recently , a large amount of chemicals are produced , which come into close contact with the human body .
agrochemicals are an certain part of modern agrotechnical procedures despite the fact that a great number of them are on the list of risk substances .
many agro - chemicals have toxic and genotoxic effects and , subsequently , after cumulative somatic mutations , also carcinogenic effects .
uncontrolled and thoughtless use of various pesticides resulted in health problems involving nervous , endocrine , reproductive and immune systems of animals and humans .
although some pesticides have been restricted or banned because they pose risks of cancer , birth defects , or neurological damage , little attention has so far been given to what may be their greatest health risk : impairment of the immune system .
hundreds of experimental studies on human cell cultures and animal models provide strong evidence that many pesticides are immunotoxic and may affect immune status and function .
cholinesterase inhibitors are a group of chemical substances precluding hydrolysis of acetylcholine and thus causing accumulation of acetylcholine in reactive sites of live organisms .
inhibition of brain acetylcholinesterase has been the major therapeutic target for alzheimer disease and other patients with dementia .
bendiocarbamate ( 2,3-isopropyledene - dioxyphenyl methylcarbamate ) is a pesticide asting upon invertebrates by irreversibly blocking the activity of the enzyme cholinesterase , which is critical in allowing muscle relaxation by removing the neuromuscular mediator acetylcholine .
it has relatively low toxicity in mammals and vertebrates in general , since it does accumulate in their tissues and the cholinesterase activity returns to norm within 24 h after acute exposure .
the clinical picture of bendiocarbamate intoxication includes diarrhoea , vomiting , dehydration , loss of weight , alopecia , hypersalivation and muscle tremor .
many studies proved that bendiocarbamate increases incidence of lymphoreticular tumours , such as lymphosarcomas , reticulosarcomas , lymphoid and myeloid leukaemia .
presently , the acute oral toxic dose for adult experimental animals is as follows : rat 34156 mg / kg body weight ( bw ) , rabbit 3540 mg / kg bw and guinea pig 35 mg / kg bb .
it is a place where the t cells precursors proliferate and mature , these are responsible for the cell - mediated immunity .
the main role of thymus - derived lymphocytes is the protection against the tumourous cells , intracellular parasites and viruses ; through cytokine secretion they activate other cells of the immune system . during the prenatal and postnatal period , patological involution of
it may be associated with environmental toxicants , trauma , severe infection , chemotherapy , smoking , hormonal therapy or malnutrition .
animal cholinesterases are widespread enzymes present in cholinergic and non - cholinergic tissues as well as in their plasma and other body fluids .
they are divided into two classes according to differing in their substrate specificity , behavior in excess substrate and susceptibility to inhibitors : acetylcholinesterase or true cholinesterase ( ache ) and butyrylcholinesterase ( bche ) .
the aim of this study was to compare the effects of bendiocarba - mate on histochemical localization of acetlycholine- and butyrylcholine - positive nerves in rabbit 's thymuses .
the study was carried out on 50 european rabbits ( oryctolagus cuniculus ) , hybrid hyla , of both sexes .
the rabbits were 54 days - old with mean bw of 1250 g. the experimental animals were kept under standard conditions in animal quarters at 1518c and natural light regimen .
bayer , leverkusen , germany ) in the form of capsules per os at a dose of 5 mg / kg bw daily for 13 days . since the animals showed a strong response to the daily administered dose ( diarrhoea , dehydration and alopecia in some animals ) , after 13 days the exposure was decreased by administering the same dose in 48 h intervals . during the experiment ,
control animals were fed standard granulated mixed feed intended for rabbits . on days 3 , 10 , 20 , 30 , 60 and 90 of the experiment
, groups of experimental animals consisting of 6 rabbits were euthanized by ether together with control group ( 8 rabbits ) of animals .
the experiment was conducted in compliance with the rules set by the ethical commission of university of veterinary medicine in kosice , slovakia and conditions for experiments on animals .
samples taken from the thymus were processed for demonstration of ache - positive and buche - positive nerve fibers . during two hours
sections were prepared on freezing microtome thick 20 m and incubated in the incubation solution during the period of 24 h at 37c .
the incubation medium ( according to method of karnovsky and roots and of el badawi and schenk ) contained acetylthiocholine iodide needed for the visualization of specific ache , tetraisopropylpyrophosphoramide ( iso - ompa ) for inhibition of non - specific ache was used . in this method acetylcholinesterase present in the cholinergic nerves
releases thiocholine from acetylthiocholine which reduces potassium ferricyanide to potassium ferrocyanide , capable of producing insoluble copper ferrocyanide with copper ions ( hatchett 's brown ) .
the method for visualization of butyrylcholinesterase is the same , but the incubation medium contained butyriltiocholine iodide as an inhibitor of specific ache instead of acetylthiocholine iodide .
the exact composition of incubation solution can be found in table 1 , the final ph of solution was 5.6 to 6.0 .
elaborated samples were assembled on slides and were examined under a light microscope jenalunar 2 ( zeiss , jena , germany ) .
we assessed visually the density of the nerve fibers in thymuses of both experimental and control rats .
the histological preparations were evaluated qualitatively under an optical microscope ( olympus provis ax ) .
we compare the histochemical localization of acetlycholine- and butyrylcholine - positive nerves in rabbit 's thymuses after administration of bendiocarbamate . 

table 1the eexact composition of incubation solution for the visualization of specific ache , according to el badawi and schenk . for visualization of buche
the medium contains butyriltiocholine iodide as an inhibitor of specific ache instead of acetylthiocholine iodide.acetylthiocholine iodide12.5 mgdistilled water2.0 mll0.82% sodium acetate15.8 mll0.6% acetic acid0.5 mll2.94% sodium citrate1.2 mll0.75% copper sulphate2.5 mll0.137% tetraisopropylpyrophosphoramide0.5 mll0.175% potassium ferrocyanide2.5 mll 

rabbit thymus of control animals contained ache- and buche - positive nerve fibers in two forms : i ) nerve fibers joining to vessels .
they formed networks around branches of vessels running in septa and , as a common bundle , headed to organ parenchyma .
the part of the cortex housing numerous lymphocytes was very poor in nerve fibers , while abundant nerve clusters could be observed at the level of cortico - medullary junction , i.e. the part which was a primary starting point of cells and early precursors ; ii ) nerve fibers independent on vessels .
they were most abundant at the cortico - medullary junction and a little less frequent in the medulla .
microscopic findings of buche - positive nerve fibers show the same density and the topography by the experimental and the control animals . on the other hand , ache - positive nerve fibers in experimental animals after administration of bendiocarbamate
microscopical findings related to ache - positive innervations of the thymus of rabbits which were administered bendiocarbamate , the inhibitor of ache , were rather indistinctive ( figures 1 and 2 ) .
one could not identify nerve structures with certainty and ache - positivity was exhibited only by non - neural cellular elements in all experimental materials examined ( figure 3 ) . in the control animals
the ache - positive nerve fibers were observed in the organ capsule in cortex layer originating from the interlobular septum .
fine nerve fibers terminated in the cortex and frequently came to close contact with lymphocytes while in the deep cortex layer and medulla they were located close to epithelial cells . 

figure 1third day of the experiment ; nerve fibers are not visualized after administration of bendiocarbamate . a , artery
third day of the experiment ; nerve fibers are not visualized after administration of bendiocarbamate .
thymus of an experimental rabbit with very poorly visible ache - positive fibers in perivascular topography .
thymus of an experimental rabbit with very poorly visible ache - positive fibers in perivascular topography .
abundant reaction product in the non - cellular part of thymus of experimental animal lacking ache - positive nerve fibers .
abundant reaction product in the non - cellular part of thymus of experimental animal lacking ache - positive nerve fibers .
the distributions of buche - positive nerve fibers after visualization were the same in experimental and control animals .
nerve fibers entered the thymus as a common bundle with arteries , as typical periarterial plexuses ( figure 4 ) . in the organ they were running in interlobular septa as a common bundle of branching off fibers .
the highest density of thicker periarterial nerve plexuses with abundance of reaction product was observed at the cortico - medullary junction ( figure 5 ) , while the finer fibers were less abundant in this area .
a characteristic feature in this topography was the presence of non - neural cellular elements with high content of colored reaction product .
the outer medullary layer contained numerous thicker and thinner nerve fibers . in the cortex layer we observed predomination of fine nerve profiles without evident direct connection to vessels . in the deep cortex topography
we were able to observe thicker nerve fibers , particularly close to the cortico - medullary border .
fine buche nerve fibers were observed also in the thymic capsule . based on our findings
we can state that buche - positive innervations of thymus in experimental and control animals show no differences , it follows bendiocarbamate in dose of 5 mg / kg bw daily not inhibit butyrylcholinesterase . 

abundant buche - positive nerve fibres ( nf ) in perivascular topography and solitary nerve fibres originating from them . the distribution and the density of nerve fibers
abundant buche - positive nerve fibres ( nf ) in perivascular topography and solitary nerve fibres originating from them .
fine buche - positive nerve fibers ( nf ) at the cortico - medullary junction .
fine buche - positive nerve fibers ( nf ) at the cortico - medullary junction .
rabbit thymus of control animals contained ache- and buche - positive nerve fibers in two forms : i ) nerve fibers joining to vessels .
they formed networks around branches of vessels running in septa and , as a common bundle , headed to organ parenchyma .
the part of the cortex housing numerous lymphocytes was very poor in nerve fibers , while abundant nerve clusters could be observed at the level of cortico - medullary junction , i.e. the part which was a primary starting point of cells and early precursors ; ii ) nerve fibers independent on vessels .
they were most abundant at the cortico - medullary junction and a little less frequent in the medulla .
microscopic findings of buche - positive nerve fibers show the same density and the topography by the experimental and the control animals . on the other hand , ache - positive nerve fibers in experimental animals after administration of bendiocarbamate
microscopical findings related to ache - positive innervations of the thymus of rabbits which were administered bendiocarbamate , the inhibitor of ache , were rather indistinctive ( figures 1 and 2 ) .
one could not identify nerve structures with certainty and ache - positivity was exhibited only by non - neural cellular elements in all experimental materials examined ( figure 3 ) . in the control animals the ache - positive nerve fibers were observed in the organ capsule in cortex layer originating from the interlobular septum .
fine nerve fibers terminated in the cortex and frequently came to close contact with lymphocytes while in the deep cortex layer and medulla they were located close to epithelial cells . 

figure 1third day of the experiment ; nerve fibers are not visualized after administration of bendiocarbamate . a , artery
third day of the experiment ; nerve fibers are not visualized after administration of bendiocarbamate . a , artery
thymus of an experimental rabbit with very poorly visible ache - positive fibers in perivascular topography .
thymus of an experimental rabbit with very poorly visible ache - positive fibers in perivascular topography .
abundant reaction product in the non - cellular part of thymus of experimental animal lacking ache - positive nerve fibers .
abundant reaction product in the non - cellular part of thymus of experimental animal lacking ache - positive nerve fibers .
the distributions of buche - positive nerve fibers after visualization were the same in experimental and control animals .
nerve fibers entered the thymus as a common bundle with arteries , as typical periarterial plexuses ( figure 4 ) . in the organ they were running in interlobular septa as a common bundle of branching off fibers .
the highest density of thicker periarterial nerve plexuses with abundance of reaction product was observed at the cortico - medullary junction ( figure 5 ) , while the finer fibers were less abundant in this area .
a characteristic feature in this topography was the presence of non - neural cellular elements with high content of colored reaction product .
the outer medullary layer contained numerous thicker and thinner nerve fibers . in the cortex layer we observed predomination of fine nerve profiles without evident direct connection to vessels .
in the deep cortex topography we were able to observe thicker nerve fibers , particularly close to the cortico - medullary border .
fine buche nerve fibers were observed also in the thymic capsule . based on our findings
we can state that buche - positive innervations of thymus in experimental and control animals show no differences , it follows bendiocarbamate in dose of 5 mg / kg bw daily not inhibit butyrylcholinesterase . 

abundant buche - positive nerve fibres ( nf ) in perivascular topography and solitary nerve fibres originating from them .
the distribution and the density of nerve fibers are similar to the control animals . a , artery
abundant buche - positive nerve fibres ( nf ) in perivascular topography and solitary nerve fibres originating from them .
fine buche - positive nerve fibers ( nf ) at the cortico - medullary junction .
fine buche - positive nerve fibers ( nf ) at the cortico - medullary junction .
the thymus played a key role in the evolution of animals during development of an adaptive immune system ; it is therefore an important element , distinguishing higher vertebrates from other animals .
our observations of ache and buche- positive nerve fibers in rabbit 's thymuses proved their presence in interlobular septa , capsule , cortex , cortico - medullary junction and in the medulla .
our results are in agreement with the observations of authors who investigated autonomic innervations of thymuses in rats and mice .
functional changes after administration of cholinesterase inhibitors are characteristic of disorders of metabolic processes responsible for regulation of acid - base balance .
it is well known that surface membranes of various subpopulations of lymphoid cells harbor a number of specific receptors of both conventional neurotransmitters and some peptides . according to mignini et al .
, released neurotransmitters may affect functions of t and b lymphocytes as well as the process of entrapping antigens by cells .
our observations of buche - positive thymus innervations that bendiocarbamate blocks specific cholinesterases but will not accumulate in the body of mammals , as after 24 h it is released from mammal tissues either by urine or by lungs or faeces , are in agreement with the results of ratner et al .
long - term administration of bendiocarbamate caused a decrease in relative proportion of medulla at the expense of cortex which contained hyperplastic reticular cells .
our results allowed us to state that buche - positive innervations of thymus did not differ significantly between experimental and control animals , i.e. bendiocarbamate administered at a dose of 5 mg .
kg bw failed to inhibit visualization of buche - positive nerve structures . on the other hand
, ache - positive nerve profiles were not visualized in experimental animals after administration of this substance .
bendiocarbamate can affect not only the thymic innervations , but also the dividing of lymphocytes .
holeckov et al . investigated induction of the unstable chromosomal aberrations , sister chromatid exchanged and stable chromosomal aberration after bendiocarbamate administration in cultured peripheral bovine lymphocytes .
varga et al . found significant positive correlation in thymic size and number of lymphocytes in peripheral blood in newborns .
it has been assumed that suppression of the immune system by some pesticides forms the basis for increased number of allergies , hypersensitivity of organisms and susceptibility to tumour growth .
one of possible mechanisms of these observations is the oxidative stress : the action of xenobiotics is frequently associated with increased level of reactive forms of oxygen .
 many pesticides used in agriculture have a negative effect on organisms . 
 the group of hazardous pesticides includes the cholinesterase inhibitor bendiocarbamate . according to literature 
 , bendiocarbamate has relatively low toxicity in mammals and vertebrates in general , since it does accumulate in their tissues and the cholinesterase activity returns to norm within 24 h after acute exposure . 
 the present study focused on the influence of bendiocarbamate on rabbit thymus after its administration at a dose of 5 mg / kg body weight ( bw ) for the period of 3 months . 
 the thymus was observed for acetylcholinesterase ( ache ) and butyrylcholinesterase ( buche)-positive nerve fibers visualized by histochemical methods . 
 microscopic findings of buche - positive nerve fibers show the same density and the topography by the experimental and the control animals . on the other hand , 
 ache - positive nerve fibers in experimental animals after administration of bendiocarbamate is only poorly identified , suggesting that bendiocarbamate inhibits ache but not buche . 
Introduction
Materials and Methods
Results
Nerve fibers of thymus
Nerve fibers after administration of bendiocarbamate
Discussion