitable for the treatment of PWS either.
PDL is the classical treatment for PWS.
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control in this study.
Therefore, 595nm PDL was used as positive
The results showed that the experimental area in comb became
white after PDL treatment (Figure 4C).
Histopathological examination showed that
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there was no significant change in the epidermis in treatment side, but dermal capillary was blocked (Figure 5C).
Immunohistochemistry showed that capillary number was
significantly reduced compared to that in the non-treatment side (Figure 6C). proved that PDL had obvious curative effect on PWS.
It was
Conclusion: There is no curative effect of the topical red-light ALA-PDT on comb dermal capillaries.
Systemic ALA-PDT could damage the dermal blood vessels, but
it also has severe effect on the epidermis and other non-targeted parts. In conclusion, either topical or systemic red-light ALA-PDT is not suitable treatment methods for PWS.
cooperation.
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Conflict of interest statement: The authors state no conflict of interest.
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Acknowledgements: The authors would like to thank all study participants for their
Funding：This work was supported by the National Natural Science Foundation of
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China (Grant numbers: 81472538, and 8140120479), and the Joint Project of New
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Frontier Technology of Shanghai Shen-kang Hospital Development Center (Grant
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number: SHDC12015123).
Reference 1. Frigerio A, Wright K, Wooderchak-Donahue W, et al. Genetic Variants Associated with Port-Wine Stains. PLoS One 2015;10(7): e133158. 2. Anolik R, Newlove T, Weiss ET, et al. Investigation into optimal treatment intervals
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of facial port wine stains using the pulsed dye laser. J Am Acad Dermatol 2012; 67 (5):985-990.
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3. Brightman LA, Geronemus RG, Reddy KK. Laser treatment of port-wine stains. Clin Cosmet Investig Dermatol,2015;8:27-33.
4. Orenstein A, Nelson JS, Liaw LH, et al. Photochemotherapy of hypervascular
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dermal lesions: a possible alternative to photothermal therapy. Lasers Surg Med
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5. Gao K, Huang Z, Yuan KH, et al. Side-by-side comparison of photodynamic
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therapy and pulsed-dye laser treatment of port-wine stain birthmarks. Br J Dermatol
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6. Kennedy JC, Pottier RH, Pross DC. Photodynamic therapy with endogenous protoporphyrin IX: basic principles and present clinical experience. J Photochem Photobiol B, 1990, 6(1-2):143-8.
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7. Marica B Ericson, Ann-Marie Wennberg, Olle Larkö. Review of photodynamic therapy in actinic keratosis and basal cell carcinoma. Ther Clin Risk Manag, 2008,
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8. Cai H, Wang YX, Zheng JC, et al. Photodynamic therapy in combination with CO2
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laser for the treatment of Bowen's disease. Lasers Med Sci2015;30(5):1505-10.
9. Gao Y, Zhang XC, Wang WS, et al. Efficacy and safety of topical ALA-PDT in the treatment of EMPD. Photodiagnosis Photodyn Ther 2015;12(1):92-7. 10. Zhang HY, Ji J, Tan YM, et al. Evaluation of 5-aminolevulinic acid-mediated photorejuvenation of neck skin. Photodiagnosis Photodyn Ther 2014;11(4):498509.
11. Lei X, Liu B, Huang Z, Wu J. A clinical study of photodynamic therapy for chronic skin ulcers in lower limbs infected with Pseudomonas aeruginosa. Arch Dermatol Res. 2015;307(1):49-55. 12. Ji J, Zhang LL, Ding HL, et al. Comparison of 5-aminolevulinic acid photodynamic therapy and red light for treatment of photoaging. Photodiagnosis Photodyn Ther
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2014; 11(2):118-21. 13. Ma L, Xiang LH, Yu B, et al. Low-dose topical 5-aminolevulinic acid
Photodiagnosis Photodyn Ther 2013;10(4):583-90.
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photodynamic therapy in the treatment of different severity of acne vulgaris.
14. Wang J, Huang X. Effects of aminolevulinic acid-based photodynamic therapy on cock combs, an animal model for port wine stains. Chin J Dermatol 2015;(48)5:333-
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337.
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15. Liu S, Yang C, Yang S, et al. Topical application of 5-aminolevulinic acid followed
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by 595-nm pulsed dye laser irradiation for the treatment of recalcitrant port wine
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stains: a primary study. J Cosmet Laser Ther 2012;14(4):189-92. 16. Chen HX, Zou XB, Zhang YJ, et al. Recent Progress in Photodynamic Therapy for
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Port Wine Stains. Chin J Derm Venereol 2014;28(6):631-633. 17. Wang XL, Wang HW, Huang Z, et al. Study of protoporphyrin IX (PpIX) pharmacokinetics after topical application of 5-aminolevulinic acid in urethral
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condylomata acuminata. Photochem Photobiol 2007;83(5):1069-73. 18. Ding HL, Wang XL, Wang HW, et al. Successful treatment of refractory facial acne
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using repeat short-cycle ALA-PDT: Case study. Photodiagnosis Photodyn Ther 2011; 8(4):343-6.
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19. Ohshiro T, Nakajima T, Ogata H, et al. Histological responses of cutaneous vascular lesion following photodynamic therapy with talaporfin sodium: a cock comb model. Keio J Med 2009; Sep;58(3):176-84. 20. Li ZH, Meng DS, Li YY, et al. Hypericin damages the ectatic capillaries in a Roman cockscomb model and inhibits the growth of human endothelial cells more potently
than hematoporphyrin does through induction of apoptosis. Photochem Photobiol. 2014 Nov-Dec ;90(6):1368-75. 21. Wang YZ, Huang Z, Yuan KH. The current situation and development in optical therapy for port-wine stains. J Pract Dermatol2015;8(5):363-366. 22. Zhang AL, Zu DM, and Yang L, et al. 5- aminolevulinic acid photodynamic therapy
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for 28 cases of nevus flammeus. Chinese Journal of Aesthetic Medicine, 2007;16(4):541-542.
23. Brightman LA, Geronemus RG, Reddy KK. Laser treatment of port-wine stains.
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Clin Cosmet Investig Dermatol 2015;8:27-33.
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Legends
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Figure 1 A: The changes of the surface fluorescence intensity after the topical application of ALA for 0~7h. * indicated P value (< 0.05) of the comparison of the fluorescence intensity at 4h with that at 1h.
B: The changes of the surface
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fluorescence intensity after the intravenous injection of ALA with the concentration of
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50mg/kg for 0~28h. ** indicated P value (< 0.01) of the comparison of the fluorescence
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intensity at 6h with that at 1h.
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Figure 2 A: The changes of the brick red fluorescent on the surface of the comb after the topical application of ALA for 0~7h.
B: The changes of the brick red fluorescent
before and after light illumination in the topical ALA-PDT group.
C: The changes of
the brick red fluorescent on the surface of the comb after the systemic application of
ALA for 0~28h.
D: The changes of the brick red fluorescent before and after light
illumination in the systemic ALA-PDT group.
E: The changes of the brick red
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fluorescent on the surface of the body after the systemic application of ALA for 0~28h.
B: The distribution of PpIX fluorescence in epidermal
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fluorescence in the epidermis.
The green fluorescence was the spontaneous
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topical application of ALA for 4h.
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Figure 3A: The distribution of PpIX fluorescence in the epidermis of the comb after the
and dermal tissues comb after the systemic application of ALA for 6h.
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and dermis situation in the blank control group.
C: Epidermis
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Figure 4 A: The morphological changes of the comb (*10) in the topical ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 B:
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J/cm2, 160 J/cm2 or 200 J/cm2 (the black circle represented the experimental area).
The morphological changes of the comb (*10) in the systemic ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 J/cm2, 160 J/cm2 or 200 J/cm2 (the black circle represented the experimental area).
C: The
morphological changes of the comb (*10) in PDL group 1,3, 5, 7 or 14 days after
treatment with the light density of 15 J/cm2 (the black circle represented the
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experimental area).
Figure 5 A: The HE staining of the comb tissue (*200) in the topical ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 J/cm2, 160 J/cm2 or 200 J/cm2. B: The HE staining of the comb tissue (*200) in the systemic
ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 J/cm2, 160 J/cm2 or 200 J/cm2.
C: The HE staining of the comb tissue
(*200) in the PDL group 1,3, 5, 7 or 14 days after treatment with different light density
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of 15 J/cm2.
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B: The immunohistochemical
staining for VEGF of the comb tissue (*200) in the systemic ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 J/cm2, 160 J/cm2 or 200 J/cm2.
C: The immunohistochemical staining for VEGF of the comb tissue
(*200) in the PDL group 1,3, 5, 7 or 14 days after treatment with different light density
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of 15 J/cm2.
Table 1 Comb vascular changes in topical ALA-PDT group after treatment(mean±s.d)
28±5.33
27±5.11
28±3.21
27±7.11
28±2.12
27±5.77
28±2.19
26±5.99
27±7.97
27±3.88
30±3.63
29±2.13
26±7.33
26±6.55
26±3.21
30±7.12
27±1.22
28±2.51
27±3.79
26±1.88
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28±3.11
27±5.99
27±6.89
25±3.13
25±7.49
24±1.45
27±2.18
27±1.73
26±3.90
25±5.91
26±3.44
25±1.82
30±1.23
26±3.21
27±7.34
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28±3.13
25±5.78
24±6.34
24±2.14
30±5.19
27±1.27
28±4.92
26±6.92
25±3.56
25±2.33
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7
29±1.67
25±1.84 26±6.17
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80J/cm2 Treatment side Non-treatment side 120J/cm2 Treatment side Non-treatment side 160J/cm2 Treatment side Non-treatment side 200J/cm2 Treatment side Non-treatment side
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Comb surface
Local ALA-PDT processing time (Day) Before 1 3 5 treatment
Table 2 Comb vascular changes in systemic ALA-PDT group after treatment(mean±s.d)
Comb surface
14
26±3.21
12±5.31* 6±5.11*
4±1.21*
3±2.84*
30±3.02
27±2.28
28±5.91
26±2.94
27±3.32
25±5.23
28±3.67
26±6.33
10±7.33* 4±7.16*
4±4.21*
30±2.44
28±4.78
27±2.59
26±1.92
27±3.17
26±3.81
Treatment side 27±6.62
25±2.97
6±2.17*
4±5.11*
3±3.01*
3±5.14*
27±1.74
28±5.90
26±3.55
26±4.01
25±2.0
24±6.33
4±2.92*
3±1.11*
2±5.01*
1±3.01*
26±2.79
27±6.11
26±6.12
26±3.03
25±2.59
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3±7.04*
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Non-treatment 28±2.02 side 200J/cm2 Treatment side 29±1.12 Non-treatment 30±2.11 side
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30±2.12
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80J/cm2 Treatment side Non-treatment side 120J/cm2 Treatment side Non-treatment side 160J/cm2
Systemic ALA-PDT processing time (Day) Before 1 3 5 7 treatment
*P<0.01 There was significant difference in P<0.01 between the treatment side and non-treatment
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side.
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Table 3 Comb vascular changes in PDL group after treatment(mean±s.d)
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595nm PDL processing time (Day) Comb surface Before 1 3 5 treatment Treatment side 29±2.77 9±2.33* 5±2.94* 4±5.11* Non-treatment 30±2.02 28±5.12 28±3.99 29±5.19 side
7
14
4±6.21*
2±4.12*
27±3.97
27±5.88
*P<0.01 There was significant difference in P<0.01 between the treatment side and non-treatment side.
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S1572-1000(17)30534-3 https://doi.org/10.1016/j.pdpdt.2018.04.017 PDPDT 1156
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
5-12-2017 23-3-2018 23-4-2018
Please cite this article as: Lai Y, Zhang H, Wei M, Ji J, Shi L, Wang P, Wang B, Huang Z, Wang X, Can red-light 5-aminolevulinic photodynamic therapy cure port wine stains on comb animal model?, Photodiagnosis and Photodynamic Therapy (2010), https://doi.org/10.1016/j.pdpdt.2018.04.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Can red-light 5-aminolevulinic photodynamic therapy cure port wine stains on comb animal model? Yongxian Lai1,2#, Haiyan Zhang2#, Minglei Wei2, Jie Ji2, Lei Shi2, Peiru Wang2, Bo
1 Shanghai skin Diseases clinical college of Anhui Medical University
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Wang2, Zheng Huang3, Xiuli Wang 2*
2 Institute of Photomedicine, Shanghai Skin Disease Hospital, Tongji University School of
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Medicine, Shanghai, China
3 MOE Key Laboratory of OptoElectronic Science and Technology for Medicine, Fujian Normal
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University, Fuzhou, China
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#Contributed equally to this work
Highlights
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*Corresponding author: [email protected];
Topical and systemic ALA was firstly used to study the curative effect of ALA-PDT
There is no curative effect of the topical red-light ALA-PDT on comb dermal
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to port wine stains(PWS) on comb animal model.
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capillaries. Systemic ALA-PDT could damage the dermal blood vessels, but it also has severe effect on the epidermis and other non-targeted parts.
Either topical or systemic red-light ALA-PDT is not suitable treatment methods for PWS.
Abstract Objective: To study the curative effect of red-light 5-Aminolevulinic photodynamic therapy(ALA-PDT) to port wine stains(PWS) on comb animal model. Methods: 160 male cocks were randomly divided into 16 groups.
Light only groups were
only given 630 nm red light irradiation with different light density.
ALA-PDT groups
were given red light after the application of topical or systemic ALA.
PDL group was
The distribution of fluorescence in tissue after topical or
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given PDL irradiation.
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group was given ALA only topical or systemic application.
The ALA only
systemic application of ALA was detected.
The morphological changes, the
pathological changes and the capillary reduction rate of the comb were observed after
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treatment for 0, 1, 3, 5, 7, 14 days.
In the topical ALA-PDT group at low light density 80J/cm2, the morphology
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ALA.
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Results: The PpIX fluorescence generated after topical and systemic application of
While under 160J/cm2 and 200J/cm2
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and the histopathology had no obvious change.
light density, severe erosion and thick scab appeared.
The immunohistochemistry showed that there was no
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epidermal necrosis and loss.
The histopathology showed
significant change in the number of capillaries under different light density (P>0.05). In the systemic ALA-PDT group under low light density 80J/cm2, only partial erosion
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and thin scab was observed on the treatment side. thick charred crust and even scar was observed.
With the increase of light density,
The histopathology showed that there
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were different degrees of damage to dermal and epidermal tissues.
And the
immunohistochemistry showed the capillary reduced significantly in the treatment side
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(P<0.01).
In control group, the comb is ruddy and plump.
Conclusion: These results suggest that either topical or systemic red-light ALA-PDT is not suitable treatment methods for PWS.
Keywords: 5-aminolevulinic acid; photodynamic therapy; red light; port wine stains； comb animal model
Introduction The nevus flammeus, also named port wine stains(PWS), is congenital venous malformation and common congenital papillary telangiectasia [1,2].
Now,
pulsed dye laser (PDL) is the ‘gold standard’ for the treatment of PWS [3].
But it is
For large area PWS, the curative effect is not ideal
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mainly used in superficial PWS.
the
due to the defects of careless operation, easy scarring and recurring.
With the
development of technology, photodynamic therapy(PDT) is used successfully to treat
for the treatment of PWS in large area.
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PWS. Compared with PDL, PDT has higher selectivity, which is especially suitable It has gradually become one of the preferred
methods for the treatment of PWS [4]. Currently, the method of intravenous injection
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of the photosensitizer hematoporphyrin monomethyl ether (HMME) is mainly used for
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PDT [5].
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5-aminolevulinic acid(ALA) is a kind of second generation porphyrin photosensitizer Under
normal circumstances, the amount of ALA in the cell is very low and non-toxic.
After
It is precursor of hemoglobin synthesis in vivo.
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developed in recent years.
the application of ALA, exogenous ALA can be selectively absorbed by proliferating cells and transform into protoporphyrin Ⅸ(PpIX) [6].
When exposed to light with the
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specific wavelength, photodynamic reaction happened [7].
Since the first report of
ALA-PDT by Kennedy in 1990, it is widely used to treat superficial skin cancer and
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precancerous lesions in the world [6].
It has been widely used in recent years.
Currently, ALA-PDT have been reported for a variety of skin diseases, including skin
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tumors, infectious skin diseases and inflammatory skin diseases [8-13].
Recently, some reports demonstrated that topical ALA-PDT on the PWS also has an effect on the treatment of PWS [14,15].
However, according to previous research, it
is found that the water solubility of ALA is too high to penetrate the dermis. And it
is only absorbed by epidermal and sebaceous gland cells [16-18].
Therefore, the
curative effect of the topical ALA-PDT on the capillaries of the dermis needs further investigation.
The comb has rich capillary network, whose pathological structure and blood
animal model of PWS [20].
Thus, it is usually used as the
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absorption characteristics are similar to PWS [19].
As the two sides of a comb are completely symmetrical,
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the untreated side of the same comb was used as control to explore the curative effect.
Here we used comb as animal model to investigate the curative effect of the red-light ALA-PDT to PWS on comb animal model by observing the changes of comb
The results will provide valuable guidance of the
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and systemic red-light ALA-PDT.
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morphology, histopathology and the number of blood vessels before and after topical
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designs and protocols for the clinical treatment of PWS.
Materials and methods
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1.Main materials and instruments
5-aminolevulinic acid (Shanghai Fudan Zhangjiang Biological Pharmaceutical Co., Ltd.); Cock derived VEGF antibody (a Research Industrial Co., Ltd. In Shanghai); Red
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light instrument (LED light source, Wuhan Yage Photoelectric Technology Co., Ltd., wavelength 630 nm, output power density 20~100mW/cm2); Vbeam laser therapeutic
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apparatus (CANDELA, USA, Wavelength 595nm, pulse width 1.5~40ms, transmitting frequency 1.5Hz); Canon PowerShot G16 digital camera (Canon Co, Japan); Curalux
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spectrum analyzer (Laser Institute, University of Munich, Germany).
2.Laboratory animal selection Six months male farm cock, a total of 170, weight 1.5~2 kg, with uniform ruddy color comb, no ulcers and necrosis.
The middle of the combs was used for this experiment.
The research was conducted in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the United National Institutes of Health.
All experimental protocols were approved by
the Review Committee for the Use of Human or Animal Subjects of Shanghai Skin Disease Hospital.
Ten of them were used to detect PpIX produced by topical or One side of the comb,
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systemic ALA, the remaining 160 for animal experiments.
mark a circular area with a diameter of 1cm using a waterproof marker pen as experimental area.
The laser penetration depth was less than 1mm. And the crest
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thickness was about 6~10mm. Thus, the non-treatment side of the comb was used as own control.
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3.Detecting the distribution of fluorescence in the comb
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The tissue absorption of ALA depends on the ALA concentration and incubation time.
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In the topical ALA-PDT group, 10% ALA cream was applied to the experimental area
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after cleaning the surface of the comb according to our previous experimental results. For the systemic ALA-PDT groups, different concentrations of ALA solution (25mg/kg,
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50mg/kg, 75mg/kg, 100mg/kg, 200mg/kg) were injected in the vein from the cock wing root, respectively.
After topical and systemic application of ALA, the curalux
spectrum analysis instrument was used to detect the protoporphyrin IX(PpIX)
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fluorescence intensity produced by ALA incubation 1 times per hour.
After incubated
for 0~7h in dark, the combs were observed by woods light for PpIX per hour.
After
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incubated for 0~28h in dark, the combs were observed by woods light for PpIX per hour.
In order to further explore the tissue absorption of ALA, the biopsy tissue of
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the comb was made for frozen section after the application of ALA topically for 4h and systematically for 6h.
The fluorescence in tissue was observed by fluorescence
microscope.
4.Grouping and therapeutic regimen of the cock
Before the experiment, 10% chloral hydrate intraperitoneal anesthesia was made on all the cocks.
160 male cocks were randomly divided into 16 groups: blank control group,
simple ALA group, red-light only 80 J/cm2 group, red-light only 120 J/cm2 group, redlight only 160 J/cm2, red-light only 200 J/cm2 group, 80 J/cm2 topical ALA-PDT group, 120 J/cm2 topical ALA-PDT group, 160 J/cm2 topical ALA-PDT, 200 J/cm2 topical
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ALA-PDT group, 80 J/cm2 systemic ALA-PDT group, 120 J/cm2 systemic ALA-PDT group, 160 J/cm2 systemic ALA-PDT, 200 J/cm2 systemic ALA-PDT group, and pulsed dye laser control group.
In the topical ALA-PDT groups, in freshly prepared
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10%ALA cream was applied to the experimental area incubation for 4h in dark
followed by the red-light irradiation at total light density 80 J/cm2, 120 J/cm2, 160 J/cm2 and 200 J/cm2, respectively.
irradiation.
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Other non-experimental areas were covered with black opaque film during In the systemic ALA-PDT groups, the ALA solution was freshly prepared
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5cm.
The distance of the red-light source to comb surface was
After incubation in dark for 6 h, the experimental area of the comb was
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root.
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with a concentration of 50mg/kg and then was injected in the vein from the cock wing
illuminated by red-light with the light density of 80J/cm2, 120J/cm2, 160J/cm2 and The light parameters were the same with those in the topical
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200J/cm2, respectively. ALA-PDT groups.
In the positive control group, the 595nm PDL was given only once
in the experiment area with the light density of 15J/cm2, the spot diameter of 7mm and The non-experimental areas were covered with black opaque
film during irradiation.
Negative control groups were divided into blank control
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the pulse width of 3ms.
The comb in the blank control group didn’t accept
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group and the red-light only group.
The comb in the red-light only group was only given the red-light
respectively.
The experimental cocks were placed avoiding light for at least 48h after
any treatment.
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irradiation with the light density of 80J/cm2, 120J/cm2, 160J/cm2 and 200J/cm2
treatment.
5.Morphological changes of the comb
Digital camera and dermatoscope were used to take photos of the morphological changes of the treated area of each comb before and after treatment.
6. Histopathological changes of the comb Six mm trephine was used to drill the whole layer of comb tissue (including the
side of experimental side was taken as control.
The experimental side was marked
with medical skin marker before the experiment.
While the control side was not
The biopsy tissues were fixed with 10% formaldehyde and embedded in
7. Immunohistochemical changes of the comb
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paraffin. Then slice sections were stained with routinely.
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marked.
The opposite
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experimental side and the contralateral side) before and after treatment.
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The biopsy tissues above were fixed with 10% formaldehyde and embedded in paraffin.
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Slice sections were immunohistochemically stained with VEGF routinely. Each slice
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randomly selected 5 magnifications; visual field (10 x 20), calculating average capillary
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number.
8.Statistical analysis
The program GraphPad Prism5.0 was used for analysis.
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presented by x ± s.
The vascular data were
The comparison between the surface fluorescence intensity
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before and after the application of ALA and the comparison of the number of blood vessels between the experimental side and the control side of the comb were detected P<0.01 was statistically significant.
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by t detection method.
Results 1. The distribution of fluorescence in comb The fluorescence intensity of the topical ALA group gradually increased with the incubation time and reached the peak at 4 h (P<0.01).
Then, the fluorescence intensity
decreased and tended to the initial level at 7h (Figure 1A).
Thus, 4h incubation time
were selected as the optimal parameters for the topical ALA-PDT treatment.
In systemic ALA groups, the fluorescence intensity gradually increased and reached the highest point at 6h (P<0.01) with the incubation time between 0~28h at the five The
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ALA concentration (25mg/kg, 50mg/kg, 75mg/kg, 100mg/kg, 200mg/kg). fluorescence intensity increased with the increase of the ALA concentration.
And
there is significant different between 50mg/kg ALA and 25mg/kg (P<0.01) (The results
And other parts of the cock also have strong
fluorescence under high ALA concentrations.
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did not provide in this paper).
It was found that the PpIX fluorescence
decreased after 6h and tended to the initial level at 22h (Figure 1B).
So, 50mg/kg
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ALA and 6h incubation time were selected as the optimal parameters for the systemic
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ALA-PDT treatment.
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Figure 1
Observed in dark field by Wood light, no fluorescence observed before topical and After the applying with ALA, the bright brick red
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systemic application of ALA. fluorescence was observed.
And the fluorescence gradually increased with the
prolongation of incubation time.
After topical application of ALA at 3 and 4h, the
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brick red fluorescence was the strongest. weakened.
The fluorescence disappeared 7h after topical application of ALA (Figure
No red fluorescence was observed on the control side.
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2A).
After 4h, the fluorescence gradually
disappeared after light illumination (Figure 2B).
After systemic application of ALA
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at 5 and 6h, the brick red fluorescence was the strongest. gradually weakened.
And the fluorescence
Then, the fluorescence
The fluorescence tended to the initial level 22h after systemic
application of ALA (Figure 2C). illumination (Figure 2D).
And the fluorescence disappeared after light
In addition, after intravenous injection of ALA, the
fluorescence was observed in both comb and the other parts of body (Figure 2E).
Figure 2
In order to further explore the tissue absorption of ALA, the biopsy tissue of the comb was made for frozen section after the application of ALA topically for 4h and systematically for 6h.
After topical application of ALA for 4h, bright brick red fluorescence
was observed in epidermis and no fluorescence in dermis.
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microscope.
The fluorescence in tissue was observed by fluorescence
In normal control groups,
there was no fluorescence in both epidermis and dermis (Figure 3A).
After systemic
and dermis (Figure 3B).
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application of ALA for 6h, diffuse brick red fluorescence was observed in epidermis
In normal control groups, there was also no fluorescence in
both epidermis and dermis.
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2.Morphological changes of the comb
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Figure 3
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All of the experimental areas showed mild swelling immediately after the light irradiation with the light density of 80J/cm2, 120J/cm2 and 160J/cm2 in the topical And the color was restored to normal after 10 min.
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ALA-PDT groups.
When the
light density was up to 200J/cm2, there was significant swelling in the experimental area immediately after the irradiation.
In the 80J/cm2 topical ALA-PDT group, there was no obvious change after
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30min.
And the color was restored to normal after
the topical ALA-PDT for 1,3, 5, 7 and 14 days.
In the 120J/cm2 topical ALA-PDT
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group, there was slight edema in the experimental area 1d after treatment. erosion and exudation was observed 5d after treatment.
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treatment.
And slight
Thin crust occurred 7d after
In the 160J/cm2 topical ALA-PDT group, edema appeared in the
experimental area 1d after treatment.
Then erosive exudation was observed 3d after
treatment and yellow callus 5d after treatment. observed 14d after treatment.
Partial epidermal necrosis was
In the 200J/cm2 topical ALA-PDT group, obvious
edema appeared 1d after treatment.
Then erosive exudation was observed 3d after
treatment, aggravated erosion 5d after treatment and yellow callus 7d after treatment. And crusts not shed with partial epidermal necrosis 14d after treatment (Figure 4A). There were no obvious changes in the control and no experimental areas in each group.
In the 80J/cm2 and 120J/cm2 systemic ALA-PDT groups, slight edema was observed
after treatment and partial shedding of eschar 14d after treatment.
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1d after treatment, slight erosion and exudation 3d after treatment, the thin crust 5d In the 160J/cm2
systemic ALA-PDT group, the experimental area appeared purple black color change
In the 200J/cm2 systemic
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1d after treatment and black crusts 3d~14d after treatment.
ALA-PDT group, purple change was observed the experiment area 1d after treatment, black crusts 3d after treatment and crust peeling off 14d after treatment (Figure 4B).
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There were no obvious changes in the control and no experimental areas in each group.
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In the PDL group, slight edema occurred in the experimental area1d after treatment.
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Thin layer of yellow and black scab was observed 1d after treatment.
And 14th day,
crust was peeling off and the experimental area showed white. There were no obvious
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changes in the control and no experimental areas in each group (Figure 4C).
The morphology of the comb was rosy and plump in negative control group.
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Figure 4
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3.Histopathological changes of the comb Comb tissue was drilled by trephine for biopsy and stained by HE after treatment.
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In the 80J/cm2 topical ALA-PDT group, there was no obvious change both in the epidermis and dermis compared with the non-treatment side.
In the 120J/cm2 topical
ALA-PDT group, slight injury, cell edema, slight exudation and edema was observed in the epidermis on the first day after treatment. more severe 3, 5, 7 or 14 days after treatment.
The changes of dermal tissue were
There was no significant change in the
epidermis and the dermal blood vessels compared with the control group and nontreatment side.
In the 160J/cm2 topical ALA-PDT group, there was a significant
change in the epidermis.
And the dermal tissue showed slight exudation and edema.
In the 200J/cm2 topical ALA-PDT group, there was more severe damage in the
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epidermis (Figure 5A).
In the 80J/cm2 systemic ALA-PDT group, the epidermal cells showed slight edema,
hyperplasia and exudation and edema of dermal tissue on the first day after treatment
Significant changes were observed in the
epidermis 3,5 ,7and 14 days after treatment.
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compared with the non-treatment side.
In the 120J/cm2 systemic ALA-PDT
group, epidermal cells showed edema and hyperplasia and dermal tissue exudation and The epidermis was crusted and the number of
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edema on the first day after treatment.
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the vessels decreased gradually 3,5 ,7 and 14 days after treatment.
In the 160J/cm2
The results of the histopathology showed epidermal cell
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epidermis at the 1st day.
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and 200J/cm2 systemic ALA-PDT group, significant changes were observed in the
edema, dermal tissue exudation, edema and dermal superficial capillary thrombosis The epidermis was black and yellow, with the exudation
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were observed on the 3rd day.
and edema of the dermis, the decrease of the number of capillaries in the superficial layer of the dermis and the decrease of the diameter of the blood vessels 5, 7, and 14
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days after treatment (Figure 5B).
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In the PDL group, the epidermal cells edematous, the dermal tissue exudative and edema, the number of capillaries in the superficial layer of the dermis decreased and
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the diameter of the vessels became smaller on the first day after treatment.
And 3, 5,
7, 14 days after treatment, tissue changes were more obvious and the epidermis was crusted (Figure 5C). There was no significant histopathological change in negative control group. Figure 5
4. Immunohistochemical changes of the comb Comb tissue was drilled by trephine for biopsy and stained by VEGF for the capillary vessels after treatment.
The results showed that there was no significant change in
the number of blood vessels between the treatment side and the non-treatment side in
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the topical ALA-PDT groups(P>0.05) (Figure 6A and Table 1).
In the systemic ALA-PDT groups, the number of blood vessels in the treatment side
And the vessels decreased
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decreased significantly on the 3rd day after treatment.
significantly 5, 7 and 14 days after treatment (P<0.01).
The number of vessels
decreased significantly with the increase of the light density (P<0.01) (Figure 6B and
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Table 2).
And the vessels decreased significantly 5, 7 and 14 days
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significantly on the 1st day.
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In the PDL group, results showed that the number of the blood vessels began to decrease
after treatment (P<0.01) (Figure 6C and Table 3).
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The immunohistochemistry showed that there were no significant changes in the number of blood vessels on both sides of the comb in negative control groups (P>0.05).
Table 1
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Table 2
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Figure 6
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Table 3
Discussion
The superficial dermis of cock combs has a rich capillary network, which has similarities with the histopathology of PWS [19].
And the optical adsorption
properties of cock blood are basically the same as that of human blood. Therefore, the comb tissue is usually used as the animal model for capillary hemangiomas by foreign research groups since the late 1960s [20]. PDL with a wavelength 585 or 595
patients [3].
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nm is now a gold standard for the treatment of PWS, which has a defined effect on But it is not suitable for the treatment of a large area of PWS [21].
In
recent years, PDT, with intravenous injection of photosensitizer HMME, is used for
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PWS for its selective specificity to target tissues [6].
Recently, some reports demonstrated that the topical ALA-PDT also have good curative Zhang Anli et al [22] used 10% ALA solution combined with topical
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effect on PWS.
The total
Liu et al [15] used 20% ALA gel combined with
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effective rate was up to 96.4%.
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application of 532nm PDL in treating 28 cases of patients with PWS.
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595nm PDL in the treatment of 35 cases of patients with PWS. 37.14% of patients improved moderately, while 28.57% had mild improvement.
effect on PWS.
However, single 532nm PDL or 595nmPDL have the therapeutic
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adverse reaction.
All patients had no
Thus, the curative effect of ALA-PDT still needs further confirmation.
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ALA is a small hydrophilic molecule, which is also a precursor of porphyrin in the synthesis of hemoglobin.
When absorbed by high proliferating cells, it will be
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converted into PpIX by enzymatic catalysis.
The results of this study and other reports
indicate that ALA only exists in the epidermis and sebaceous gland, not in the basement
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membrane [16-18].
Our experiment also confirmed that the absorption and
transformation of ALA was found only in the epidermis after the applying of ALA. And the fluorescence intensity reached the peak 4h after topical ALA application, but no red fluorescence was observed in the dermis (Figure 3A). absorbed by the blood vessels in the dermis.
That is, ALA was not
While the blood vessels closure is the
primary purpose of the treatment of PWS.
We also confirmed that PpIX fluorescence
can be observed in the dermis after systemic application of ALA. fluorescence intensity peaked at 6h.
And the
While we also detect the PpIX fluorescence in
other parts of the cock body as well as the epidermis of the comb (Figure 2E, 3B). Then, systemic ALA-PDT caused the severe damage of the other parts of the cock body,
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especially the epidermis.
The best absorption light source of ALA is about 410nm blue light and the red light
penetrability.
Clinicaly, red light is selected as ALA-PDT light source for its deeper
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around 630nm.
In this experiment, 4 light density of 80J/cm2, 120J/cm2, 160J/cm2 and
200J/cm2 were used to further explore the curative effect of topical ALA-PDT for PWS.
When the light density was 120J/cm2,
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the comb under low light density of 80J/cm2.
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The results showed that there was no change in the morphology and histopathology of
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the comb appeared slight exudation and thin crust.
And the reaction was more severe
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under the light density of 160J/cm2 and 200J/cm2 (Figure 4A). histopathology showed epidermal necrosis and loss (Figure 5A).
The results of Therefore, as the
severe.
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energy density increased, the damage of topical ALA-PDT on the epidermis was more VEGF is a marker of vascular endothelial cell.
We use VEGF
immunohistochemistry to observe the change in the number of vessels after ALA-PDT It was found that there was no statistical difference in the number of
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treatment [21].
vessels between the treatment side and the non-treatment side (Figure 6A, table 1).
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Thus, the results demonstrated that topical ALA-PDT has direct damage on the epidermis with increased energy density, while there is no damage on the blood vessels
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in the dermis.
So, the topical ALA-PDT had no curative effect on PWS.
In order to further explore the influence of ALA-PDT on the blood vessels of PWS, systemic injection of ALA combined with red light was used for the treatment of comb. Previously few studies on systemic ALA-PDT were reported.
Thus, we do some
experiments to explore the concentration ALA.
We found that the fluorescence
intensity increased with the increase of ALA concentration.
The fluorescence
intensity in 50mg/kg was significantly different from that of 25mg/kg (P<0.01). However, with the increasing of the ALA concentration, the other parts of the cock also had a strong fluorescence generation, which would cause severe side effects after
for our experiment.
Therefore, the ALA concentration of 50mg/kg was chosen
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illumination (Figure 1B).
And the same light density as the topical ALA-PDT was chosen.
It was found that there was only partial erosion and thin crust appeared under low light With the increase of light density, charred thick crust and scar was observed
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density.
on the treatment side of the comb (Figure 4B). Histopathological examination showed the dermal and epidermal tissues were damaged correspondingly with the increase of
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light density (Figure 5B). Immunohistochemistry showed that there was a significant
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reduction in capillaries of the treatment side (Figure 6B, Table 2).
Based on the above
But it also had severe damage to the epidermis.
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energy.
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results, it showed systemic ALA-PDT had the effect on blood vessels under certain And when the ALA in
vascular endothelial cells transformed into other tissues and epidermis, there would be
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adverse reactions on other non-targeted tissues.
And we also found that there was still
fluorescence 28h after the application of intravenous ALA.
Therefore, systemic
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ALA-PDT was not suitable for the treatment of PWS either.
PDL is the classical treatment for PWS.
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control in this study.
Therefore, 595nm PDL was used as positive
The results showed that the experimental area in comb became
white after PDL treatment (Figure 4C).
Histopathological examination showed that
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there was no significant change in the epidermis in treatment side, but dermal capillary was blocked (Figure 5C).
Immunohistochemistry showed that capillary number was
significantly reduced compared to that in the non-treatment side (Figure 6C). proved that PDL had obvious curative effect on PWS.
It was
Conclusion: There is no curative effect of the topical red-light ALA-PDT on comb dermal capillaries.
Systemic ALA-PDT could damage the dermal blood vessels, but
it also has severe effect on the epidermis and other non-targeted parts. In conclusion, either topical or systemic red-light ALA-PDT is not suitable treatment methods for PWS.
cooperation.
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Conflict of interest statement: The authors state no conflict of interest.
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Acknowledgements: The authors would like to thank all study participants for their
Funding：This work was supported by the National Natural Science Foundation of
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China (Grant numbers: 81472538, and 8140120479), and the Joint Project of New
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Frontier Technology of Shanghai Shen-kang Hospital Development Center (Grant
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number: SHDC12015123).
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3. Brightman LA, Geronemus RG, Reddy KK. Laser treatment of port-wine stains. Clin Cosmet Investig Dermatol,2015;8:27-33.
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5. Gao K, Huang Z, Yuan KH, et al. Side-by-side comparison of photodynamic
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6. Kennedy JC, Pottier RH, Pross DC. Photodynamic therapy with endogenous protoporphyrin IX: basic principles and present clinical experience. J Photochem Photobiol B, 1990, 6(1-2):143-8.
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7. Marica B Ericson, Ann-Marie Wennberg, Olle Larkö. Review of photodynamic therapy in actinic keratosis and basal cell carcinoma. Ther Clin Risk Manag, 2008,
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laser for the treatment of Bowen's disease. Lasers Med Sci2015;30(5):1505-10.
9. Gao Y, Zhang XC, Wang WS, et al. Efficacy and safety of topical ALA-PDT in the treatment of EMPD. Photodiagnosis Photodyn Ther 2015;12(1):92-7. 10. Zhang HY, Ji J, Tan YM, et al. Evaluation of 5-aminolevulinic acid-mediated photorejuvenation of neck skin. Photodiagnosis Photodyn Ther 2014;11(4):498509.
11. Lei X, Liu B, Huang Z, Wu J. A clinical study of photodynamic therapy for chronic skin ulcers in lower limbs infected with Pseudomonas aeruginosa. Arch Dermatol Res. 2015;307(1):49-55. 12. Ji J, Zhang LL, Ding HL, et al. Comparison of 5-aminolevulinic acid photodynamic therapy and red light for treatment of photoaging. Photodiagnosis Photodyn Ther
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2014; 11(2):118-21. 13. Ma L, Xiang LH, Yu B, et al. Low-dose topical 5-aminolevulinic acid
Photodiagnosis Photodyn Ther 2013;10(4):583-90.
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photodynamic therapy in the treatment of different severity of acne vulgaris.
14. Wang J, Huang X. Effects of aminolevulinic acid-based photodynamic therapy on cock combs, an animal model for port wine stains. Chin J Dermatol 2015;(48)5:333-
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337.
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15. Liu S, Yang C, Yang S, et al. Topical application of 5-aminolevulinic acid followed
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by 595-nm pulsed dye laser irradiation for the treatment of recalcitrant port wine
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stains: a primary study. J Cosmet Laser Ther 2012;14(4):189-92. 16. Chen HX, Zou XB, Zhang YJ, et al. Recent Progress in Photodynamic Therapy for
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Port Wine Stains. Chin J Derm Venereol 2014;28(6):631-633. 17. Wang XL, Wang HW, Huang Z, et al. Study of protoporphyrin IX (PpIX) pharmacokinetics after topical application of 5-aminolevulinic acid in urethral
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condylomata acuminata. Photochem Photobiol 2007;83(5):1069-73. 18. Ding HL, Wang XL, Wang HW, et al. Successful treatment of refractory facial acne
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using repeat short-cycle ALA-PDT: Case study. Photodiagnosis Photodyn Ther 2011; 8(4):343-6.
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19. Ohshiro T, Nakajima T, Ogata H, et al. Histological responses of cutaneous vascular lesion following photodynamic therapy with talaporfin sodium: a cock comb model. Keio J Med 2009; Sep;58(3):176-84. 20. Li ZH, Meng DS, Li YY, et al. Hypericin damages the ectatic capillaries in a Roman cockscomb model and inhibits the growth of human endothelial cells more potently
than hematoporphyrin does through induction of apoptosis. Photochem Photobiol. 2014 Nov-Dec ;90(6):1368-75. 21. Wang YZ, Huang Z, Yuan KH. The current situation and development in optical therapy for port-wine stains. J Pract Dermatol2015;8(5):363-366. 22. Zhang AL, Zu DM, and Yang L, et al. 5- aminolevulinic acid photodynamic therapy
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23. Brightman LA, Geronemus RG, Reddy KK. Laser treatment of port-wine stains.
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Clin Cosmet Investig Dermatol 2015;8:27-33.
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Legends
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Figure 1 A: The changes of the surface fluorescence intensity after the topical application of ALA for 0~7h. * indicated P value (< 0.05) of the comparison of the fluorescence intensity at 4h with that at 1h.
B: The changes of the surface
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fluorescence intensity after the intravenous injection of ALA with the concentration of
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50mg/kg for 0~28h. ** indicated P value (< 0.01) of the comparison of the fluorescence
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intensity at 6h with that at 1h.
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Figure 2 A: The changes of the brick red fluorescent on the surface of the comb after the topical application of ALA for 0~7h.
B: The changes of the brick red fluorescent
before and after light illumination in the topical ALA-PDT group.
C: The changes of
the brick red fluorescent on the surface of the comb after the systemic application of
ALA for 0~28h.
D: The changes of the brick red fluorescent before and after light
illumination in the systemic ALA-PDT group.
E: The changes of the brick red
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fluorescent on the surface of the body after the systemic application of ALA for 0~28h.
B: The distribution of PpIX fluorescence in epidermal
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fluorescence in the epidermis.
The green fluorescence was the spontaneous
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topical application of ALA for 4h.
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Figure 3A: The distribution of PpIX fluorescence in the epidermis of the comb after the
and dermal tissues comb after the systemic application of ALA for 6h.
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and dermis situation in the blank control group.
C: Epidermis
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Figure 4 A: The morphological changes of the comb (*10) in the topical ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 B:
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J/cm2, 160 J/cm2 or 200 J/cm2 (the black circle represented the experimental area).
The morphological changes of the comb (*10) in the systemic ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 J/cm2, 160 J/cm2 or 200 J/cm2 (the black circle represented the experimental area).
C: The
morphological changes of the comb (*10) in PDL group 1,3, 5, 7 or 14 days after
treatment with the light density of 15 J/cm2 (the black circle represented the
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A
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experimental area).
Figure 5 A: The HE staining of the comb tissue (*200) in the topical ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 J/cm2, 160 J/cm2 or 200 J/cm2. B: The HE staining of the comb tissue (*200) in the systemic
ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 J/cm2, 160 J/cm2 or 200 J/cm2.
C: The HE staining of the comb tissue
(*200) in the PDL group 1,3, 5, 7 or 14 days after treatment with different light density
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of 15 J/cm2.
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B: The immunohistochemical
staining for VEGF of the comb tissue (*200) in the systemic ALA-PDT group 1,3, 5, 7 or 14 days after treatment with different light density of 80 J/cm2, 120 J/cm2, 160 J/cm2 or 200 J/cm2.
C: The immunohistochemical staining for VEGF of the comb tissue
(*200) in the PDL group 1,3, 5, 7 or 14 days after treatment with different light density
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of 15 J/cm2.
Table 1 Comb vascular changes in topical ALA-PDT group after treatment(mean±s.d)
28±5.33
27±5.11
28±3.21
27±7.11
28±2.12
27±5.77
28±2.19
26±5.99
27±7.97
27±3.88
30±3.63
29±2.13
26±7.33
26±6.55
26±3.21
30±7.12
27±1.22
28±2.51
27±3.79
26±1.88
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28±3.11
27±5.99
27±6.89
25±3.13
25±7.49
24±1.45
27±2.18
27±1.73
26±3.90
25±5.91
26±3.44
25±1.82
30±1.23
26±3.21
27±7.34
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14
28±3.13
25±5.78
24±6.34
24±2.14
30±5.19
27±1.27
28±4.92
26±6.92
25±3.56
25±2.33
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7
29±1.67
25±1.84 26±6.17
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80J/cm2 Treatment side Non-treatment side 120J/cm2 Treatment side Non-treatment side 160J/cm2 Treatment side Non-treatment side 200J/cm2 Treatment side Non-treatment side
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Comb surface
Local ALA-PDT processing time (Day) Before 1 3 5 treatment
Table 2 Comb vascular changes in systemic ALA-PDT group after treatment(mean±s.d)
Comb surface
14
26±3.21
12±5.31* 6±5.11*
4±1.21*
3±2.84*
30±3.02
27±2.28
28±5.91
26±2.94
27±3.32
25±5.23
28±3.67
26±6.33
10±7.33* 4±7.16*
4±4.21*
30±2.44
28±4.78
27±2.59
26±1.92
27±3.17
26±3.81
Treatment side 27±6.62
25±2.97
6±2.17*
4±5.11*
3±3.01*
3±5.14*
27±1.74
28±5.90
26±3.55
26±4.01
25±2.0
24±6.33
4±2.92*
3±1.11*
2±5.01*
1±3.01*
26±2.79
27±6.11
26±6.12
26±3.03
25±2.59
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A
3±7.04*
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Non-treatment 28±2.02 side 200J/cm2 Treatment side 29±1.12 Non-treatment 30±2.11 side
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30±2.12
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80J/cm2 Treatment side Non-treatment side 120J/cm2 Treatment side Non-treatment side 160J/cm2
Systemic ALA-PDT processing time (Day) Before 1 3 5 7 treatment
*P<0.01 There was significant difference in P<0.01 between the treatment side and non-treatment
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side.
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Table 3 Comb vascular changes in PDL group after treatment(mean±s.d)
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595nm PDL processing time (Day) Comb surface Before 1 3 5 treatment Treatment side 29±2.77 9±2.33* 5±2.94* 4±5.11* Non-treatment 30±2.02 28±5.12 28±3.99 29±5.19 side
7
14
4±6.21*
2±4.12*
27±3.97
27±5.88
*P<0.01 There was significant difference in P<0.01 between the treatment side and non-treatment side.
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