Optical Technology for Fibrotic Skin Changes Objectification in

Experimental Systemic Scleroderma

Yulia Chursinova

, Dmitriy Kulikov

, Dmitry Rogatkin

, Irina Raznitsyna

1, 2

Darya Mosalskaya

and Maksim Bobrov

Moscow Regional Research and Clinical Institute "MONIKI", 61/2, Shchepkina str.,

Moscow, RF, 129110, Russian Federation

National Research Nuclear University MEPhI, 31, Kashirskoe highway, Moscow, RF, 115409, Russian Federation

Keywords: Skin Fibrosis, Inflammation, Fluorescence, Saturation, Non-invasive, Diagnostics, in Vivo.

Abstract: Currently the examination of skin fibrosis is based on subjective non quantitative methods and requires

invasive procedures. Optical techniques abled to evaluate different quantitative parameters of ordered

tissues can be used to solve these problem. Measurements of endogenous fluorescence intensity, regional

tissue oxyhemoglobin saturation, and blood filling volume allowed to define the high endogenous

fluorescence intensity of porphyrin in skin fibrosis. Besides that, the decrease in oxygen intake parameters

together with the fluorescence intensity increase of collagen was determined. Consequently the optical

diagnostic techniques can become an effective method for skin fibrosis evaluation.

1 INTRODUCTION

Fibrosis of different organs and systems is one of

severe medical issues as it affects a significant

proportion of the human population (Wermuth,

2015; Rockey et al., 2015). It is the main

pathological process not only in such autoimmune

disorders as scleroderma, rheumatoid arthritis,

Crohn’s disease, ulcerative colitis, and systemic

lupus erythematosus (Wynn et al., 2011), but also in

liver, kidney, lung diseases and heart failure

(Bataller et al., 2005; Wynn, 2008).

Currently there are a lot of studies on fibrosis.

The pathogenesis of tissue fibrosis is considered to

be a dynamic and reversible process connected with

inflammation and hypoxia (Driskell et al., 2013;

Manresa et al, 2014). Nevertheless, the histological

examination still remains the reference method for

diagnosis, which invasiveness is the main

disadvantage as it impairs the examined tissues state

(Monstrey et al., 2008).

Fibrotic skin changes are the defining features of

all scleroderma forms (Gabrielli et al., 2009). The

degree and the rate of fibrosis progression correlate

with patients’ death rate (Clements et al., 2000;

Khanna et al., 2010). In clinical practice the

modified Rodnan skin score (mRSS) that measures

the skinfold thickness is widely used for skin

fibrosis evaluation. However, the information

received when using this method is rather subjective.

The application of mRSS is essentially limited as it

requires special knowledge and skills from

physicians. Subcutaneous fat changes developed in

some patients suffering from this disease can also

lead to diagnostic pitfall. Furthermore skin score is

insensitive to initial presentation of a disease that is

however clinically significant (Maurer et al., 2014).

Non-invasive methods of skin fibrosis diagnosis

such as ultrasound scan, elastography, confocal

microscopy, and optical coherence tomography are

still of limited use in routine clinical practice due to

the lack of the accurate criteria for fibrosis

assessment (Kang et al., 2014). The microvascular

damage dominates in the pathogenesis of skin

fibrosis. That causes endothelial cell activation

leading to the hypoxia and the increase in the

amount of inflammation triggers that starts

uncontrolled inflammation response. As a result of it

fibroblasts excessively differentiate into

myofibroblasts, responsible for extracellular matrix

synthesis, which major component is collagen

(Jinnin, 2010; Hinz B. et al., 2012; Ho et al., 2014).

The excess of collagen is known to be detected by

laser fluorescence spectroscopy as this substance

fluoresces under UV light (Smirnova et al., 2012).

194

Chursinova, Y., Kulikov, D., Rogatkin, D., Raznitsyna, I., Mosalskaya, D. and Bobrov, M.

Optical Technology for Fibrotic Skin Changes Objectiﬁcation in Experimental Systemic Scleroderma.

DOI: 10.5220/0006633101940199

In Proceedings of the 11th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2018) - Volume 1: BIODEVICES, pages 194-199

ISBN: 978-989-758-277-6

Fluorophores responsible for inflammation and

hypoxia can also be detected in red and green

spectrum range (Franco et al., 2016).

In modern medicine the development of the rapid

and non-invasive method enabled to give a

quantitative assessment of skin fibrosis is absolutely

necessary. In such a case optical techniques have a

diagnostic potential to become the basis for a

fundamentally new approach to fibrosis

comprehensive assessment.

The aim of our study was to examine diagnostic

capabilities of optical technologies in animal skin

fibrosis evaluation. Animal models are still of a

great importance for skin fibrosis pathogenesis

investigation, and the results obtained can either be

reproduced in clinical researches or give a

meaningful data for understanding the pathogenesis

of this process (Avouac et al., 2013; De Langhe,

2015).

2 MATERIALS AND METHODS

The study was performed on the outbred white male

mice aged 6 weeks with a mass of 25-30 grams, N =

47. Animals were kept in vivarium standard

conditions in a 14 hour natural light at a temperature

of 21-23 °C and a humidity of 50 - 65%. They

received balanced granulated feed, that didn’t

include fluorophores and had a free water access.

The experiment was conducted in compliance

with the welfare of animals used in experiment

(Declaration of Helsinki), EU Directive 86/609/EEC

on the protection of animals used in experiments,

and European Convention for the Protection of

Vertebrate Animals Used for Experimental and other

Scientific Purposes (ETS 123) Strasbourg, 1986).

The relevant animal model of scleroderma was

used for fibrosis development. We chose bleomycin-

induced fibrosis as it allows to represent the initial

presentation of this disease (Avouac, 2014).

Animals were divided into two groups. In the

first one (N = 30) subcutaneous injections of

bleomycin (BLM) in a dose of 0,1 ml (100μL of

bleomycin preliminary dissolved in 0.9% NaCl,

concentration 0.5 mg/ml) were administered. In the

second one (control group, N = 17) subcutaneous

injections of 0,1 ml of 0,9 % NaCl (PBS) were

administered. All animals were daily injected in

shaved skin of interscapular region during 21 days.

The first four injections were made in different

angles of a marked square with a size of 1 cm², the

fifth one was done in its middle.

On the 0, 7, 14 and 21 day the endogenous

fluorescence intensity, regional tissue

oxyhemoglobin saturation, and blood filling volume

were measured in vivo, on the skin surface just

above the experimental area. All measurements were

taken using non-invasive multifunctional laser

diagnostic system “LAKK-M” (SPE ‘LAZMA’ Ltd,

Russia) (Figure 1) on the operating regimes

“Fluorescence” and “Microcirculation” (Rogatkin et

al., 2009). Scheme of diagnostic system, the mouse

locations in the setup and sensor localization are

presented at figure 2.

Figure 1: Diagnostic system “LAKK-M”.

Figure 2: Scheme of the diagnostic system.

Low-power lasers with a wave length of λ

= 365,

535 and 635 nm were used for fluorescence

excitation. The output power at the distal end of the

Optical Technology for Fibrotic Skin Changes Objectiﬁcation in Experimental Systemic Scleroderma

195

fiber-optical probe was about 2 – 3 mW for each

light source. The wavelengths on which the

fluorescence had a maximum value were marked

with λ

. Thus for collagen λ

= 455 nm, for porphyrin

= 610 nm. It should be noted, that it is hard to

separate the fluorescence of collagen and elastin, so

in the following we considered that the fluorescence

on the wave length of λ

= 455 nm represents both of

fluorophores. In this study the intensity dynamics on

this wavelength (later on – “the fluorescence

intensity”) in controlled equivalent intensity of

irradiation was evaluated.

In “Microcirculation” operating regime laser

Doppler flowmetry and tissue reflectance oximetry

were used enabling to continuously register tissue

saturation and blood filling volume values in

percent.

The relative oxygen consumption rate (U)

characterized by the oxygen intake per tissue blood

flow volume unit was assessed according to the

time-averaged (15 s) measurements using the

following formula (Rogatkin et al., 2013):

U= (S

- S

)/ V

(1)

, means tissue oxyhemoglobin saturation, V

means blood filling volume. In this case, S

is the

functional pulse saturation of the oxyhemoglobin

fraction in the arterial peripheral blood. It was

assumed equal to 98%.

Histological samples were taken on 0, 7, 14, and

21 day. Skin fragments 1,0 cm  1,0 cm in size were

separated from the research region followed by

material examination according to a standard

protocol. An epidermis condition, inflammatory

changes in dermis, subcutaneous fat, and panniculus

carnosus, as well as dermis thickness and collagen

fibers structure were assessed.

Furthermore, a noncompetitive enzyme-linked

immunosorbent assay method was used to evaluate

C-reactive protein in control period. For this purpose

we used a mice blood serum having been

centrifuged in 1500 g mode during 15 minutes.

Researches were performed on microplate

photometer for enzyme immunoassay Stat Fax 2100,

Awareness Technology, USA. We also used Mouse

CRP (an enzyme immunoassay kit for the

quantitative measurement of mouse CRP), Czech

Republic.

Statistical analysis was carried out in Microsoft

Excel 2016. A hypothesis for the difference between

two groups was tested by the comparison of

arithmetic means and the construction of 95%

confidence intervals for them.

3 RESULTS AND DISCUSSION

In BLM animal group a skin fibrosis was reproduced

by the 21

day of experiment. The histological

pattern of tissue in the injection area in both groups

on the 21

day of experiment is shown in the Figure

Figure 3: Skin histology on the 21

day, haematoxylin and

eosin stain at a magnification of x 100. In BLM group:

flatness of epidermis, thickened derma, homogenisation of

collagen fibres, hyperplasia of hair follicles, dermal

adipose layer depletion, inflammatory infiltrate under

panniculus carnosus. In PBS group: the structure of the

epidermis and dermis is unchanged, inflammatory

infiltrate under panniculus carnosus is detected.

Examples of measured fluorescence spectrum

from the injection area at λ

= 365 nm on the 21

day

of the experiment is shown in the Figure 4.

Figure 4: The example of the fluorescence spectrum in

BLM and PSB groups on the 21

day of the experiment; λ

= 365 nm.

Obtained spectra are characterized by the

presence of two maxima corresponding to the

backscattering peak (365 nm) and to the

fluorescence of the collagen and elastin (455 nm).

Figure 5 illustrates the results of optical

measurements and laboratory analysis.

BIODEVICES 2018 - 11th International Conference on Biomedical Electronics and Devices

196

Figure 5: Dynamic by days: А. - Fluorescence intensity

rate of collagen and elastin (λ

= 365 nm, λ

= 455 nm); B.-

Fluorescence intensity rate of porphyrin (λ

= 535 nm, λ

610 nm); C. - Relative oxygen consumption rate in tissues;

D. - C-reactive protein rates. 95% confidence interval is

shown in the chart.

Figure 5A shows the dynamics of the

t6yrteaverage fluorescence intensity rate of collagen

and elastin in both groups. On the 21

day a

statistically significant difference in BLM and PBS

groups appeared.

The results of porphyrin fluorescence (Figure 5B)

demonstrate the increase in the average fluorescence

intensity as compared to the 0 day of the experiment

in both animal groups. Besides that, a statistically

significant difference in BLM and PBS groups were

received on the 21

day.

The results of relative oxygen consumption rate

demonstrate its statistically significant decrease by

the 21

day in BLM group (Figure 5C).

Figure 5D shows the increase in C-reactive

protein rate of animal blood serum in both groups.

Statistically significant differences in BLM and PBS

groups were assessed on the 7

day of experiment.

All data obtained during the experiment leads to

a number of conclusions and assumptions. Thus, we

believe, that the increase in endogenous fluorescence

intensity rate of collagen and elastin on the 21

day

in BLM group is due to its accumulation in the

histologically confirmed fibrosis area. Whereas

collagen is the main extracellular substance of

connective tissue in skin fibrosis (Ho et al., 2014),

we consider, that the impact of elastin fluorescence

is imperceptible.

Tissues in which collagen fibers are extensively

synthesized are known to have a high oxygen

requirement. Nevertheless, the formed fibrosis

decreases it due to the reduction in the number of

cell elements (Lokmic et al., 2012). The relative

oxygen consumption rate on the 21

day in BLM

group is representative of this causation. (Figure 5A

and 5C). Some researchers consider that fibrosis is

permanent when a tissue becomes few-celled and

has a lack of biologically active molecules essential

to the extracellular substance deterioration (Iredale,

2007; Wynn, 2008, Rockey, 2015). We believe that

contemporary examination of collagen fluorescence

and the relative oxygen consumption rate in tissues

will allow to establish the synchronous nature of this

process. The information received is essential to

clinicians as the data on the degree and the rate of

skin fibrosis progression in systemic scleroderma

enable to diagnose this form of disease, to determine

a management strategy in time, and to predict the

clinical course.

Porphyrins are well known to response on

metabolic changes in tissues quickly. Their synthesis

is particularly enhanced in cells during inflammation

and hypoxia (Petritskaya et al., 2015). Vasculopathy

is known to cause the oxygen delivery decrease in

Optical Technology for Fibrotic Skin Changes Objectiﬁcation in Experimental Systemic Scleroderma

197

cells at the beginning of scleroderma. In the

following, fibrosis tissue induces perfusion defect

and becomes the main cause of hypoxia (Van Hal et

al, 2011). Since bleomycin-induced model better

represents tissue fibrotic changes (Yamamoto et al,

2011), we suppose that statistically significant

magnification in porphyrin fluorescence rate on the

day indicates a chronic hypoxia and is based on

the perfusion defect.

We also assumed the development of

inflammation of the dermis during fibrosis formation

in experimental model. High levels of blood serum

C-reactive protein in BLM group on the 7th day

were determined in support of it. That implies the

disease high activity and is a significant predictor of

complications and premature mortality (Muangchan

et al., 2012; Darby et al., 2016). Hence, daily

subcutaneous injections also were associated with

inflammation under panniculus carnosus in both

animal groups. Probably, the increase in porphyrin

fluorescence intensity in both groups was due to it.

Nevertheless, to prove our assumption a separate

study needs to be carried out providing with the

method development that will make it possible to

distinguish chronic hypoxia and inflammation of

different sites.

4 CONCLUSIONS

The use of optical technologies in the experiment

enabled to determine the increment in endogenous

fluorescence intensity of collagen and the decrease

in tissue oxygen intake in the fibrosis area. We also

registered the increase in endogenous fluorescence

intensity rate of the porphyrins as a potential chronic

hypoxia and inflammatory marker.

It is important that all optical methods used in

this study were non-invasive. Nevertheless we

managed to obtain quantitative and impersonal

information. Considering the fact that the animal

scleroderma model is relevant, the data obtained can

be reproduced in man.

The results of the experiment, of course,

demonstrate the necessity of a research continuation

in this direction, but already now it is possible to

predict great opportunities for optical technologies

in the diagnosis of skin fibrosis.

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