Patulin is a mycotoxin produced by several Penicillium, Aspergillus, and Byssachlamys species growing on food products. In this study, we investigated the effects of patulin on the thymus of growing ...male rats aged five to six weeks. The rats were receiving it orally at a dose of 0.1 mg kg -1 bw a day for either 60 or 90 days. At the end of the experiment, the thymus was examined for histopathology by light microscopy and for epidermal growth factor (EGF) and its receptor (EGFR) by immunolocalisation. For morphometry we used the Bs200prop program to analyse images obtained with the Olympus BX51 light microscope. Cell ultrastructure was studied by electron microscopy. In rats treated with patulin, the thymus showed haemorrhage, plasma cell hyperplasia, a dilation and fibrosis in the cortex, enlarged interstitial tissue between the thymic lobules, enlarged fat tissue, thinning of the cortex, and blurring of the cortico-medullary demarcation. Electron microscopy showed signs of cell destruction, abnormalities of the nucleus and organelles, and loss of mitochondrial cristae. However, no differences were observed in thymus EGF and EGFR immunoreactivity between treated and control rats.
Patulin je mikotoksin koji proizvode plijesni sojeva Penicillium, Aspergillus i Byssachlamys na razli _itim prehrambenim proizvodima kao podlozi. U _inke patulina istražili smo na timusu mužjaka štakora u razvoju (dobi 5 do 6 tjedana). Mikotoksin je životinjama davan per os u dnevnoj dozi 0,1 mg kg -1 tj. t. 60 odnosno 90 dana. Na kraju pokusa štakori su žrtvovani, timus je podvrgnut histološkim analizama s pomoću svjetlosne mikroskopije, a imunocitokemijskim je metodama istražena stani _na lokalizacija epidermalnog faktora rasta (EGF) i njegova receptora (EGFR). Morfometrijska analiza provedena jes pomoću ra _unalnog programa Bs200prop povezanog u sustav sa svjetlosnim mikroskopom Olympus BX51. Elektronskomikroskopski je istražena ultrastruktura stanica timusa. Utvrđeno je da patulin izaziva krvaranja u timusu, hiperplaziju plazma-stanica, dilataciju i fibrozu u kortikalnoj regiji timusa, širenje intersticijskog tkiva između režnjeva timusa, povećanje masnih stanica, smanjenje debljine kore timusa te nestanak kortiko-medularne demarkacije. Elektronskomikroskopski u tkivu timusa štakora tretiranih patulinom uo _eni su znakovi raspadanja stanica, abnormalnosti jezgre i organela te gubitak mitohondrijskih krista. Unato _ navedenomu, na presjecima tkiva kontrolnih štakora i štakora tretiranih patulinom nismo utvrdili razlike u imunoreaktivnosti EGF i EGFR, što bi trebalo dodatno istražiti osjetljivijim molekularnim metodama.
The aim of the study was the effect of acute heat stress on the histological, immunohistochemical, ultrastructural, stereological and biochemical characteristics of corticotroph pituitary cells and ...cells of the adrenal glands of rats as a target organ of the hypothalamic-pituitary-adrenal axis in response to the effects of stressors. Wistar male rats were exposed to a temperature of 38°C in the chamber for 60 minutes. Animals were sacrificed by decapitation after treatment in the morning, at about the same time, in order to avoid the influence of circadian rhythm on the studied parameters. Corticotroph pituitary cells were analyzed at the level of light and
by transmission electron microscopy using immunohistochemical and stereological methods. The adrenal glands were studied by histological and ultrastructural characteristics of the cells of cortex and medulla. Quantitative characteristics of the adrenal glands were determined using stereological and morphometric methods at light and electron microscopy. Peptide neurons (VIP, SP and NPY) in the cortex and medulla of the adrenal glands were assessed by immunohistochemical methods. To show the flow of blood to the adrenal glands a Novelli histological method of staining was used. The cells in proliferation were assessed by Ki-67 antigen, whereas the detection of apoptotic cells of the adrenal gland was enabled by propidium-iodide staining. The concentration of pituitary adrenocorticotropic hormone (ACTH) and corticosterone, aldosterone, adrenaline and noradrenaline in the rat adrenal gland circulation were determined. In the pituitary, the applied treatment resulted in increased secretion of ACTH from corticotroph cells. On the level of light microscopy, it was demonstrated by decreased number of immunopositive ACTH cells, and at the level of electron microscopy by reduced number of granules in the ACTH cells. Accordingly, biochemical data showed that the concentration of ACTH in the circulation increased. The blood vessels of the pituitary gland in platelet aggregation were present. In the adrenal glands changes were evident at both light and electron microscopy level. In the ZG, there was an increased synthesis and secretion of aldosterone what was showed by significant reduction in the number of lipid droplets, increasing the number of mitochondria and high levels of
circulating aldosterone. The increase in concentration of circulating aldosterone is a response to dehydration caused by the exposure of animals to high temperature of the external environment. In the ZF a significant increase in the surface profile and volume of the cells were observed, particularly in cytoplasmic components, together with reduction in the number of lipid droplets, increasing number of mitochondria, and significant increase in corticosterone levels in the blood at the same time. Enlargement
of cells involved in the synthesis and secretion of the stress hormone corticosterone, changing of relative abundance of components in the cells in a direction which indicates a greater involvement in the synthesis of hormones, and biochemical data on the increase in circulating corticosterone levels suggested an active participation of ZF cells in response to stress caused by acute exposure of rats to elevated environmental temperatures. In the ZR an extremely high accumulation of lipid droplets was found, which
would point to a slowdown in the synthesis of hormones and secretion of this zone. In all zones of adrenal cortex, after acute exposure to the heat stress, the increase in the number of apoptotic cells, proliferation of connective tissue fibers, the reduction of NPY, VIP and SP immunoreactive nerve fibers and the apparent aggregation of platelets in the capillaries
were found. All this suggests that the acute heat stress, beside inspiring synthetic activity of parenchyma cells of ZG and ZF, had a general negative effect on the crust of the adrenal glands. Aggregation of platelets is probably the result of dehydration, while the occurrence of apoptosis and proliferation of connective tissue may be the predecessor of parenchymal fibrosis. In the adrenal medulla the largest changes were found in the adrenaline cells of which the profile surface was significantly reduced with
the decrease of cytoplasm and nuclei of cells. A significant reduction of full granules, while significantly increase in the other two categories of mobilized granules (altered and empty) and the presence of whole adrenaline granules in capillaries indicate strong ejection of adrenaline, which was confirmed by its high levels in the circulation. In noradrenaline cells of the adrenal gland there was redistribution in the presence of different types of granules, the decrease of full granules, and the increase of the number of
the other two types of granules. The concentration of noradrenaline in the systemic circulation was significantly increased. Given the fact that adrenaline and noradrenaline are directly involved in the reaction of "fight or flight", the above data complete the picture of the applied stress-inducing effect of experimental treatment. In various ganglionic, neural and catecholamine cells of the adrenal glands, as well as in the region of the cortex, reduced NPY, VIP and SP immunoreactivity were found. Based on the foregoing, it is clear that the acute exposure of rats to elevated environmental temperature for 60 minutes, it was enough to trigger the mechanisms involved in the stress response, both on the biochemical and cellular level. In addition to increased levels of circulating hormones,
which can be detected almost immediately after the commencement of stress acting stimuli, adjustment of the fine structure of cells is evident, requiring more time and basing itself on more complex mechanisms. In addition, according to the obtained results it appears that the adrenal glands, even after this acute treatment aim at complex hystomorphologic alterations. In that case, it may not be possible to ignore the serious pathological consequences such as embolism of blood vessels caused by increased platelet aggregation.
Ispitivan je uticaj akutnog toplotnog stresa na histološke,
imunohistohemijske, ultrastrukturne, stereološke i biohemijske karakteristike
adrenokortikotropnih ćelija hipofize i ćelija nadbubrežnih žlezda pacova kao
ciljnih organa hipotalamo-hipofizne-adrenalne osovine u odgovoru na dejstvo
stresora. Pacovi mužjaci soja Wistar izlagani su temperaturi od 38°C u komori
u trajanju od 60 minuta. Životinje su nakon tretmana žrtvovane dekapitacijom
u prepodnevnim časovima, u približno isto vreme, da bi se izbegao uticaj
cirkadijalnog ritma na proučavane parametre. Kortikotropne ćelije
adenohipofize su analizirane na nivou svetlosne i transmisione elektronske
mikroskopije primenom imunohistohemijskih i stereoloških metoda. U
nadbubrežnim žlezdama ispitivane su histološke i ultrastrukturne
karakteristike ćelija kore i srži. Kvantitativne odlike nadbubrežnih žlezda
određivane su primenom stereoloških i morfometrijskih metoda na nivou
svetlosne i elektronske mikroskopije. Peptidergički neuroni (VIP, SP i NPY) u
kori i srži nadbubrežnih žlezda procenjivani su imunohistohemijskim metodama.
Za prikazivanje protoka krvi u nadbubrežnim žlezdama korišćena je Noveli
histološka metoda bojenja. Ćelije u proliferaciji procenjivane su preko Ki-67
antigena, dok je za detekciju apoptotskih ćelija nadbubrežnih žlezdi
primenjeno bojenje propidijum-jodidom. Određivane su i koncentracije
adrenokortikotropnog hormona hipofize (ACTH), kao i kortikosterona,
aldosterona, adrenalina i noradrenalina nadbubrežnih žlezda pacova u
cirkulaciji. U hipofizi, primenjeni tretman doveo je do povećanog izlučivanja
ACTH iz kortikotropnih ćelija. Ovo se na nivou svetlosne mikroskopije
manifestovalo smanjenjem broja ćelija imunopozitivnih na ACTH, a na nivou
elektronske mikroskopije smanjenjem broja granula u ACTH ćelijama. U skladu
sa tim, biohemijski podaci pokazali su da je koncentracija ACTH u cirkulaciji
povećana. U krvnim sudovima hipofize prisutna je agregacija krvnih pločica. U nadbubrežnim žlezdama promene su bile izraženije kako na nivou svetlosne tako i na nivou elektronske mikroskopije. U ZG kore nadbubrežnih žlezda došlo je do povećane sinteze i sekrecije aldosterona na šta su ukazali značajno smanjenje broja lipidnih kapljica, uvećanje broja mitohondrija i visok nivo aldosterona u cirkulaciji. Porast koncentracije aldosterona u cirkulaciji
predstavlja odgovor organizma na dehidrataciju izazvaju izlaganjem životinja visokoj temperaturi spoljne sredine. U ZF zapaženo je značajno povećanje površine profila i volumena ćelija, posebno citoplazmatične komponente, zajedno sa smanjenjem broja lipidnih kapi, porastom broja mitohondrija, uz značajno povećanje nivoa kortikosterona u krvi. Uvećanje ćelija uključenih u
sintezu i sekreciju hormona stresa, kortikosterona, promena relativne zastupljenosti subćelijskih komponenata u pravcu koji ukazuje na veću angažovanost u sintezi hormona, kao i biohemijski podaci o povišenju nivoa kortikosterona u cirkulaciji ukazuju na aktivno učešće ćelija ZF u odgovoru na stres izazvan akutnim izlaganjem pacova povišenoj temperaturi spoljne sredine. U ZR je nađeno izuzetno veliko nagomilavanje lipidnih kapljica što bi ukazivalo na zastoj u sintezi i sekreciji hormona ove zone. U svim zonama
kore nadbubrežnih žlezda, posle akutnog izlaganja toplotnom stresu, nađeni su uvećanje broja apoptotskih ćelija, proliferacija vlakana vezivnog tkiva, smanjenje NPY,VIP i SP imunoreaktivnosti nervnih vlakana i primetna agregacija krvnih pločica u kapilarima. Sve ovo upućuje na zaključak da je akutni toplotni stres, pored toga što je podstakao sintetsku i sekretnu aktivnost parenhimskih ćelija ZG i ZF, imao opšte nepovoljno dejstvo na koru nadbubrežnih žlezda. Agregacija krvnih pločica verovatno je posledica
dehidratacije, dok pojava apoptoze i proliferacija vezivnog tkiva mogu biti prethodnice fibrozi parenhima.
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