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REVERSIBILITY OF MORPHOLOGICAL CHANGES IN THE MESENTERIC LYMPH
NODES AFTER PERORAL ADMINISTRATION OF GOLD
NANOPARTICLES
Olga V. Zlobina, Saratov State Medical University , RussiaSvetlana S. Pakhomy, Saratov State Medical University , RussiaAlla B. Bucharskaya, Saratov State Medical University , Russia
Irina O. Bugaeva, Saratov State Medical University , RussiaGalina N. Maslyakova, Saratov State Medical University , Russia
Nikolai G. Khlebtsov, IBPPM RAS, RussiaBoris N. Khlebtsov, IBPPM RAS, Russia
Vladimir A. Bogatyrev, IBPPM RAS, Russia
To study the severity and reversibility of structural and functional changes in the mesenteric lymph nodes of rats with regard to the size and duration of oral administration of gold nanoparticles
The purpose of the study
Reactive center of cortical substance of lymphoid follicle (x1000)
1. To study the effect of the size of orally administered gold nanoparticles on the cellular composition of the mesenteric lymph nodes
2. To analyze the recovery rate of the initial cellular composition of mesenteric lymph nodes after cessation of administration of gold particles of different sizes
3. Determine the recovery time the initial cellular composition of mesenteric lymph nodes after oral administration of gold nanoparticles
Objectives
Materials and methods48 mature outbred male rats weighing 180-220 g (12 animals per group)
Gold nanoparticles size of 1-3, 15, 50 nm (IBPPM RAS, Saratov)
3 experimental groups (oral administration of gold nanoparticle with size of 1-3, 15, 50 nm for 8 days )
Control group (oral administration 1 ml of saline for 8 days )
The lymph node biopsies were taken for histological examination a day after cessation of
oral administration (6 animals per group)
The lymph node biopsies were taken for histological examination 14 days after cessation of oral
administration (6 animals per group)
1. Hematoxylin-eosin (for counting cell components).
2. Toluidine blue at pH Vinogradov - 4.8(to identify metachromatic mast cells).
3. Methyl green pyronine by Brachet (for the detection of DNA and RNA).
4. Smears, painting Romanovsky-Giemsa.
Methods of study of histological preparationsmesenteric lymph nodes
1. Computer system to control the size of the micro-and nanometer-scale structures Image Analyzer.
2. The standard method of counting the cell elements at a magnification of 200, 400, 900, 1350 using specialized morphometric grid (Avtandilov GG, 1972, 1992).
3. Morphometric image analysis program - ImageJ.
Methods of study of histological preparationsmesenteric lymph nodes
1. The reactions of the cellular elements of the mesenteric lymph
nodes a day after introduction of nanoparticles were size
dependent.
2. The statistically significant dynamics was not observed in the
group treated with gold nanoparticles of 1-3 nm.
3. The introduction of nanoparticles of 15 nm and 50 nm led to
significant changes in the content of the cellular elements of the
lymph nodes and was more reflected in the paracortical zone than
in areas of lymphoid follicles and brain strands.
Results: a day after administration
1. The presence of nanoparticles in animal organism intensified the process
of blast transformation of lymphocytes
2. The control group - immunoblasts were absent in paracortical area of
lymph nodes; their number increased to at administration of 15 nm and 50
nm nanoparticles (to 2,6 ± 0,4 and 2,9 ± 0,1 respectively) .
3. Mast cells present only in the area of brain strands in control and
experimental groups after introduction of nanoparticles, whose number
increases in direct proportion to the size of the nanoparticles.
4. In all the studied areas of lymph nodes the increased frequency of mitotic
figures was noted after nanoparticle administration.
Results: a day after administration
Changes in the number of small lymphocytes after 8 dayintroduction of nanoparticles of different sizes
Control
Zone of lymph.follicles
Paracortical zone
Zone of brain cords
1-3 nm 15 nm 50 nm
The changing of immunoblasts number in zone of lymphoid follicles after oral administration of nanoparticles
50 nm
15 nm
1-3 nm
control
The data indicate the marked immune modulating effect of gold
nanoparticles in animals, depending on the nanoparticle size and
organ specificity. A pilot study revealed a certain temporal dynamics of
cellular responses.
Results: a day after administration
Lymph node after nanoparticle administration (a) 1-3 nm; (b) 15 nm (х400).
a b
1. The cellular composition of the lymph nodes after the introduction of
gold nanoparticle size of 1-3 nm for 8 days corresponded the control
values
2. The number of lymphocytes and immunoblasts decreased less
significant and was not reaching the control values after cessation of
15 nm nanoparticles administration. It may be due to incomplete
restoration of the original state of the immune system 14 days after
cessation of nanoparticle administration
Results - the reversibility of the morphological changes
Regression analysis of the recovery rate of the cellular composition of lymph nodes after cessation of 15 nm nanoparticle administration
Objects of study
(number on 6400
mkm2)
Zones of
lymph
nodes
Regression analysis results
Coefficients
of linear regression
The projected terms of
achievement of the
control values
Large lymphocytes
Lymph.foll.
Paracortical
Brain cords
8,5
0,05
0,14
18
20
16
Immunoblasts
Lymph.foll.
Paracortical
Brain cords
0,04
0,08
0,12
20
16
18
1. Duration of initial recovery of cellular composition of the lymph
nodes does not exceed 21 days, in connection with which it can be
concluded that 15 nm nanoparticles have not immunotoxicity
actions at 8 days oral administration
2. 14 days after cessation of administration of 50 nm nanoparticles
it may be noted that cellular composition did not corresponds to
the control values. By regression analysis it was found that 21-st
day is predicted to achieve the control values of cellular
composition.
Regression analysis of the recovery rate of the cellular composition of lymph nodes after cessation of 15 nm nanoparticle administration
Conclusions
1. Oral administration of gold nanoparticles leads to changes in the cellular composition of the mesenteric lymph nodes, the severity and reversibility of the reaction is determined by the size of nanoparticles
2. Administration of gold nanoparticles with size 15 nm and 50 nm leads to a change in the cellular composition of nodes on the 8th day. The number of medium and large lymphocytes are almost doubles in brain cords, the content of plasma cells is increasing, degranulation signs of mast cells are appearing in subcapsular zone
3. 14 days after cessation of 1-3 nm gold nanoparticle administration the number of small and medium lymphocytes, plasma cells, mast cells, and mitotic figures corresponds to the initial values. According to a regression analysis the number of immunoblasts and large lymphocytes corresponds to the control values after 16 days, indicating the absence of immunotoxicity.