MORPHOLOGICAL CHANGES IN ORGANS OF IMMUNOGENESIS AND INDICATORS OF

PERIPHERAL BLOOD UNDER THE INFLUENCE OF GOLD NANOPARTICLES IN THE EXPERIMENTAlla B. Bucharskaya, Saratov State Medical University , RussiaSvetlana S. Pakhomy, Saratov State Medical University, Russia

Olga V. Zlobina, Saratov State Medical University, RussiaGalina N. Maslyakova, Saratov State Medical University, Russia

Olga V. Matveeva, Saratov State Medical University, RussiaIrina O. Bugaeva, Saratov State Medical University, Russia

Nikita A. Navolokin, Saratov State Medical University, RussiaBoris N. Khlebtsov, Institute of Biochemistry and Physiology of Plants

and Microorganisms RAS, RussiaVladimir A. Bogatyrev, Institute of Biochemistry and Physiology of

Plants and Microorganisms RAS, RussiaNikolai G. Khlebtsov, Institute of Biochemistry and Physiology of Plants and Microorganisms RAS, Saratov State University, Russia

Valery V. Tuchin, Saratov State University, Russia

The aim of the work was to study in vivo the effects of gold nanoparticles (GN) of different sizes on the morphological structure of immunogenesis organs and the peripheral blood indicators in laboratory animals

PURPOSE OF THE RESEARCH

Colors of various sized monodispersed gold nanoparticleshttp://www.mi2g.com/cgi/mi2g/frameset.php?pageid=http%3A//www.mi2g.com/cgi/mi2g/press/110210.php

2 nm GN, peroral administration during

15 days (n=6)

15 nm GN, peroral administration during

15 days(n=6)

50 nm GN, peroral administration during

15 days (n=6)

Сontrol group, peroral administration of 0,9% saline solution during 15 days (n=6)

Design of experiment

The experiments were conducted accordingly “International guiding principles for biomedical researches involving animals” (CIOMS, ICLAS, 2012).

1. Morphological method: Hematoxylin and eosin staining

2. Hematological test 0f peripherical blood

3. Morphological method of bone marrow examination: May-

Grunwald staining

4. Morphometric analysis of the histological samples was

conducted using the digital image analysis system Mikrovizor

medical μVizo-103 (LOMO, Russia).

Materials and methods

Results

At the analysis of peripheral blood indicators of laboratory animals the decrease of leukocytes number was found. After administration of 15 nm gold nanoparticles the significant reduction of leukocytes and lymphocytes number were noted, the reducing of number of all types of leukocytes were detected in the group with administration of 50 nm gold nanoparticles.

ResultsThe bone marrow examination after administration of nanoparticles the signs of irritation of leukocyte germ hematopoiesis were noted in all groups – the increasing of number of myelocytes, metamyelocytes and stab leukocytes. The most pronounced changes were observed in the groups with 15 nm and 50 nm nanoparticles administration, the decrease of leuco-erythroblastic ratio was noted, probably due to the decrease of segmented neutrophils.

Fig.1 Bone marrow. May-Grunwald staining

The indicators of bone marrow

Control group

Experimental groups with GN administration

1-3 nm 15 nm 50 nm

Myeloblasts 1,0±0,67 1.33±0.67 0.75±0.47 1.66±0.57Neutrophil myelocytes 1.16±0.28 6±2.1 6.75±1.5* 7±1.73*

Neutrophil metamyelocytes

3±0.67* 13.3±2.88* 10.75±2.36* 11.3±3.3*

Neutrophil stab leycocytes

7.5±1.8 17±1* 17.5±1.73* 13±1.73*

Neutrophil segments 32.8±8.31 22±6.08 23.25±3.86 23±2.645

Eosinophils 1.66±0.67 1.66±0.57 1.25±0.957 0.66±0.57

Basophils 0,25±0,25 0,75±0,32 0,33±0,17 0,25±0,25

Monocytes 0.66±0 2±1.73 1.75±1.258 1.33±1.15

Lymphocytes 16±0 14.33±3.05 16.5±4.04 14.3±3.21erythroblasts 0 0 0.5±1 0.33±0.57

Basophilic normocytes 1±0 2±0 1.75±1.25 2.66±0.57

Polychromatic normocytes

12 17.66±4.16 13.5±6.4 20±4.35

Oxyphilic normocytes 3±0 2.66±1.15 3.25±0.5 4.66±1.52Leukocytic-

erythroblastic ratio2.6±0 1.7±0.73 2.05±0.37* 1.19±0.32*

Results

After administration of gold nanoparticles the morphokinetics of cellular elements of mesenteric lymph nodes had size-dependent character and was characterized by lymphocytopoiesis activation. The most striking changes occurred after administration of 15 and 50 nm gold nanoparticles compared with the control group.

The number of test objects (on 6400 square)

Zones of lymphatic

nodes

Control group

Experimental groups with GN administration

1-3 nm 15 nm 50 nm

Small

lymphocytes

Lymph follicules

64.2±2.1 62.3±1.6 71.2±1.2* 83.3±1.7*

Paracortical 60.1±1.2 61.2±1.2 66.2±1.1* 72.4±1.1*

Brain cords 20.2±1.3 18.3±1.0 12.6±0.1 20.8±0.6

Middle

lymphocytes

Lymph follicules

18.2±0.4 16.6±1.2 30.1±2.1* 35.3±1.0*

Paracortical 12.1±1.1 14.3±0.8 19.1±0.8 26.6±0.8

Brain cords 10.2±1.7 10.4±1.0 12.7±1.1 22.3±1.2

Large lymphocytes

Lymph follicules

0 0.5±0.01 2.2±0.7 9.61±1.2

Paracortical 0.75±0.1 0.67±0.1 1.2±0.3 1.2±0.23Brain cords 10.2±1.7 0.3±0.1 0.5±0.1 3.6±1.2

Immunoblasts

Lymph follicules

2.5±0.3 2.9±0.1 4.8±0.3* 10.4±0.3*

Paracortical 0 0 0.7±0.1* 2.9±0.1*Brain cords 2.3±0.3 2.0±0.3 3.5±0.4 5.2±1.0

Plasmocytes

Lymph follicules

0 0.1±0.02 0 0.35±0.5

Paracortical 0.71±0.1 0 1.2±0.08 1.75±0.4Brain cords 15.3±0.6 17.9±1.2 26.4±3.4* 38.4±3.4*

Mastocytes

Lymph follicules

0 0 0.6±0.4 0.8±0.7

Paracortical 0 0 0.3±0.01 2.0±0.6Brain cords 0.3±0.01 0.1±0.07 1.4±0.03 3.4±0.5

Mitotic figures

Lymph follicules

0.2±0.01 0.7±0.1 4.0±0.3* 4.6±0.07*

Paracortical 0.8±0.16 0.9±0.2 1.2±0.06 2.1±0.16Brain cords 0.7±0.1 0.9±0.16 3.5±0.1 4.2±0.2

Conglomerates of 50-nm GNPs in the cytoplasm of macrophages. G.-E. x1500

Results

After GN administration the morphological changes in spleen were presented in the form of circulatory disorders of varying severity and activation of proliferative processes.Normal blood supply of the spleen was observed only in 25% animals after 15 nm GN administration, in other cases the lack of blood supply was noted. The lymphoid follicles contain bright germinative centers represented by large blast cells, which indicates activation of proliferative processes.

Spleen. Increasing of lymphoid follicles at 16-day administration of 50 nm GN . Hematoxilin-eosin.

Conclusions

At study of peripheral blood indicators the size-dependent decrease in the number of neutrophils and lymphocytes was noted, especially pronounced in rats after administration of 50 nm GN. At bone marrow examination the stimulation of lymphocytic and myelocytic germs of hematopoiesis was recorded after GN administration. The signs of strengthening of cell maturation processes was noted in mesenteric lymph nodes, which was reflected in increased amounts of immunoblast and large lymphocytes. The quantitative changes of cellular components in immunogenesis organs indicate the activation of differentiation and proliferation processes. We suggest the presence of immunomodulating effect of 15 nm and 50 GN on on lymphocyte subpopulations in spleen and lymph nodes.