In Vivo Experimental Detection of Inflammatory Process in Tissues

by Fluorescence Spectroscopy

Irina Guseva

1,2

, Dmitriy Rogatkin

, Polina Kulikova

and Dmitriy Kulikov

Moscow Regional Research & Clinical Institute "MONIKI" named after M.F. Vladimirskiy,

Shepkina str., Moscow, Russian Federation

National Research Nuclear University MEPhI, Kashirskoe highway, Moscow, Russian Federation

Key

words: Fluorescence, Non-invasive, Diagnostics, Inflammatory Process, In Vivo.

Abstract: Laser fluorescence spectroscopy (LFS) is widely used in medicine. Today, oncology and oncosurgery are

considered as the most promising fields of its application. It is known that cancerous tissues are able to

accumulate different porphyrins, both endogenous and exogenous, in enhanced amount due to increased

metabolism in cancerous cells. So, LFS can be used in vivo for detection of malignant tumours as well as for

real-time intraoperative imaging or diagnostics at a photodynamic therapy. One of the reason of the

enhanced accumulation of porphyrins in tissues is a chronic hypoxia. Therefore, it was hypothesized, that

LFS could also be used for diagnosis of local inflammation in tissues. Recently, some indirect data

confirming the hypothesis was obtained when observed inflammation due to invasion of external substances

into tissues. This study proves the hypothesis in a direct experiment with animals and laboratory tests.

Enhanced fluorescence intensity of the exogenous photosensitizer was found in inflamed tissues. The direct

association between intensity of the fluorescence, histological characteristics and blood test results was

shown. It was found that the registered fluorescence signal correlates with neutrophil counts in blood of

tested animals. It proves that LFS could be an effective tool for registration of local inflammation.

1 INTRODUCTION

Laser fluorescence spectroscopy (LFS) is currently

one of the promising methods for a non-invasive (in

vivo) characterisation of biological tissues and its

functional conditions (Johansson et al., 2008),

(Tuchin, 2002), (Mycek et al., 2003), (Rogatkin et

al., 2013). LFS is based on a registration of

fluorescence spectra and (or) fluorescence intensities

of endogenous or exogenous fluorophores on a

surface of the tested tissues. A great interest of

researchers to the method can be explained by its

advantages over other methods of assessment of soft

biological tissues. LFS differs from other methods

by noninvasive modality (minimally invasive, if

special preparations are used). It gives a possibility

of in vivo diagnosis in real time, and is safe for the

body (the method implies low-power laser light).

Among others areas, oncology and oncosurgery

are considered as the most promising fields of

application of noninvasive LFS in medicine of today

(Tuchin, 2002). It is known, that cancerous tissues

accumulate different porphyrins and its derivations,

both endogenous and exogenous, in enhanced

amount due to the increased metabolism in

malignant cells (Mycek et al., 2003). Therefore, LFS

can be used in vivo for a detection of malignant

tumors as well as for real-time intraoperative

imaging in oncosurgery or for a diagnostics at a

photodynamic therapy. A great number of medical

publications deal with the application of LFS for

early detection of malignancies in skin (Calin, et al

2013), oral mucosa (De Veld et al., 2005),

gastrointestinal tract (Duraipandian et al., 2012),

(Koizumi et al., 2013), and urogenital system (Stenzl

et al., 2011), (Karaoglu et al., 2014), as well as for

cancer diagnostics at a photodynamic therapy

(Andersson-Engels et al., 1995). In oncology LFS

can help surgeons to visually distinguish healthy

tissue from the cancerous one and to perform a

precise ablative process (Vahrmeijer et al., 2013).

Also, LFS can be used to identify sentinel lymphatic

nodes by providing their realtime intraoperative

imaging. Furthermore, it can be used to prevent

iatrogenic damage to vital structures, such as the

ureter or nerves (Handgraaf, 2014). There are many

Guseva, I., Rogatkin, D., Kulikova, P. and Kulikov, D.

In Vivo Experimental Detection of Inﬂammatory Process in Tissues by Fluorescence Spectroscopy.

DOI: 10.5220/0005659301390144

In Proceedings of the 9th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2016) - Volume 1: BIODEVICES, pages 139-144

ISBN: 978-989-758-170-0

139

publications on the use of this method for

intraoperative imaging in the breast surgery,

gynecology, neurology and other medical specialties

(Handgraaf, 2014), (Tummers, 2014), (Sugie, 2013).

Application of LFS seems also to be very promising

in the robotic assisted surgery (Hellan,2014). All

these application of LFS are based on the fact of the

abnormal fluorescence of both endogenous and

exogenous fluorophores in tumorous tissues.

Meanwhile, it was shown previously, that one of

the reason of the enhanced accumulation of

porphyrins in tissues is a chronic hypoxia (Rogatkin

et al., 2009). Therefore, it can be hypothesized, that

LFS could also be used for diagnosis of a local

inflammation in tissues. Currently, the search of

noninvasive methods of local inflammation

diagnosis is an extremely actual task, especially in

the regenerative medicine, when local regenerative

processes are under investigations and control. Also,

it is important in oncology. It is well-known that a

lot of pathological changes in tumorous tissues, in

particular in cancerous ones, are accompanied by a

number of local and systemic inflammatory

responses (Diakos, 2014). What if the enhanced

accumulation of porphyrins in a tumour is caused

not only by malignat processes, but also by

inflammatory ones, and it is not specific for

cancerous cells? Today, there is a few data only

devoted to the differentiation of the fluorescence

spectra for a cancer and inflammation. So, this issue

requires further attention (Lv, 2015), (Zhang, 2013),

because of potential errors can exist in LFS due to

this phenomenon.

Generally, in clinical practice of today, a

leukogram response test is widely used for the

inflammation diagnosis. Leukocytosis, neutrophilia,

left shift of the leukogram, etc. are the frequent

blood hallmarks of inflammations (Marshall, 2006).

However, the leukogram is nonspecific indicator of

local inflammations. It is not able to specify the

location of the inflammatory process. Biopsy and

subsequent histological examination can accurate

and reliable detect of any local inflammations, but it

is an invasive method. There are publications on the

use of thermography as a noninvasive quantitative

imaging method for assessing the local inflammation

(Christensen, 2014), (Arfaoui, 2012). However, the

increase in temperature is only one of signs of local

inflammation and, therefore, may be considered as

an auxiliary method. The rise in temperature is not

pathognomonic for local inflammation and may

indicate a normal physiological reactions (enhanced

functional activity of muscles, heating of skin, etc.).

Another modern method for local inflammation

diagnosis is a scintigraphy (Love, 2013). However,

this method involves an introduction into the

organism of radioactive isotopes, so it is associated

with dangerous ionizing radiation exposure. Thus,

the search for new, noninvasive or minimally

invasive, and real-time instrumental methods for

diagnosis of a local inflammation is an extremely

important task.

Recently, some indirect data confirming the

hypothesis of enhanced accumulation of exogenous

fluorophores of an aluminum phthalocyanine series

in inflammed tissues were published (Petritskaya et

al., 2014). Enhanced fluorescence was observed at

local inflammation due to invasion of external

substances into tissues. But in the reffered study the

histological or a blood test confirmation of the

inflammation process wasn’t been done. The aim of

our current study was to confirm possibilities of LFS

to detect in vivo a local inflammation in tissues in a

direct experiment with animals and laboratory tests.

2 MATERIALS AND METHODS

The study was performed in white laboratory mice

(N=12) and was conducted in accordance with all

ethical principles formulated in the Declaration of

Helsinki on the care and use of animals in research

and the Regulations of the European Science

Association (86/609/ЕС).

Local inflammation was provoked as follows.

All procedures were carried out under general

anesthesia (Zoletil + Xylazine). An incision was

made in the lateral part of the inguinal fold, then

skin was separated from the fascia by blunt

dissection, and the underlying muscle was clamped

at a distance of 7 mm from the incision by a

Mosquito clamp. The size of the resulting zone of

injury was 3х3х3 mm (Figure 1). Then, the

photosensitizer “Photosens” based on aluminum

phthalocyanine was injected intraperitoneally in the

dose of 2 mg/kg. It is known, that the fluorescent

signal of this photosensitizer in tissues can be

detected during 4-5 weeks, so it is possible to use the

aluminum phthalocyanine based photosensitizer in

prolonged experiments without any additional

injections. To prevent side effects, experimental

animals were not exposed to direct sun radiation

during the experiment, so any phototoxic effects did

not affect the laboratory mice.

BIODEVICES 2016 - 9th International Conference on Biomedical Electronics and Devices

140

Figure 1: Position of the inflamed region relative to the

incision.

To confirm the fact of inflammation, hematology

tests as well as a conventional histological analysis

of the affected area of the hind limb of mice were

done prior the provocation and on Days 3 and 10

after the provocation of the local inflammation. For

this purpose, two mice every time points (Days 0, 3,

10) were withdrawn from the experiment. Since our

interest was only to confirm the presence of local

inflammation without assessment of any specialties

of the inflammation, it was sufficient to carry out

both histological examination and a blood test only

in these three points of time (Days 0, 3, 10) as the

most expressive points of the evolution of such

inflammation. In hematology test results we firstly

took into account the percentage ratio of neutrophils

(neutrophil counts) as the most informative

parameter at a local inflammation in animals.

Figure 2: The diagnostic system “LAKK-M”.

In all experiments, fluorescence was recorded in

vivo with the use of laser diagnostic system LAKK-

M in the “Fluorescence” operation regime. The

system is equipped with fiber optical probe (Figure

2). Excitation of tissue fluorescence was made in the

continuous wave (CW) mode at the wavelength 635

nm (a semi-conductor laser). Power of the laser

radiation on a distal end of the optical fiber probe

(on a surface of tissues) was around 5 mW.

Fluorescence intensity was measured at 670 nm – in

a maximum of the fluorescent spectrum of the used

photosensitizer “Photosens”. Subsequently, the

intensity at this wavelength will be called

“fluorescence intensity”.

Measurements of the fluorescence intensity in

animals were carry out before the provocation of the

local inflammation (before the injection of the

photosensitizer and on Days 2, 3, 6, 8, 10 and 16

after the provocation and the injection.

Examples of fluorescence spectra from inflamed

tissues and from a contralateral region are shown in

Figure 3.

Figure 3: Examples of fluorescence spectra from the

contralateral region and from inflamed tissues (3 days

after the injection of the photosensitizer).

To study the dynamics of photosensitizer

accumulation in inflamed tissues, the peak value of

the measured fluorescence intensities was tracked in

time. Also, to clarify the influence of the initial laser

radiation power as well as of local optical properties

of tissue on the registered intensities, we compared

the dynamics of the measured fluorescence

intensities and the dynamics of the coefficient of

fluorescence contrast K

(Rogatkin, et al 2013),

which calculated using the intensities as follows:





1





∙







∙



where K

is the coefficient of fluorescence contrast

(0<K

<2); I

is the maximum of the fluorescence

intensity; I

is the measured intensity of the

backscattered radiation at the excitation wavelength;

β is an instrumental reducing coefficient (β≈1000 to

diminish I

to a level which is comparable with the

level of I

). In this coefficient I

is normalized both

In Vivo Experimental Detection of Inﬂammatory Process in Tissues by Fluorescence Spectroscopy

141

by local optical properties of tissues and by a power

of excitation radiation, so it is less sensitive to theirs

changes.

To compare the fluorescence intensity from

inflammation and contralateral areas, an index of

inflammation intensity μ(λ

) was calculated as

follows:



























where I

is the fluorescence intensity from the

inflamed area, I

is the fluorescence intensity from

the contralateral region, and λ

is the fluorescence

wavelength (in our case λ

= 690 nm).

3 RESULTS AND DISCUSSION

All experimental results showed an enhanced

intensity of the fluorescence signal in the injured

tissues, compared to that in the contralateral region.

Figure 4 presents the averaged data for the group of

mice.

Figure 4: Dynamics of fluorescence intensities.

Earlier it was shown (Rogatkin et al., 1998), that

uncertainty of results of such measurements in the

laser fluorescence diagnostics amounts to 40% of the

measured value. Therefore, the experimentally

observed differences between signals from the intact

area and the inflamed tissues are significant. This

suggests that inflammatory processes can be in vivo

detected by LFS.

The dynamics of K

is shown in Figure 5. It is

easy to see that there are no fundamental differences

in the behavior of the curves in Figures 4 and 5. It

confirms, that the influence of the laser power

fluctuation or an influence of local optical properties

of tissues on registered intensities is small enough at

so high fluorescence of the photosensitizer used.

Figure 5: Dynamics of the coefficient of fluorescence

contrast.

Dynamics of the index of inflammation intensity

μ(λ

) versus days is shown in Figure 6. For clarity,

the time points of hematology and histology tests are

denoted by crosses.

Figure 6: Dynamics of the inflammation intensity index

μ(λ

All data of laboratory blood tests showed an

increase in the neutrophil counts (Figure 7) and a

relative increase in the band neutrophil counts

(Figure 8), i.e. a shift of the leukogram to the left.

These parameters indicate exactly the occurrence of

inflammatory process in organism.

Histological examination performed at Day 0

before the provocation of inflammation was normal.

Histological examination performed at Day 3 after

the provocation revealed an acute inflammatory

response in the injured muscle tissue, dermis and

subcutaneous adipose tissue, namely: there were

edema, leukocyte inflammatory infiltration of

tissues, and necrotic foci in muscle. Reduction of

inflammatory activity and some regenerative muscle

tissue changes were observed at Day 10. But the

inflammatory infiltrate, indicating a continuing

inflammatory process, remained in the dermis and

subcutaneous adipose tissue.

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Figure 7: Changes of blood neutrophil counts in laboratory

mice.

Figure 8: Changes of blood band neutrophil counts in

laboratory mice.

Based on these results (Figures 7 and 8), it can

be assumed that the inflammation intensity index

μ(λ

) in inflammation regions correlates well with

blood neutrophil counts. The increase of the index

μ(λ

) on Day 3 corresponds to an acute local

inflammatory response, which was evident in both

hematology tests and at histological assessment. On

Day 10, the µ(λ

) remained high, correlating with

histologically confirmed persistence of the

inflammation in subcutaneous adipose tissue and

dermis, and with the increase in the blood neutrophil

counts.

4 CONCLUSIONS

The aim of the current study was to confirm

possibilities of LFS to detect in vivo a local

inflammation in tissues. In a direct experiment with

animals and confirmational laboratory tests it was

shown and proved that non-malignant inflamed

tissues can have an enhanced accumualtion of

exogenious photosensitizer and, therefore, can have

an enhanced superficial fluorescence of it like

several cancerous tissues have. Furthermore, it was

shown that the intensity of the fluorescent signal in

the inflamed tissues correlated with blood neutrophil

counts and was associated with changes in histology.

It is of special note, that this result is very

important both for an experimental research and for

a practical medicine. Many processes in a human

body are associated with development of local

inflammations. First of all, these are different

processes related to mechanical and thermal tissue

damage, to introduction of foreign agents into a

tissue, at a transplantation, for example. So, a

diagnosis of a local inflammation by LFS technique

in vivo can be used as a navigation method in

surgery and, also, as an intraoperative assessment of

tissue conditions at a regenerative surgery. These

data is important for the fluorescence diagnostics in

oncology, as well. It was shown that the exogenous

photosensitizer can be accumulated not only in

malignant cells, but also in the area of inflammation.

It makes us reassess the applicability limits of LFS

at a photodynamic therapy.

Radiation therapy is an effective and accepted

treatment modality in oncology. Various radiation-

induced reactions, including inflammation, could be

its side effects, which is the reasons why radiation

doses are often fractionated. However, fractionation

procedures are poorly individualized, and standard

radiation regimens are used due to a lack of

affordable instrumental method for assessment of

individual local inflammations. The proposed

technique could become such a method. So, in a

view of the foregoing, it can be assumed that this

method of diagnostics of a local inflammation may

have a broad clinical application.

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