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.