Milica Kovačević Filipović

Stereological and Immunohistochemical Study of the Spleen in Hypothyroid Juvenile Rats

Abstract The purpose of this work was to investigate the influence of hypothyroidism on spleen tissue morphology and immune cell density in fourteen-day-old juvenile rats. Hypothyroidism in pups (n=10) was induced by administration of propylthiouracil (PTU) in drinking water (1.5 mg/L) to their mothers during pregnancy and period of lactation. Fourteen-day-old pups were sacrificed and the thyroid-stimulating hormone (TSH) serum concentration and thyroid activation index (Ia) were determined. Increased serum level of TSH and increased Ia showed that pups from PTU treated mothers were hypothyroid. White and red spleen pulp, marginal zone and connective tissue volume density has been assessed by using the stereological method. Using immunohistochemistry, the present CD3+ T lymphocytes, CD45RA+ B lymphocytes and CD68+ macrophages were quantified. A significant reduction of volume density of the periarteriolar lymphocyte sheath (VvPALS) and lymphatic follicles (Vvf) due to depletion of T and B lymphocytes respectively, was observed in the spleens of hypothyroid pups compared to controls. The volume density of the red pulp (Vvrp), marginal zone (Vvmz) and connective tissue (Vvct) was increased, as well as the number of CD68+ macrophages in the spleens of hypothyroid pups compared to controls. These results indicate that thyroid hormones might be important for normal development of both, specific and innate immune cells in the spleen during prenatal and early postnatal period. Cilj ovog istraživanja je bio da ispita uticaj hipotireoidizma na morfologiju tkiva slezine i zastupljenost ćelija imunskog sistema kod dve nedelje starih juvenilnih pacova. Hipotireoidizam mladunaca (n=10) je indukovan aplikacijom propiltiouracila (PTU) u vodi za piće (1,5 mg/L), majkama tokom perioda graviditeta i laktacije. Dve nedelje stari mladunci su žrtvovani i određena im je koncentracija tireostimulirajućeg hormona (TSH) u serumu, kao i indeks aktivacije štitaste žlezde (Ia). Na osnovu povišenog nivoa TSH i povišenog Ia, utvrđeno je da je PTU aplikovan majkama, doveo do pojave hipotireoidizma kod njihovih potomaka. Volumenska gustina bele i crvene pulpe, marginalne zone i vezivnog tkiva slezine mladunaca ispitivana je stereološkom analizom. Zastupljenost CD3+ T limfocita, CD45RA+ B limfocita i CD68+ makrofaga u slezini je određena primenom imunohistohemijske metode. Kod hipotireoidnih mladunca postojalo je značajno smanjenje volumenske gustine periarterijskog limfocitnog omotača (VvPALS) i limfnih folikula (Vvf), kao i deplecija CD3+ T limfocita i CD45RA+ B limfocita, u poređenju sa mladuncima iz kontrolne grupe. Za razliku od bele pulpe, volumenska gustina crvene pulpe (Vvcp), marginalne zone (Vvmz) i vezivnog tkiva (Vvct), kao i broj CD68+ makrofaga je bio povećan kod hipotireoidnih mladunaca u odnosu na kontrolnu grupu. Ovi rezultati ukazuju da tireoidni hormoni mogu da budu važni za normalan razvoj ćelija specifi čnog i nespecifi čnog imunskog sistema tokom prenatalnog i ranog postnatalnog perioda razvoja.

Acute-phase response in Babesia canis and Dirofilaria immitis co-infections in dogs

Babesia canis and Dirofilaria immitis are emerging and geographically overlapping vector-borne pathogens in dogs. Infection with B. canis leads to acute-phase response (APR) that can be mild to severe and results in either non-complicated or complicated forms of the disease. The aim of this study was to determine whether acute B. canis infection is more severe in dogs with underlying asymptomatic D. immitis infection. Dogs of both sexes, different ages and breeds, with naturally occurring mono-infections with B. canis (n=13) and D. immitis (n=18) and co-infected dogs (n=7) were enrolled as well as healthy controls (n=15). Routine haematology and biochemistry, agarose gel electrophoresis (agEF) protein fraction separation and enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA) were performed. Based on clinical and laboratory findings, sepsis was diagnosed in the majority of dogs with acute B. canis infection with or without underlying asymptomatic D. immitis infection. Overall, haematology, biochemistry and agEF pattern changes were induced and dictated by acute B. canis infection whether or not the dogs had an asymptomatic D. immitis infection. D. immitis infection slightly influenced the level of anaemia, slightly aggravated the level of dehydration and increased the concentration of γ-globulins in acute-phase B. canis infection. D. immitis infection prevented B. canis-induced leukopenia. SAA equally increased in dogs with acute B. canis infection with or without underlying D. immitis infection. The level of SAA was not changed in dogs with asymptomatic D. immitis when compared to the controls. In conclusion, asymptomatic D. immitis infection does not influence overall APR after acute B. canis infection.

Altered state of primordial follicles in neonatal and early infantile rats due to maternal hypothyroidism: Light and electron microscopy approach

Thyroid hormones (TH) are one of the key factors for normal prenatal development in mammals. Previously, we showed that subclinical maternal hypothyroidism leads to premature atresia of ovarian follicles in female rat offspring in the pre-pubertal and pubertal periods. The influence of decreased concentration of TH on primordial follicles pool formation during neonatal and early infantile period of rat pups was not investigated previously. Maternal hypothyroidism during pregnancy has irreversible negative influence on primordial follicles pool formation and population of resting oocytes in female rat offspring. The study was done on neonatal and early infantile control (n-10) and hypothyroid (n-10) female rat pups derived from control (n-6) and propylthiouracil (PTU) treated pregnant dams (n-6), respectively. Ovaries of all pups were removed and processed for light and transmission electron microscopy (TEM). Number of nests, oogonia and oocytes per nest, primordial, primary, secondary and preantral follicles were determined. Screening for overall calcium presence in ovarian tissue was done using Alizarin red staining. Morphology and volume density of nucleus, mitochondria and smooth endoplasmic reticulum (sER) in the oocytes in primordial follicles was also assessed. Caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), both markers for apoptosis, and proliferating cell nuclear antigen (PCNA) for proliferation were determined in oocytes and granulosa cells in different type of follicles. In neonatal period, ovaries of hypothyroid pups had a decreased number of oogonia, oocytes and nests, an increased number of primordial follicles and a decreased number of primary and secondary follicles, while in early infantile period, increased number of primary, secondary and preantral follicles were found. Alizarin red staining was intense in hypothyroid neonatal rats that also had the highest content of dilated sER. Number of mitochondria with altered morphology in both groups of hypothyroid pups was increased. Apoptosis markers have not shown significant difference between groups but PCNA had an increased expression in the oocytes and granulosa cells in primordial follicles of hypothyroid rats. Light and electron microscopy analysis indicate that previously detected premature ovarian follicular atresia in pre-pubertal and pubertal hypothyroid rats is preceded with premature formation of primordial follicles followed by slight changes on sER and mitochondria in examined oocytes, and increased expression of PCNA.

The Blood is Rich in Different Types of Mesoderm Derived Stem and Progenitor Cells

The blood and bone marrow have been thoroughly investiagated for more than a century, but we are still gaining surprising new informations. Blood transports different mature cells such as erythrocytes, platelets and granulocytes, but curiously, the blood is also transporting a number of non-differentiated cells of various mesodermal lineages: hematopoietic and mesenchymal stem and progenitor cells, endothelial progenitor cells and very small embryonal like cells are some of the most impressive examples. In adults the bone marrow is the source of practiclly all cells that could be found in the blood. Stem and progenitor cells egress from the bone marrow and home to the bone marrow or various tissues in a highly regulated manner. The fact that the hematopoetic stem and progenitor cells traffi c through the blood and repopulate the bone marrow niche is largely explored in stem cell therapy in human medicine. In this review we will briefl y describe the main characteristics of stem and progenitor cells, the mechanisms of their mobilization from the bone marrow and homing to target tissues. Also, the history and importance of the fact that different stem, progenitor and precursor cells could be isolated from the blood circulation will be discussed in the light of informations concerning their use in human and veterinary medicine.

Effect of Subclinical and Overt Form of Rat Maternal Hypothyroidism on Offspring Endochondral Bone Formation

Abstract Maternal hypothyroidism in its overt form affects skeletal development of the offspring, but these data are not available for the subclinical form which is becoming very frequent among pregnant women. We hypothesized that the subclinical form of hypothyroidism in rat dams, infl uences the process of offspring endochondral ossifi cation affecting proliferation and differentiation of chondrocytes, osteoclasts and osteoblasts in pups. Seven-day-old male pups (n=18) derived from control dams and dams treated with a low dose (1.5 mg/L) or high dose (150 mg/L) of propylthiouracil in drinking water during pregnancy and lactation were used. Histomorphometric analysis of pups’ tibia proximal growth plate, expression of mRNA, immunohistochemical and histochemical visualization of extracellular matrix components was performed. The length of the tibia was reduced in hypothyroid pups. Secretion of type 2 and 10 collagens in the subclinical and overt form were lower while the amount of glycosaminoglycans was higher when compared with controls. Down-regulated tartrate resistant acid phosphatase mRNA indicated altered osteoclasts function while lower expression of dentin matrix acid protein-1 mRNA and reduced synthesis of type 1 collagen accentuated a compromised bone formation in the overt form of hypothyroidism. The subclinical form of maternal hypothyroidism had a negative effect on the differentiation of hypertrophic chondrocytes and calcifi ed cartilage removal in 7-day-old pups. In addition, overt hypothyroidism had a negative effect on the proliferation of chondrocytes and deposition of osteoid. Both forms of hypothyroidism resulted in a decrease of tibia length due to changes in growth plate formation.

Z-cells and oogonia/oocytes in the advanced process of autophagy are the dominant altered cells in the ovaries of hypothyroid newborn rats

Abstract Induced prenatal hypothyroidism in rat pups leads to accelerated primordial follicle assembly and premature follicular atresia with ovary failure. This work investigates the influence of maternal hypothyroidism induced with 6-n-propyl-2-thyouracil (PTU) on the number and morphology of oogonia/oocytes in newborn rat pups with light and transmission electron microscopy. Expression of apoptosis and autophagy markers in oogonia/oocytes were examined using immunohistochemistry. Hypothyroid newborn pups had a decreased number of mitotic and resting oogonia, while the number of altered oogonia/oocytes was increased. Ultrastructural observations revealed the increased presence of degenerated pachytene oocytes (Z-cells) and oogonia/oocytes undergoing autophagy, apoptosis and combined apoptosis and autophagy, in this group. The most abundant altered oogonia/oocytes in the hypothyroid group were those with morphological features of advanced autophagy and Z-cells. The percentage of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positive oogonia/oocytes was significantly lower in the hypothyroid group. No significant difference was recorded in the expression of caspase-3, ATG7 and LC3 possibly reflecting that these proteins were not involved in the oogonia/oocyte alteration process during prenatal rat hypothyroidism. The obtained results indicate that developmental hypothyroidism in the offspring enhances the number of Z-cells and oogonia/oocytes altered with the advanced process of autophagy.