Study of early stages of lymphedemausing multiphoton and THz microscopy

Yury V. Kistenev, 1,2 Alexey V. Borisov, 1,2 Ekaterina A. Sandykova,1,2 Darya K. Tuchina,1,3 Polina A.Timoshina,1,3 Anastasya I.Knyazkova1

1Tomsk State University, Russian Federation

2Siberian State Medical University, Russian Federation

3Saratov State University, Russian Federation

e-mail: katrin_nemchenko@mail.ru

Introduction

• Lymphedema is a chronic progressive disease of the lymphatic system caused by abnormal accumulation of

tissue fluid with a high protein content. Early diagnosis of this disease helps to choose the right treatment

and prevent its further development. The existing methods of lymphedema diagnosing at early stages are

not strict and consistent. The invention of multiphoton and THz microscopy opens up new possibilities for

lymphedema tissue analysis in vivo.

• Multiphoton laser microscopy (multiphoton laser tomography, MPT) provides instantaneous imaging of

living skin at the cellular and subcellular levels. MPT uses second harmonic generation (SHG) from matrix

tissue components, such as collagen, providing functional and structural label-free tissue imaging.

Description of used experimental biology method

In this study we developed a rat model of lymphedema.

Based on the experience of previous experiments [1], a

model of lymphedema on the rat’s tail was

implemented. The main lymphatic manifolds were

injected with a solution of methylene blue

subcutaneously in the middle of the tail. After staining

the lymphatic vessels, we recede 5 mm from the base of

the tail and perform excision of a strip of skin 2 mm

wide along the entire perimeter of the tail. Further,

under optical magnification (x3.5), we find the main

colored lymphatic collectors. We coagulate them and

cross them, thus breaking the outflow of lymph from the

deep lymphatic system of the rat's tail.

1. Real-Time In Vivo Imaging Collagen in Lymphedematous Skin Using

Multiphoton Microscopy / X. Wu, Sh. Zhuo, J. Chen, N. Liu // Pub. Med.

- 2011. – Vol. 33, Issue 6. – P. 463–467.

Cross-section of the rat tail. The main lymphatic collectorsrun along the sides of the tail, along the large veins, courseof which is shown on the figure.

Source: http://rat911.ru/krysy/dlya-chego-krysam-dlinnyj-xvost/.

Description of used experimental biology method

A, Microlymphatic network consists of hexagonal initiallymphatics and two deep collecting lymphatic vessels.Fluorescent tracer is injected with constant pressure in theinterstitium of the distal end of the mouse tail. B, In thephysiological situation, the tracer is transported through theinitial and collecting lymphatics. The latter have a muscular walland intraluminal valves. C, A constricted state of the collectinglymphatics under eNOS inhibition increases resistance anddecreases fluid velocity in the lymphatic network. D, Proximalligation of the collecting lymphatics leaves the initial lymphaticsas the only route for fluid flow. Loss of control of lymph fluidtransport and decrease in total resistance, to which lymphvessel diameter is inversely proportional, leads to increasedfluid velocity and injection flow rate.

Source: Endothelial Nitric Oxide Synthase Regulates Microlymphatic Flow via Collecting Lymphatics / J. Hagendoorn, T.P. Padera, S. Kashiwagi et al.// Pub. Med. - 2004; 95(2) – P. 204-209.

Schematic representation of the effects of eNOS blockade and ligation on the mouse tail lymphatic network.

Description of used experimental biology method

A rat with tail after surgery.

The keeping, nutrition, care about animals and their

removal from the experiment was carried out in

accordance with the requirements of GOST R ISO

10993-2-2009 and "Euroguide on the

accommodation and care of animals used for

experimental and other scientific purposes".

The equipment Qualitative comparisons of healthy tissue and tissue with lymphedema(on the 7th and 14th days after surgery) were carried out.Measurements have been done using the MPTflex microscope byJenlab and THz time-domain spectrometer T-Spec by EXPLA.

Time-domain THz spectrometer T-SPEC

MPTflex microscope

Technical data:

- Tuning range 0.2-3.5THz

- Dynamic range > 60 dB

Technical data:

laser pulse width: 200 fs

repetition frequency: 80 MHz

in situ laser power: 2-50 mW

wavelength range. 710-920 nm

Multiphoton laser microscopy images The examples of MPT images of subsurface skin layer of patients with lymphedema in comparison with healthy volunteers are presented in the Fig. 1. The study involved the group of patients with II stage extremities lymphedema at the age of 23 to 68 years (n=8) and the group of healthy volunteers at the age of 19 to 65 (n=8). The papillary layer of the skin with a penetration depth of about 100 μm was examined. Evidently, that a healthy participant’s collagen morphological structure has a fine mesh with a well-regulated distribution and a well-defined orientation (Fig. 1a), while as lymphedema patient’s tissue collagen fibers are disorganized (Fig. 1b).

Fig. 1 MPT images: a - collagen of healthy

volunteer; b – collagen of patient with II

stage of lymphedema.

These results are consistent with studies that have been carried out by other authors, using the animal model (Fig. 2).

Multiphoton laser microscopy images

Fig. 2 a – SHG image of collagen in normal rat tail skin;

collagen is arranged in an organized manner and largely

oriented in the same direction. b - the corresponding histological

image of normal rat tail skin. c – SHG image of collagen in

lymphedematous rat tail skin on the 7-th day after surgery;

collagen appears less compacted and disorganized. d – the

corresponding histological image of lymphedematous rat tail

skin on the 7-th day after surgery. Reprinted under permission

of the "John Wiley and Sons" (License No. 4230091173063,

15.11.2017)

Source: Real-Time In Vivo Imaging Collagen in Lymphedematous Skin Using Multiphoton

Microscopy / X. Wu, Sh. Zhuo, J. Chen, N. Liu // Pub. Med. - 2011. – Vol. 33, Issue 6. – P. 463–

467.

THz absorption spectraTHz absorption spectra of the lymphedema tissue (Fig.3b) show the development of lymphedema with greataccuracy at a given frequency. This is also confirmed by the PCA method. The absorption spectra of the healthytissue (Fig. 3a) are shown for comparison.

Fig. 3. THz wide-ranging absorption spectra: a) of the healthy tissue, b) of the lymphedema tissue.

Data Principal Component Analysis

• The PCA applications for 2D THz image analysis was done on animalmodel (rats) lymphedema tissue. Using the optimization algorithm wecarried out the classification of THz spectra of the most informativeareas obtained in-vivo from the lymphedema affected leg tissue(result of surgery) and obtained from healthy leg tissue.

• The principal components are built using the THz spectra in the 0.8-1.0 THz spectral range. Note that the separation of the groups usingTHz imaging became possible after three weeks from thelymphedema surgery initiation.

Data Principal Component Analysis

The results show good enough separation of lymphedema tissues and healthy tissues in thespace of the principal components at the end of experiments (see Fig. 4).

Fig. 4. Lymphedema tissues and healthy tissues THz spectra spatial distribution in the principal component space a)

second week after surgery, b) fourth week after surgery.

Conclusion

• The small animal model of lyphedema was created

• During experiments, the change of collagen in the skin of tail and legsof the rat with lymphedema obtained as a result of surgery wasobserved in vivo.

• Characteristics of THz and MPM image patterns of the lymphedematissue were identified.

The work was carried out under partial financial support of the Russian Foundation for Basic Research (Project No. 18-42-703012).